tecnai 12 alignments - uzh · tecnai on-line help alignments 1 tecnai 12 software version 4.0...

Tecnai on-line help Alignments 1 Tecnai 12 Software version 4.0

Tecnai on-line help manual -- Alignments Table of Contents 1 Alignments in the Tecnai microscope.................................................................................................. 4 2 Alignment procedures ......................................................................................................................... 5 3 Introduction to electron optics ............................................................................................................. 9

3.1 Electron gun ................................................................................................................................. 9 3.1.1 Thermionic gun ..................................................................................................................... 9 3.1.2 Field Emission Gun.............................................................................................................10

3.2 Lenses........................................................................................................................................ 11 3.3 Deflection coils ........................................................................................................................... 12

3.3.1 Pivot points ......................................................................................................................... 13 3.3.2 Gun coils ............................................................................................................................. 14 3.3.3 Beam coils .......................................................................................................................... 14 3.3.4 Image coils.......................................................................................................................... 15

3.4 Stigmators .................................................................................................................................. 15 3.5 Focusing the diffraction pattern.................................................................................................. 16 3.6 The diffraction shadow image .................................................................................................... 17

4 Gun procedure .................................................................................................................................. 19 4.1 Gun tilt ........................................................................................................................................ 19 4.2 Gun tilt pivot points..................................................................................................................... 19 4.3 Gun shift ..................................................................................................................................... 20 4.4 Spot-size dependent gun shift.................................................................................................... 21

5 Beam HM-TEM procedure ................................................................................................................ 22 5.1 Preparation Beam HM-TEM alignment ...................................................................................... 22 5.2 Adjust Minicondenser lens ......................................................................................................... 22 5.3 Pivot point beam shift HM .......................................................................................................... 22 5.4 Pivot point beam tilt HM ............................................................................................................. 23 5.5 Dynamic conical dark field pivot point HM (STEM systems only) .............................................. 23 5.6 Dynamic conical dark field distortion HM (STEM systems only) ................................................ 24 5.7 HM-TEM rotation center............................................................................................................. 24 5.8 Calibrate Trackball ..................................................................................................................... 24 5.9 Beam shift calibration HM .......................................................................................................... 25 5.10 Beam tilt (dark field) calibration HM ........................................................................................... 25 5.11 Dynamic conical dark field beam tilt calibration HM (STEM systems only)................................ 25 5.12 Spot size-intensity calibration..................................................................................................... 26

6 Image HM-TEM Procedure ............................................................................................................... 27 6.1 Preparation Image HM-TEM alignment...................................................................................... 27 6.2 Pivot point image shift HM ......................................................................................................... 27 6.3 Pivot point diffraction shift SA .................................................................................................... 27 6.4 Objective Lens preset SA........................................................................................................... 28 6.5 Pivot point diffraction shift Mh .................................................................................................... 29 6.6 Mh objective-lens preset and image shift ................................................................................... 29 6.7 Image shifts SA .......................................................................................................................... 30 6.8 Pivot point diffraction shift Mi ..................................................................................................... 31 6.9 Mi objective lens preset and image shift .................................................................................... 31 6.10 Mi cross-over shift ...................................................................................................................... 32 6.11 Align diffraction pattern .............................................................................................................. 32 6.12 Align camera lengths.................................................................................................................. 32 6.13 Align camera length Mh ............................................................................................................. 33


6.14 Image shift calibration HM.......................................................................................................... 33 6.15 Diffraction shift calibration HM ................................................................................................... 34 6.16 Beam shift - image shift calibration HM...................................................................................... 34 6.17 Off-axis TV HM image alignment (only if off-axis TV installed) .................................................. 35 6.18 Off-axis TV diffraction alignment (only if off-axis TV installed) ................................................... 36

7 Beam LM Procedure ......................................................................................................................... 37 7.1 Preparation Beam LM alignment................................................................................................ 37 7.2 Pivot point beam shift LM........................................................................................................... 37 7.3 Pivot point beam tilt LM.............................................................................................................. 37 7.4 LM rotation center ...................................................................................................................... 38 7.5 Calibrate Trackball ..................................................................................................................... 38 7.6 Beam shift calibration LM........................................................................................................... 38 7.7 Beam tilt (dark field) calibration LM............................................................................................ 39 7.8 Spot size-intensity calibration LM............................................................................................... 39

8 Image LM Procedure......................................................................................................................... 41 8.1 Preparation Image LM alignment ............................................................................................... 41 8.2 Pivot point image shift LM .......................................................................................................... 41 8.3 Pivot point diffraction shift LM .................................................................................................... 41 8.4 LM images.................................................................................................................................. 42 8.5 Align LAD diffraction pattern ...................................................................................................... 42 8.6 Image shift calibration LM .......................................................................................................... 43 8.7 Diffraction shift calibration LAD .................................................................................................. 43 8.8 Beam shift - image shift calibration LM ...................................................................................... 44 8.9 Off-axis TV LM image alignment (only if off-axis TV installed)................................................... 45

9 Beam Nanoprobe procedure ............................................................................................................. 46 9.1 Preparation Beam Nanoprobe alignment ................................................................................... 46 9.2 Pivot point beam shift Nanoprobe .............................................................................................. 46 9.3 Pivot point beam tilt Nanoprobe ................................................................................................. 46 9.4 Dynamic conical dark field pivot point Nanoprobe (STEM systems only) .................................. 47 9.5 Dynamic conical dark field distortion Nanoprobe (STEM systems only) .................................... 47 9.6 Nanoprobe rotation center ......................................................................................................... 47 9.7 Calibrate Trackball ..................................................................................................................... 48 9.8 Beam shift calibration Nanoprobe .............................................................................................. 48 9.9 Beam tilt (dark field) calibration Nanoprobe ............................................................................... 49 9.10 Dynamic conical dark field beam tilt calibration Nanoprobe (STEM systems only).................... 49 9.11 Spot size-intensity calibration Nanoprobe.................................................................................. 50

10 Image Nanoprobe Procedure ........................................................................................................ 51 10.1 SA objective lens preset Nanoprobe.......................................................................................... 51 10.2 Mh objective lens preset Nanoprobe.......................................................................................... 51 10.3 Mi objective lens preset Nanoprobe........................................................................................... 51 10.4 Align diffraction pattern (Nanoprobe) ......................................................................................... 51 10.5 Beam shift - image shift calibration Nanoprobe.......................................................................... 52

11 Stigmator Procedure...................................................................................................................... 53 11.1 Condenser stigmator calibration ................................................................................................53 11.2 Objective stigmator calibration ................................................................................................... 53 11.3 Diffraction stigmator calibration.................................................................................................. 54

12 HM-STEM Procedure .................................................................................................................... 55 12.1 HM-STEM Preparation............................................................................................................... 55 12.2 HM-STEM Objective / Intensity preset ....................................................................................... 55 12.3 HM-STEM Beam tilt pivot points ................................................................................................ 56 12.4 HM-STEM Rotation center ......................................................................................................... 57 12.5 HM-STEM Beam-shift pivot points ............................................................................................. 58 12.6 HM-STEM Align diffraction pattern............................................................................................. 58


12.7 HM-STEM Detector alignment ................................................................................................... 59 12.8 HM-STEM Distortion adjustment................................................................................................ 61 12.9 HM-STEM Default rotation ......................................................................................................... 62

13 LM-STEM procedure ..................................................................................................................... 64 13.1 LM-STEM Preparation ............................................................................................................... 64 13.2 LM-STEM Intensity preset.......................................................................................................... 64 13.3 LM-STEM Beam-shift pivot points.............................................................................................. 64 13.4 LM-STEM Align diffraction pattern ............................................................................................. 65 13.5 LM-STEM Detector alignment .................................................................................................... 65 13.6 LM-STEM Distortion adjustment ................................................................................................ 67 13.7 LM-STEM Default rotation.......................................................................................................... 68

14 HM-EFTEM procedure................................................................................................................... 69 14.1 HM-EFTEM Preparation............................................................................................................. 69 14.2 HM-EFTEM Image-shift pivot points .......................................................................................... 69 14.3 HM-EFTEM SA Diffraction-shift pivot points .............................................................................. 70 14.4 HM-EFTEM Mh Diffraction-shift pivot points .............................................................................. 70 14.5 HM-EFTEM Mi Diffraction-shift pivot points ............................................................................... 71 14.6 HM-EFTEM Mh Pre-alignment ................................................................................................... 72 14.7 HM-EFTEM SA Image-shift pre-alignment................................................................................. 72 14.8 HM-EFTEM Mi Image-shift pre-alignment.................................................................................. 73 14.9 HM-EFTEM Mh Image shift........................................................................................................73 14.10 HM-EFTEM SA Image shift ........................................................................................................74 14.11 HM-EFTEM Mi Image shift ......................................................................................................... 74 14.12 HM-EFTEM SA and Mi Cross-over correction ........................................................................... 75 14.13 HM-EFTEM Camera-length pre-alignment................................................................................. 78 14.14 HM-EFTEM Camera-length alignment ....................................................................................... 78

15 LM-EFTEM Procedure................................................................................................................... 80 15.1 LM-EFTEM Preparation ............................................................................................................. 80 15.2 LM-EFTEM Image-shift pivot points ........................................................................................... 80 15.3 LM-EFTEM Diffraction-shift pivot points..................................................................................... 80 15.4 LM-EFTEM Image-shift pre-alignment ....................................................................................... 81 15.5 LM-EFTEM Image-shift alignment ............................................................................................. 82

16 EFTEM Nanoprobe Procedure ...................................................................................................... 83 16.1 SA objective lens preset EFTEM Nanoprobe............................................................................. 83


1 Alignments in the Tecnai microscope When a user logs into the microscope (by starting the Tecnai user interface), the microscope will recall the necessary alignments. The microscope will follow a fixed procedure in restoring these alignments: • Look if a particular alignment exists for the user and, if so, load it. • If no user alignment exists, look if the alignment exists for the supervisor and, if so, load it. • If no supervisor alignment exists, look if the alignments exists for service and, if so, load it. • If no service alignment exists, look if the alignments exists for factory and, if so, load it. • If no factory alignments exists, load default settings. Alignments will exist for a particular user if the user has ever executed (part) of an alignment procedure. Alignments are as much as possible saved as a single parameter (except in the case of linked parameters like pivot points or x-y values which are always kept together). A complete alignment for a user may thus consist of a mix of user-aligned values and values from other levels (supervisor, ...). A user who has never done a gun alignment thus inherits the alignment from supervisor or higher. If the filament (on LaB6 or W) or FEG tip has been changed and a new alignment done by the higher level, the user will automatically get the new, correct alignment. When alignments are stored, they are stored completely (thus not only the user's own values). Upon restore the microscope will compare the values being reloaded. If the values are identical to the values in the next higher existing level, the values are not stored in the user's own alignments.


2 Alignment procedures Below is a list of alignment procedures. Some of the procedure steps may not be visible (dependent on user level 'user' or 'expert'). Some alignments like STEM, EFTEM and Lorentz may also be absent, since they depend on the hardware configuration of the instrument. General notes: • In principle some alignments could be combined in single steps, thereby reducing the number of

steps. In practice, it is then often forgotten to one of the possible alignments, with the consequence that one alignment is then misaligned. Alignment procedure steps generally therefore perform only a single alignment at a time.

• In an alignment procedure some steps may be skipped when using Next and Previous. This is done to skip those alignments that are not sensitive to changes in operating conditions (like pivot points), so that the procedure only follows the more often-used alignments. The visual indication which alignments are skipped and which are executed, takes the form of two different icons in front of the subprocedure step. Where the icon is a blue arrow on a white background, pointing to the right (into the subprocedure) the subprocedure is not skipped. Where the arrow points down on a yellow background, the subprocedure is skipped.

• Some alignment procedures start with a preparation step. This step can be necessary because otherwise the entry point for the whole procedure would be in a 'skipped' subprocedure (see previous point).

The following alignment procedures exist: Gun • Gun tilt • Gun tilt pivot point • Gun shift • Spot-size dependent gun shift Beam HM-TEM • Preparation • Minicondenser lens • Beam shift pivot point • Beam tilt pivot point • Dynamic conical dark field pivot point HM (STEM systems only) • Dynamic conical dark field distortion HM (STEM systems only) • Rotation center • Calibrate Trackball • Beam shift calibration • Beam tilt calibration • Dynamic conical dark field beam tilt calibration HM (STEM systems only) • Spot size-intensity calibration • Coma-free amplitude • Coma-free pivot points • Coma-free alignment


Image HM-TEM • Preparation • Image shift pivot point HM • Diffraction shift pivot point SA • SA objective lens preset • Diffraction shift pivot point Mh • Mh preset and alignment • SA magnifications alignment • Diffraction shift pivot point Mi • Mi preset and alignment • Align diffraction pattern • Align camera lengths • Image shift calibration • Diffraction shift calibration • Beam shift - image shift calibration • Off-axis TV HM image alignment (only if off-axis TV installed) • Off-axis TV diffraction alignment (only if off-axis TV installed) Beam LM • Preparation • Beam shift pivot point • Beam tilt pivot point • Rotation center • Calibrate Trackball • Beam shift calibration • Beam tilt calibration • Spot size-intensity calibration Image LM • Preparation • Image shift pivot point • Diffraction shift pivot point • LM magnifications alignment • Align LAD pattern • Image shift calibration • Diffraction shift calibration • Beam shift - image shift calibration • Off-axis TV LM image alignment (only if off-axis TV installed)


Beam Nanoprobe • Preparation • Beam shift pivot point • Beam tilt pivot point • Dynamic conical dark field pivot point Nanoprobe (STEM systems only) • Dynamic conical dark field distortion Nanoprobe (STEM systems only) • Rotation center • Calibrate Trackball • Beam shift calibration • Beam tilt calibration • Dynamic conical dark field beam tilt calibration Nanoprobe (STEM systems only) • Spot size-intensity calibration Image Nanoprobe • SA objective lens preset • Mh objective lens preset • Mi objective lens preset • Align diffraction pattern • Beam shift - image shift calibration Stigmators • Condenser • Condenser stigmator shunt • Objective • Diffraction HM-STEM • Preparation • Objective / Intensity preset • Beam tilt pivot points • Rotation center • Beam shift pivot points • Align diffraction pattern • Detector alignment • Scan distortion adjustment • Default scan rotation • AC shunt • Calibration LM-STEM • Preparation • Intensity preset • Beam shift pivot points • Align diffraction pattern • Detector alignment • Scan distortion adjustment • Default scan rotation


EFTEM HM • Preparation • HM Image-shift pivot points • SA Diffraction-shift pivot points • Mh Diffraction-shift pivot points • Mi Diffraction-shift pivot points • Mh Pre-alignment • SA Image-shift pre-alignment • Mi Image shift pre-alignment • Mh Image-shift alignment • SA Image-shift alignment • Mi Image-shift alignment • SA and Mi Cross-over correction alignment • Camera length pre-alignment • Camera length alignment EFTEM LM • Preparation • Image-shift pivot points • Diffraction-shift pivot points • Image-shift pre-alignment • Image-shift alignment EFTEM Nanoprobe • SA preset


3 Introduction to electron optics The microscope consists essentially of three parts: 1. The electron gun where the beam is generated. 2. The lenses, deflection coils and stigmators that make the image and project it on the screen. 3. The projection chamber with one or more types of electron detectors to record images, diffraction

patterns, ... (plate camera, TV, ...). The first two topics will be covered in this section.

3.1 Electron gun The electron beam is generated in the electron gun. Two basic types of gun can be distinguished: the thermionic gun and the field emission gun (FEG). • Thermionic guns are based on two types of filaments: tungsten (W) and lanthanum-hexaboride

(LaB6) (cerium-hexaboride, CeB6, can also be used instead of LaB6; its performance is roughly the same as that of LaB6). On modern instruments the different types of thermionic filaments can be used interchangeably.

• The FEG employs either a (thermally-assisted) cold field emitter - as on the Philips EM 400-FEG - or a Schottky emitter - as on the more recent generations of FEG microscopes (CM20/CM200 FEG, CM30/CM300 FEG, Tecnai F20 and F30).

3.1.1 Thermionic gun The thermionic gun (so-called triode or self-biassing gun) consists of three elements: the filament (cathode), the Wehnelt and the anode. The Wehnelt has a potential that is more negative - the bias voltage - than the cathode itself. The bias voltage is variable (controlled by the Emission parameter) and is used for controlling the emission from the filament. A high bias voltage restricts the emission to a small area, thereby reducing the total emitted current, while lowering the bias voltage increases the size of the emitting area and thus the total emission current.

The emitted electrons that pass through the Wehnelt aperture are focused into a cross-over between the cathode and anode. This cross-over acts as the electron source for the optics of the microscope.

The size of the cross-over is determined by the type of filament, the electric field between cathode and anode and by the exit angles of the electrons from the filament. At low bias voltages, electrons are emitted from a larger area of the curved tip of the filament, causing a higher divergence of emission angles and thus a larger source size. Higher emission therefore not necessarily improves the brightness (a performance parameter of the emitter, measured in A/cm²srad). In addition, higher emission increases the Coulomb interaction between electrons - the so-called Boersch effect - (some get accelerated, others decelerated) which increases the energy spread.


3.1.2 Field Emission Gun In the case of a Field Emission Gun (abbreviated FEG), electron emission is achieved in a different way than with thermionic guns. Because a FEG requires a different gun design as well as much better vacuum in the gun area (~10e-8 Pa instead of the ~10e-5 Pa necessary for thermionic guns), it is found only on dedicated microscopes (Tecnai F20, F30). The FEG consists of a small single-crystal tungsten needle that is put in a strong extraction voltage (2-5 kV). In the case of a cold FEG or thermally-assisted cold FEG, the needle is so sharp that electrons are extracted directly from the tip. For the Schottky FEG (as used on the Tecnai microscopes) a broader tip is used which has a surface layer of zirconia (ZrO2). The zirconia lowers the work function of the tungsten (that is, it enhances electron emission) and thereby makes it possible to use the broader tip. Unlike the thermionic gun, the FEG does not produce a small cross-over directly below the emitter, but the electron trajectories seemingly originate inside the tip itself, forming a virtual source of electrons for the microscope.

The FEG emitter is placed in a cap (suppressor) which prevents electron emission from the shaft of the emitter and the heating filament (very similar to the Wehnelt of the thermionic gun). Electron emission is regulated by the voltage on the extraction anode. Underneath the extraction anode of the FEG is a small electrostatic lens, the gun lens. This lens is used to position the first cross-over after the gun in relation to the beam-defining aperture (usually the C2 aperture). If the gun lens is strong, the cross-over lies high above the aperture while a weak gun lens positions the cross-over close to the aperture, giving a high current but at the expense of aberrations on the beam. A strong gun lens is therefore used where small, intense and low-aberration electron probes are needed (diffraction, analysis and scanning), while a weak gun lens is used when high currents are important (TEM imaging). In the latter case, the beam is spread and the aberrations do not affect the area within the field of view. The high brightness of FEGs comes about because of two reasons: 1. The small size of the tip ensures that large numbers of electrons are emitted from a small area (high

A/cm²). 2. The electrons come out of the tungsten crystal with a very restricted range of emission angles (high

A/srad). FEGs also have a low energy spread due to their low working temperature and emission geometry (small virtual source size, but much larger actual size of the emitting area).


3.2 Lenses The lenses in electron microscopes are electromagnetic lenses (the only exception being the gun lens in the FEG instruments, which is an electrostatic lens). These lenses all consist of a coil, through which an electrical current flows, and a magnetic circuit, which is a piece of magnetic alloy with a specific shape. The current flowing through the coil generates a magnetic field in the magnetic circuit. Where the circuit is interrupted (the gap), the magnetic field goes out into the vacuum and creates the lens field that is used for focusing the electron beam. How the lens works is determined by the shape of the pole piece (the part of the magnetic circuit where the bore and gap are). Water flows through pipes to remove the heat generated by the electron current in the lens coil.

Changing the current through the lens coil changes the magnetic field and thus the strength of the lens. Although electromagnetic lenses and electrons behave quite differently from light lenses and light, the general principles of light optics can be applied and the electromagnetic lenses can be described for convenience like the lenses of light optics. The TEM usually contains two condenser lenses: • The first condenser lens, or C1, determines the demagnification (size reduction) of the electron

source onto the specimen and thus the spot size. Its control is found under the spot size control, which has 11 steps.

• The second condenser lens, or C2, determines how strongly the beam is focused onto the specimen. As a consequence it varies the intensity of the beam on the viewing screen. The C2 lens is controlled through the Intensity knob. Inside or close to the second condenser lens there is an aperture (the second-condenser or C2 aperture), which is used as the beam-defining aperture (it limits the amount of the beam convergence for a fully focused beam).

The magnification system of the microscope consists of a set of five lenses: the objective, diffraction, intermediate, projector 1 and projector 2 lenses. Except in low-magnification (LM) mode, the objective lens is always the strongest lens in the microscope, magnifying between about 20 and 50x, depending on the type of objective lens. The individual lenses of the magnification (or projector) system are not controlled directly by the operator, but instead the microscope contains a number of magnifications for image and diffraction mode, each with its own settings of the magnifying lenses. The only lenses that are controlled directly by the operator are the objective lens (for focusing the image) and the diffraction lens (for focusing the diffraction pattern). In LM mode the objective lens is switched (nearly) off in order to achieve the smallest magnifications. With the objective lens off, the diffraction lens is used for focusing the image. The electron-optical configuration in LM is reversed with respect to the high-magnification range: the functions of the


objective and diffraction lenses and stigmators switch as do the functions of the objective and selected-area apertures. Lens and aperture functions in HM (objective lens on) and LM (objective lens off) High Magn Low Magn Obj. lens Image focus Diffraction (LAD) focus Diff. lens Diffraction focus Image focus Obj. aperture Contrast forming Area selection SA aperture Area selection Contrast forming Obj. stigmator Image stigmation Diffraction stigmation Diff. stigmator Diffraction stigmation Image stigmation TWIN-type objective lenses Most Tecnai microscopes are equipped with a TWIN-type objective lens (the variants BioTWIN, TWIN, S-TWIN and U-TWIN). The lens design and the two resulting basic optical modes, the microprobe and nanoprobe modes, are discussed separately in more detail.

3.3 Deflection coils Throughout the microscope, the path followed by the electron beam is affected by a number of deflection coils, mounted in different locations. Deflection coils play an essential role in the alignment of the microscope and are used for aligning the gun, beam, objective lens, magnification system (image and diffraction shifts to the screen center) and detector alignments (image or diffraction shifts to a detector that is situated off the optical axis). Most of the steps in the alignment procedures either align the deflection coils themselves or use the deflection coils to align another electron-optical element. In principle a single deflection coil is sufficient for a particular action, provided that it is mounted at the level where its action is needed. In practice, such arrangements are not feasible due to space limitations or other constraints. All deflections are done therefore through double deflection coils that are situated at another level in the microscope.

A deflection coil is a set of coils on either side of the electron beam. If one is given a positive magnetic field and the other one a negative one, the electrons in the beam will be attracted by the positive field and repelled by the other, leading to a deflection towards the positive coil. The actual coils are extended over arcs of 120°. The arcs are used to generate a homogeneous magnetic field. By arranging the coils in sets of two, mounted perpendicular to each other (X and Y directions), the beam can be deflected into any direction by a suitable combination of x and y. The deflection coils are always mounted in sets of two above another (so-called double deflection coils). Use of double deflection coils involves the important concept of pivot points as explained below.

Each microscope has three sets of double deflection coils: the gun coils just underneath the electron gun (or underneath the high-tension accelerator in case of 200 or 300 kV instruments); the beam deflection coils above the objective lens; and the image deflection coils below the objective lens. An additional, more simple, one-directional coil forms the microscope shutter that is used for exposure of the negatives.


3.3.1 Pivot points Double deflection coils are capable of two completely independent actions, a tilt and a shift. These two actions should be decoupled, that is, when a shift is intended only a shift and no tilt should occur (a pure shift) and vice versa (pure tilt). Examples of the importance of pure shift are: • high-resolution imaging, where a beam tilt would undo all the effort spent in correctly aligning the

objective lens; • scanning, where a tilt in addition to the beam shift will change the magnification; • TEM dark-field imaging, where a beam shift with an additional beam tilt would change the incident-

beam direction and thus the nature of the diffracting condition. Because of the importance of pure shift and pure tilt, considerable effort is spent in correctly aligning the deflection coils. No two electron microscope columns are exactly identical and slight differences that exist between deflection coils make it necessary to align the coils by means of setting pivot points. A pivot point is simply a point around which the beam will pivot (like the analogue of the seesaw in the children's' playground). The alignment of the pivot point determines the relation between the two coils used, making sure that the beam pivots around the correct point.

The concept of the pivot point is probably easiest to understand for beam deflection coils in a simplified microscope consisting of a double deflection coil followed by a lens with equal distances between the deflection coils and between the lower coil and the image plane above the lens.

A beam shift comes about by deflecting the beam through an angle a by the upper coil and then doing the reverse (-a) with the lower coil. In a perfect system the beam would come out parallel to its initial direction but displaced sideways. Since all beams that are parallel at the image plane must go through a single point in the back-focal plane, shifting the beam should have no effect on the location of the beam in the back-focal plane. A beam tilt comes about by deflecting the beam through an angle a with the upper deflection coil and then deflecting by -2a by the lower coil. A beam tilt will result in a beam shift in the back-focal plane but should cause no shift in the image plane.


If a combination of beam shift and beam tilt is needed, then the settings for these are simply added. In the example above, setting beam tilt plus beam shift would involve setting an angle 2a on the upper coils and -3a on the lower coil. Setting the pivot points is done by deflecting the beam with a wobbler and minimising any movement - of the beam in the diffraction plane in the case of beam shift (no tilt should occur) and of the beam in the image in the case of beam tilt (no shift should occur). A wobbler is a mechanism for rapidly switching a microscope element or function from a negative value to an identical but positive value; it can thus be on beam shift or beam tilt, image shift, a stigmator, objective-lens current, high tension, etc., even though the traditional meaning is the beam-tilt aid for focusing the TEM image. Since a beam tilt is visible in diffraction as a diffraction shift, beam shift pivot points are set in diffraction mode, while beam tilt pivot points are set in image mode - where a beam shift will be visible. Where it is important, pivot point alignment has two adjustable directions - a main one and the perpendicular correction. If the coils were perfect, the latter would not be necessary. In practice a small correction may be needed, because the lower coils is rotated slightly relative to the upper one. If the perpendicular correction is unnecessary (e.g. for the gun tilt pivot points), then only the main direction is adjustable (only the Multifunction X knob works).

3.3.2 Gun coils The gun deflection coils are situated directly underneath the anode in the case of a Tecnai 10 or 12 and below the high-tension accelerator in the case of Tecnai 20, F20, 30 and F30. These coils perform two functions. They make sure that the electron beam enters the microscope (that is, the C1 lens) parallel to the optical axis by means of the gun tilt and that the beam goes through the center of the C1 lens by means of the gun shift.

3.3.3 Beam coils The beam deflection coils, situated above the objective lens, serve many purposes. They shift and tilt the beam, both static and dynamic (the latter in most of the scanning modes), are used for aligning the objective lens, and correct beam movement caused by the condenser stigmator. They play a role therefore in many alignment steps. In addition, the beam deflection coils can be used coupled to the image deflection coils in a number of instances, for example for image shift or descanning.


3.3.4 Image coils The image deflection coils, situated below the objective lens, have many uses. They shift the image and the diffraction pattern, to align various magnifications, camera lengths and modes (such as TEM and STEM), they correct image or diffraction-pattern movement caused by the objective and diffraction stigmators, respectively, and set the Detector alignments that move the image or diffraction pattern to a detector that is situated off the microscope axis (STEM BF/DF, TV). In addition, the image deflection coils can be used coupled to the beam deflection coils in a number of instances, for example for image shift or descanning.

3.4 Stigmators Even though considerable effort is spent in order to ensure high lens quality, none of the lenses in a microscope is 100 percent perfect. Small inhomogeneities remain or can come about later, for instance by dust adhering to a pole piece or by magnetism or charging of the specimen itself. These imperfections cause a loss of rotational symmetry of the lens. In one direction the lens will therefore focus more strongly than in the perpendicular direction, causing an asymmetry called astigmatism. This image defect is corrected by the stigmator. The stigmator consists of a quadrupole, which basically is a lens whose astigmatism can be varied continuously. The quadrupole has four elements, arranged at 90 degrees around the beam. These elements are used together in two sets, with each set lying on opposite sides of the beam. If one set is given a positive value and the other a negative, then the positive elements will attract the electrons and have a defocusing effect, while the negative elements repel the electrons and focus (green arrows). The resulting astigmatism (dark red ellipse) cancels the astigmatism in the electron lens (making the beam round: red circle). The actual design of the stigmators inside the microscope is - as with the deflection coils - more complicated and based on a magnetic field (field direction and strength shown by blue arrows). Each stigmator consists of two of the elements, one mounted above the other and rotated by 45° with respect to each other. Each of these elements is controlled by one of the Multifunction knobs (X and Y directions). The combination of two elements allows correction of the astigmatism in any direction.

Microscopes have three sets of stigmators: the condenser stigmator to make the focused beam circular; the objective stigmator to correct astigmatism in the high-magnification (M, SA) image and the low-angle diffraction (LAD) pattern; and the diffraction stigmator to correct astigmatism in the diffraction pattern and the low-magnification (LM) image. The quadrupoles used as stigmator can only correct second-order astigmatism. Fortunately (or perhaps logically), this is the strongest astigmatism found. Third-order astigmatism is usually apparent only in the so-called caustic image. This type of image is obtained when a strongly convergent beam is focused into


a small spot, as can be the case for a diffraction pattern or nanoprobe. Occasionally, fourth-order astigmatism is observed when small, dirty objective apertures are used. Because the stigmator settings vary from one mode to another and between various spot sizes, a number of independent stigmator values are stored by the microscope.

3.5 Focusing the diffraction pattern Unlike the image, the diffraction pattern does not have a clear criterion for establishing when it is in focus. Often it is presumed to be 'in focus' when the pattern has spots that are as small as possible. This is not strictly true. The diffraction pattern is in focus when the focus lies at the back-focal plane of the objective lens. Only when the incident beam is parallel does the cross-over lie in the back-focal plane.

With a parallel beam incident on the specimen (grey), the objective lens focuses the electrons into a cross-over whose position coincides with the back-focal plane (and thus the true diffraction focus). When the beam is convergent (but not wholly focused), there still is a cross-over but it is displaced from the back-focal plane upwards (in the extreme case, a fully focused beam, the cross-over lies at the image plane). When the beam is divergent, the cross-over is displaced downward from the back-focal plane.


If the diffraction pattern is not focused properly, there are a number of consequences: • The camera length can be wrong • The diffraction will be rotated away from its proper orientation • The pattern may be distorted • Alignments such as beam shift pivot points can be wrong • The scanning magnification can be wrong due to misaligned pivot points Due to the absence of a clear criterion, we end up with a chicken-and-egg situation (what was first, the chicken or the egg?). For example, if it can be assumed that the shift pivot points are correct, then it is easy to establish the correct diffraction focus by wobbling a beam shift and minimising diffraction-pattern movement. However, the pivot points can only be aligned correctly if the diffraction pattern is focused properly. In order to resolve this situation, we have determined Intensity settings for the different modes (LM, HM-TEM and Nanoprobe) for a parallel beam. These Intensity settings are preset in the alignment procedures, making it easy to find diffraction focus (the spot-pattern condition). After the alignment have been done (camera length focus), the diffraction focus can also be found by simply pressing the Eucentric focus button (this resets the variable diffraction focus to zero). With this method for establishing diffraction focus, the SA aperture is not (and should not be) used.

3.6 The diffraction shadow image When the diffraction pattern is focused properly at the back-focal plane, the pattern is either a spot pattern in SAED or a disk pattern in CBED (with a fully focused beam). In either case the diffraction pattern contains no image information at all (the magnification of the image in the diffraction pattern is infinite). It is also possible to defocus the diffraction pattern slightly (see below). When this is done, the (by now expanded) spots or disks do contain image information (the magnification is no longer infinite) and so we have obtained a mixture of diffraction and image information. This is called a shadow image. The shadow image (in this case from SAED diffraction) can be understood from the diagram below. When a parallel illuminates an area of the specimen, all transmitted (bright-field) beams converge in a single cross-over in the back-focal plane of the objective lens. In this cross-over we cannot distinguish between beams coming from the different parts of the specimens, because all beams go through a single point (ideally). However, above or below the back-focal plane, the beams do not go through a single point but - in three dimensions - they form a disk and each point in the disk corresponds to an area of the specimen.


In SAED the shadow image is obtained by changing the diffraction focus (FOCUS). In CBED the shadow image is obtained by defocusing the beam on the specimen (INTENSITY). The shadow image is often used when working with crystals: • During tilting it allows observation of both crystal orientation (from the diffraction pattern) and position

(the shadow image), making it easier to correct (with X-Y stage movement) for apparent image shift during tilting (especially with the non-eucentric b tilt).

• It can be used to create multiple dark-field images (the pattern contains the bright-field disk with the bright-field image and several diffraction disks, each with its own dark-field image).

• It can be used to position, focus and stigmate the focused beam accurately while in diffraction (or STEM).

In the shadow image, there are a couple of effects dependent on the direction of defocusing (under- or overfocus). In going from under- to overfocus the shadow image: • Flips by 180°. • Inverts the contrast. Because of these effects, one should work consistently (either always underfocus or always overfocus).


4 Gun procedure

4.1 Gun tilt Purpose: The gun tilt makes sure that the electron beam from the gun comes down parallel to the optical axis, so that no electrons from the beam are lost before they can be used for imaging, etc. Importance: ESSENTIAL for having sufficient beam intensity. Method: Obtain maximum intensity. Procedure The gun tilt alignment consists of two steps: • A step for preparation and finding light. • A step to set the gun tilt itself. Description Gun alignment consists of two parts: gun tilt and gun shift. The gun tilt alignment makes sure that the electron beam enters the microscope (that is, the C1 - spot size - lens) parallel to the optical axis, while gun shift ensures that the beam goes through the center of the C1 lens.

The gun tilt is aligned simply by maximizing the intensity (with a useful guide often being the exposure time measured on the screen). For fine-tuning at high magnifications use the direct alignments and the criteria outlined under FEG alignment. Since gun tilt alignment implies no gun shift (which would be visible as a beam shift), it should be possible to align the gun tilt without movement of the beam. If this is not the case, the gun tilt pivot points are not aligned properly.

4.2 Gun tilt pivot points Purpose: Minimize movement of the beam during gun tilt alignment. Importance: CONVENIENCE for not having to correct beam position while doing the gun tilt alignment. Method: Minimize beam movement.


Procedure The gun tilt pivot point alignment consists of four steps: • A step for preparation. • Two steps to align the pivot points for the x and y directions. And a final step to redo the gun tilt itself (because the pivot points affect the gun tilt, the latter step can be necessary).

4.3 Gun shift Purpose: Shift the electron beam sideways so that it comes down along the optical axis. Importance: ESSENTIAL for minimizing movements between different spot sizes and for having the beam correctly along the optical axis for all spot sizes. Method: Minimize spot displacement when spot size (that is focal length of C1 lens) is changed. Procedure The gun shift alignment consists of two steps: • A first step in which spot 9 is centered with the beam deflection coils. • A second step in which spot 3 is centered with the gun deflection coils. The two steps are repeated until the change in gun shift is very small. In the gun shift procedure the spot-size dependent gun shift values for spots 3 and 9 are reset to zero (for proper alignment the spot-size dependent gun shift should therefore be done after the gun shift procedure). Description Gun alignment consists of two parts: gun tilt and gun shift. The gun tilt alignment makes sure that the electron beam enters the microscope (that is, the C1 - spot size - lens) parallel to the optical axis, while gun shift ensures that the beam goes through the center of the C1 lens.

When spot size 9 is used (or any spot size above that), the C1 lens is strong. Under these conditions all aberrations of the microscope system above it are demagnified by the lens (C1 works in the opposite way of the magnification system; instead of magnifying the image, it makes the image of the source - the electron gun - smaller). Thus the demagnification of the misalignment of the gun shift is much smaller for spot 9 than for spot 3, so spot 9 gives the reference ('no' gun shift) and spot 3 defines the gun shift itself.


These diagrams show schematically how the beam position would change as a function of spot number when spots 3 and 9 are used for gun shift alignment (left) and spots 1 and 11 (right). For spots 3 and 9 the overall shift is smaller. The remaining deviations are corrected by the spot-size dependent gun shift.

4.4 Spot-size dependent gun shift Purpose: Set the exact gun shift for all spots individually. Importance: ESSENTIAL for minimizing movements between different spot sizes and for having the beam correctly along the optical axis for all spot sizes. Method: Align all spots relative to spot 9. Procedure The spot-size dependent gun shift alignment consists of three steps: • A first step in which spot 5 is centered with the beam deflection coils. • A second step in which spot 9 is centered with the beam deflection coils (spot 5 is done first because

spot 9 may be difficult to find, especially if the beam is defocused). • A third step in which all spots except 11 are centered (setting the spot-dependent gun shift). • A fourth step in which spot 11 is centered (as far as it will go - it may become blocked by a fixed

aperture when shifted too far).


5 Beam HM-TEM procedure

5.1 Preparation Beam HM-TEM alignment Purpose: Set up microscope for aligning the upper part of the column (the illumination system) in HM-TEM. Importance: ESSENTIAL to make sure that the alignment is done for the correct conditions: centered C2 aperture, eucentric height and specimen in focus. Method: C2 aperture centering: • Focus spot and center it on the screen • Turn INTENSITY overfocus (clockwise) • Center aperture until illuminated area is symmetrical around the screen center. Eucentric height: with the CompuStage switch on the Alpha wobbler and minimize image movement by changing the Z height.

5.2 Adjust Minicondenser lens Purpose: Set the focal length of the Minicondenser lens at the front-focal plane of the objective lens. Importance: ESSENTIAL for proper nanoprobe and scanning operation. Method: First set up by focusing the diffraction pattern (smallest diffraction spots). Then minimize the size of the diffraction spots with the Minicondenser lens. Procedure The alignment procedure consists of three steps: • Two preparation steps for setting up the image and diffraction pattern, respectively. • A step in which the minicondenser lens can be adjusted. Note: The Minicondenser lens can be used to change the optics of the condenser system (e.g. the convergence angles in microprobe and nanoprobe modes depend on the minicondenser lens setting). It should be noted, however, that changing the minicondenser lens has a number of consequences: • Because the Minicondenser lens is located below the beam deflection coils, changes of the lens

affect many beam alignments. • Strong changes of the Minicondenser lens may result on strong beam drift (the lens doesn't have any

water-cooling, since it releases its heat to the objective lens itself). • Changing the Minicondenser lens affects the field of view that can be illuminated, especially when

small objective apertures are used.

5.3 Pivot point beam shift HM Purpose: Align beam shift pivot point = make sure that the beam does not tilt when it is shifted. Importance: ESSENTIAL for keeping the beam parallel to the optical axis when shifting. Method: Shifting a beam parallel to itself means that it must always go through the front-focal point (= shift pivot point) of the objective lens. This plane is conjugate to the back-focal (diffraction) plane and the alignment of the pivot point can thus be seen in diffraction. The shift 'wobble' done by the microscope should give no beam tilt, so the two central spots in the diffraction pattern should overlap.


Procedure The alignment procedure consists of four steps: • Two preparation steps for setting up the image and diffraction pattern, respectively. • Two steps in which the X and Y pivot points are aligned. Notes: • The shift 'wobble' may have one beam position blocked by the specimen. If no second beam is

visible when turning MF-X, then (re)move the specimen. • Diffraction focus: the Intensity setting is preset to a fixed value that gives a parallel incident beam

which in turn should give a spot diffraction pattern. If the pattern is not focused, it should be focused with the diffraction lens (FOCUS), not Intensity.

5.4 Pivot point beam tilt HM Purpose: Align beam tilt pivot point = make sure that the beam does not shift when it is tilted. Importance: ESSENTIAL for keeping the beam centered during rotation-center alignment, focusing with the wobbler and dark-field imaging. Method: A tilting beam must remain centered on the specimen (so the tilt pivot point coincides with the specimen). The tilt wobble done by the microscope should give no beam shift, so only one spot should be visible in the image. Procedure The alignment procedure consists of three steps: • One preparation step for setting up the image. • Two steps in which the X and Y pivot points are aligned. Note: Unlike the shift pivot point (previous subprocedure), the tilt pivot point is sensitive to objective-lens focus.

5.5 Dynamic conical dark field pivot point HM (STEM systems only) Purpose: Align beam tilt pivot point = make sure that the beam does not shift when it is tilted. Importance: ESSENTIAL for keeping the beam centered during dynamic conical dark-field imaging. Method: A tilting beam must remain centered on the specimen (so the tilt pivot point coincides with the specimen). The tilt wobble done by the microscope should give no beam shift, so only one spot should be visible in the image. Procedure The alignment procedure consists of three steps : • One preparation step for setting up the image. • Two steps in which the X and Y pivot points are aligned. Notes: • The AC beam tilt pivot point is sensitive to objective-lens focus. • The AC beam tilt pivot point is used only for Dynamic Conical Dark Field. • Because TIA drives the beam in Dynamic Conical Dark Field, TIA must be running during execution

of this alignment.


5.6 Dynamic conical dark field distortion HM (STEM systems only) Purpose: Make sure that the beam tilt describes a circle as seen in diffraction. Importance: ESSENTIAL for proper dynamic conical dark-field imaging. Method: A tilting beam must remain centered on the specimen (so the tilt pivot point coincides with the specimen). The tilt wobble done by the microscope should give no beam shift, so only one spot should be visible in the image. Procedure The alignment procedure consists of five steps : • One preparation step for setting up the image. • One step in which the diffraction pattern is centered. • One step in which the static beam tilt on the AC coils is adjusted until the beam is at the 4 cm circle. • Two steps in which the beam scans around and the distortion is adjusted until the movement is

circular. The major part of the distortion should be corrected in the first step, any residual distortion in the second. Repeat the last two steps as often as needed.

5.7 HM-TEM rotation center Purpose: Make sure that the beam is along the optical axis of the objective lens. Importance: ESSENTIAL for minimizing lens aberrations and image movement during focusing. Method: The microscope 'wobbles' the objective lens current, making the image go through focus. Make the sideways movement of the image as small as possible with the rotation center (= tilting the beam to the optical axis). The 'focus wobble' can be made smaller or larger with the Focus Step Size knob. Procedure The alignment procedure consists of two steps: • One preparation step for setting up the image. • A step in which the rotation center is aligned. Notes: • The rotation center is an alignment that is based on a beam tilt, hence its appearance in the Beam

alignment procedure and not the Image alignment procedure. • For proper high-resolution alignment of the objective lens on 200 and 300 kV instruments, rotation

center is only suitable as a first step. Coma-free alignment should be used as the final objective-lens alignment.

5.8 Calibrate Trackball Purpose: Define the rotation between the trackball and beam-shift coordinates. Importance: CONVENIENCE for having the beam shift move in the same direction as the trackball. Method: Move beam with the trackball, going from left to right and back, change direction with the MF-Y if the beam does not move in the same direction on the screen. Procedure The alignment procedure consists of two steps : • In the first step the beam is shifted to the center of the screen. For this purpose the alignment value

is used while the user value is reset to zero. • In the second step, the direction of the beam shift is aligned with respect to the movement by the

trackball. When the trackball is moved from left to right, the beam should also move from left to right


on the screen. If the beam moves in a different direction, adjust the direction with the Multifunction Y knob.

5.9 Beam shift calibration HM Purpose: Calibrate the beam shift to physically meaningful values. Importance: CONVENIENCE. Method: Move the focused beam to the 40 mm circle with Multifunction X and adjust the displayed value of the image shift using Multifunction Y. Procedure The alignment procedure consists of four steps: • In the first step the beam is accurately centered on the screen. • In the second step, the beam is shifted with Multifunction X to the 40 mm circle. Then the displayed

value for the beam shift is adjusted to the correct value with the Multifunction Y. • The third and fourth steps repeat the first and second steps but now for the Y direction of the beam

shift.

5.10 Beam tilt (dark field) calibration HM Purpose: Calibrate the beam tilt (dark field) to physically meaningful values. Importance: ESSENTIAL for meaningful beam tilt values in dark field. Method: Tilt the beam and adjust the displayed value of the beam tilt using Multifunction X,Y. Procedure The alignment procedure consists of five steps: • The first step is a preparation step for the diffraction mode. • In the second step the diffraction pattern must be centered accurately (on the center of the viewing

screen or the tip of the beam stop). • In the third step, the beam is tilted (this is, the diffraction pattern is shifted) with Multifunction X to

bring a ring to the center and the beam tilt value is adjusted with Multifunction Y to the correct value. • The fourth and fifth steps repeat the procedure of the second and third steps for the Y diffraction

shift. Description The beam tilt is converted through the calibration procedure into to physically meaningful units. The beam tilt can be read off in the flap-out of the Alignment Control Panel and is used in the Dark Field Control Panel.

5.11 Dynamic conical dark field beam tilt calibration HM (STEM systems only) Purpose: Calibrate the AC beam tilt (dynamic conical dark field) to physically meaningful values. Importance: ESSENTIAL for meaningful beam tilt values in dynamic conical dark field and for ensuring a match between static and dynamic conical dark field. Method: Tilt the beam and adjust the displayed value of the beam tilt using Multifunction X,Y.


Procedure The alignment procedure consists of three steps : • The first step is a preparation step for the diffraction mode. • In the second step the diffraction pattern must be centered accurately (on the center of the viewing


bring a ring to the center and the beam tilt value is adjusted with Multifunction Y to the correct value. Description The beam tilt is converted through the calibration procedure into to physically meaningful units. The beam tilt can be read off in the flap-out of the Alignment Control Panel and is used in the Dark Field Control Panel.

5.12 Spot size-intensity calibration Purpose: Make sure that a focused beam remains focused when spot size is changed. Importance: CONVENIENCE for keeping spot focus the same for all spot sizes, ESSENTIAL for proper operation of Intensity Zoom and Intensity Limit. Method: After focusing spot 3, all spots are focused in turn. The deviations in intensity setting from spot focus are stored for all spots. Procedure The alignment procedure consists of two steps: • One preparation step in which the beam is focused for spot size 3. • A step in which all spot sizes are focused. Note: The condenser system (C1 and C2 lenses) is normalized when the spot size is changed to make the spot setting better reproducible. Description The Intensity (C2 lens) and spot size (C1 lens) settings are not independent. In order to give the same effect for all spot sizes, the Intensity is changed whenever spot size is changed. In addition to the preprogrammed changes, individual instruments differ slightly in their relation between C1 and C2. The spot size-intensity calibration allows adjustment for this individual behavior. For the Intensity Zoom and Intensity Limit functions this procedure defines the Intensity settings at which the beam is focused, which is essential for proper operation of these functions.


6 Image HM-TEM Procedure

6.1 Preparation Image HM-TEM alignment Purpose: Set up microscope for aligning the lower part of the column (the imaging system) in HM-TEM. Importance: ESSENTIAL to make sure that the alignment is done for the correct conditions: centered C2 aperture, eucentric height and specimen in focus. Method: C2 aperture centering: • Focus spot and center it on the screen • Turn INTENSITY overfocus (clockwise) • Center aperture until illuminated area is symmetrical around the screen center. Eucentric height: with the CompuStage switch on the Alpha wobbler and minimize image movement by changing the Z height.

6.2 Pivot point image shift HM Purpose: Align image shift pivot point = make sure that the diffraction pattern does not shift when the image shift is changed. Importance: ESSENTIAL for the proper functioning of image shifts. Method: Minimize the movement of the diffraction pattern. The microscope 'wobbles' the image shift which should cause no diffraction shift. Procedure The alignment procedure consists of four steps: • Two preparation steps for setting up the image and diffraction pattern, respectively. • Two steps in which the X and Y pivot points are aligned. Note: Diffraction focus. The Intensity setting is preset to a fixed value that gives a parallel incident beam which in turn should give a spot diffraction pattern. If the pattern is not focused, it should be focused with the diffraction lens (FOCUS), not Intensity. Description The alignment of the pivot points of the image deflection coils is essential to the proper functioning of many other alignments and calibrations: image shifts, diffraction shifts, detector alignments and measurement functions. The image coils pivot points (image shift and diffraction shift) alignment is reasonably insensitive to the operating conditions and normally needs to be done only once. It is important that the image coils pivot points are aligned prior to aligning other parts of the microscope where the image coils are used, otherwise the latter alignments may have to be redone. Alignments where the image coils are used are those where images or diffraction patterns are moved (image shift, diffraction shift/diffraction alignment).

6.3 Pivot point diffraction shift SA Purpose: Align the diffraction shift pivot point = make sure the image does not move when the diffraction shift is changed. Importance: ESSENTIAL for the proper functioning of image and diffraction shifts. Method: Minimize the movement of the image. The microscope 'wobbles' the diffraction shift which should cause no image shift.


A common consequence of a diffraction shift that has not been aligned is a mismatch between the image detail selected with the selected area aperture and the area actually contributing to the diffraction pattern. When the image coils have not been aligned properly, image and diffraction shift are not uncoupled and the diffraction shift will also cause an image shift, thereby moving the image detail out of the selected area aperture. Procedure The alignment procedure consists of three steps: • A preparation step for setting up the image. • Two steps in which the X and Y pivot points are aligned. Description The alignment of the pivot points of the image deflection coils is essential to the proper functioning of many other alignments and calibrations: image shifts, diffraction shifts, detector alignments and measurement functions. The image coils pivot points (image shift and diffraction shift) alignment is reasonably insensitive to the operating conditions and normally needs to be done only once. It is important that the image coils pivot points are aligned prior to aligning other parts of the microscope where the image coils are used, otherwise the latter alignments may have to be redone. Alignments where the image coils are used are those where images or diffraction patterns are moved (image shift, diffraction shift/diffraction alignment).

Misaligned diffraction shift pivot point. Aligned diffraction shift pivot point.

6.4 Objective Lens preset SA Purpose: Setting the eucentric focus preset at the eucentric height. Importance: CONVENIENCE for having the eucentric focus at the eucentric height. Method: Focus the image for the highest SA magnification. Procedure • The alignment procedure consists of two steps: • A preparation step in which the SA image is focused at an intermediate magnification. • A step in which the highest SA magnification must be focused.


6.5 Pivot point diffraction shift Mh Purpose: Align the diffraction shift pivot point = make sure the image does not move when the diffraction shift is changed. Importance: ESSENTIAL for the proper functioning of image and diffraction shifts. Method: Minimize the movement of the image. The microscope 'wobbles' the diffraction shift which should cause no image shift. A common consequence of a diffraction shift that has not been aligned is a mismatch between the image detail selected with the selected area aperture and the area actually contributing to the diffraction pattern. When the image coils have not been aligned properly, image and diffraction shift are not uncoupled and the diffraction shift will also cause an image shift, thereby moving the image detail out of the selected area aperture. Procedure The alignment procedure consists of three steps: • A preparation step for setting up the image. • Two steps in which the X and Y pivot points are aligned. In the preparation step the P1 lens is maximized (with MF-X) to reduce the effective magnification. Still, the beam (when wobbling in the next two steps) may be difficult to find. Description The alignment of the pivot points of the image deflection coils is essential to the proper functioning of many other alignments and calibrations: image shifts, diffraction shifts, detector alignments and measurement functions. The image coils pivot points (image shift and diffraction shift) alignment is reasonably insensitive to the operating conditions and normally needs to be done only once. It is important that the image coils pivot points are aligned prior to aligning other parts of the microscope where the image coils are used, otherwise the latter alignments may have to be redone. Alignments where the image coils are used are those where images or diffraction patterns are moved (image shift, diffraction shift/diffraction alignment). For images, see section 6.3.

6.6 Mh objective-lens preset and image shift Purpose: Aligning the Mh-magnification images with SA and finding the difference in focus. Importance: CONVENIENCE for being able to find the image at high magnifications. Method: Focus the image for the lowermost Mh magnification and center the image relative to the highest SA image. Repeat for all Mh magnifications. Procedure The alignment procedure consists of three steps: • A preparation step in which an image feature is centered (with the specimen stage) in the highest SA

magnification. • A step in which the lowest Mh magnification is focused and aligned relative to the highest SA

magnification. • A step in which all other Mh magnifications are focused and aligned.


Notes: • The magnification system (projector lenses) is normalized when the magnification is changed to

make the image position better reproducible. • The instruction 'center illumination' with MF-X,Y is correct ! When the magnification is changed from

the highest SA to the lowermost Mh, the beam will remain in the same position but the whole image may be shifted. Bringing the beam to the center first by shifting the image - not the beam - allows one to see the image feature and then center it.

Using the P1 lens Because of the high magnifications used (and the resulting small field of view), it can be difficult to align the Mh magnifications (no light visible). The alignment procedure therefore provides a trick. It is possible to change the P1 lens (increase its current) by toggling the MF-X control to it (press the R2 button). When the P1 lens is made stronger, it reduces the effective magnification but has very little effect on image position and focus. This makes it possible to spread the beam (and actually see it more easily because of the lower magnification) and center it. Use the following procedure if the beam is not visible at the normal Mh magnification: • Toggle the MF-X control to the P1 lens (press R2). • Turn the MF-X knob a bit clock-wise. • Change the Intensity setting and see if the beam can be found. • Repeat the previous two steps until the beam is seen. Note that at very much changed P1 setting,

the image is partly blocked by an aperture (the differential pumping aperture between the column and the projection chamber) so not the whole screen can be illuminated.

• If the image is out of focus, focus it. • Toggle back to MF-X,Y control to the image shift and center the beam (and image). • Turn the magnification once up and down (this resets the P1 lens to its proper value). • Focus and center the image.

6.7 Image shifts SA Purpose: Aligning all SA images with each other. Importance: CONVENIENCE so that the image remains centered when the magnification is changed. Method: Center a recognizable image with the specimen stage. Lower the magnification one step, center the image with the Multifunction X,Y knobs. Repeat for all magnifications. Procedure The alignment procedure consists of two or four steps, dependent on the presence of absence of Parfocal Magnification Series (last two steps): • A preparation step in which an image feature is centered (with the specimen stage) in the highest SA

magnification. • A step in which all SA magnifications are aligned relative to the highest SA magnification. • A preparation step in which the highest SA magnification is focused accurately. • A step in which all other SA magnifications are focused accurately. Parfocal magnification series The parfocal magnification series applies an automatic correction to each SA magnification so as to make the focus difference between the SA magnification steps as small as possible. This correction factor is independent of focus settings and applies only to the SA magnifications. Similar correction factors for the M (Mi, and Mh) magnifications as well as diffraction (D) are already present as standard in the Tecnai software.


Notes: • The magnification system (projector lenses) is normalized when the magnification is changed to

make the image position better reproducible. • The highest SA magnification is the reference image for the whole microscope, with regard to focus

(eucentric focus preset) and image shift.

6.8 Pivot point diffraction shift Mi Purpose: Align the diffraction shift pivot point = make sure the image does not move when the diffraction shift is changed. Importance: ESSENTIAL for the proper functioning of image and diffraction shifts. Method: Minimize the movement of the image. The microscope 'wobbles' the diffraction shift which should cause no image shift. A common consequence of a diffraction shift that has not been aligned is a mismatch between the image detail selected with the selected area aperture and the area actually contributing to the diffraction pattern. When the image coils have not been aligned properly, image and diffraction shift are not uncoupled and the diffraction shift will also cause an image shift, thereby moving the image detail out of the selected area aperture. Procedure The alignment procedure consists of three steps: • A preparation step for setting up the image. • Two steps in which the X and Y pivot points are aligned. Description The alignment of the pivot points of the image deflection coils is essential to the proper functioning of many other alignments and calibrations: image shifts, diffraction shifts, detector alignments and measurement functions. The image coils pivot points (image shift and diffraction shift) alignment is reasonably insensitive to the operating conditions and normally needs to be done only once. It is important that the image coils pivot points are aligned prior to aligning other parts of the microscope where the image coils are used, otherwise the latter alignments may have to be redone. Alignments where the image coils are used are those where images or diffraction patterns are moved (image shift, diffraction shift/diffraction alignment). For images, see section 6.3.

6.9 Mi objective lens preset and image shift Purpose: Aligning the Mi-magnification images with SA and finding the difference in focus. Importance: CONVENIENCE for finding the same image feature centered and the image in focus when going from SA to Mi and vice versa. Method: Move the required feature in the specimen to the center with the image shift and focus the image. Procedure The alignment procedure consists of three steps: • A preparation step in which an image feature is centered (with the specimen-stage) in the lowest SA

magnification. • A step in which the highest Mi magnification is focused and aligned relative to the lowest SA

magnification. • A step in which all Mi magnifications are aligned relative to the highest Mi magnification.


Note: The magnification system (projector lenses) is normalized when the magnification is changed to make the image position better reproducible.

6.10 Mi cross-over shift Purpose: Aligning the Mi cross-over shift to ensure that the differential pumping aperture does not (partially) block the beam and causes a cut-off of the image. Importance: ESSENTIAL for avoiding partial image cut-offs at low Mi magnifications. Method: Move the shadow of the differential pumping aperture so it becomes centered around the screen center. Procedure The alignment procedure consists of one step in which a shadow from the aperture (if visible) is moved so it does no longer obstruct the image. Notes: • If an aperture casts a shadow in the image for the lowermost Mi magnification, but this shadow does

not move with the change of the Multifunction knob settings, and instead another aperture comes into view and starts blocking the beam, the original shadow is not that of the differential pumping aperture, but of the fixed SA aperture. The latter aperture cannot be "moved" by changing the optics.

• Because of the need to have the beam (which in this case means the diffraction pattern) centered properly in the differential pumping aperture, it is not possible to change the position of the diffraction pattern when you enter diffraction from an Mi magnification (because that would ruin this alignment). In principle you should always go to diffraction from an SA magnification.

6.11 Align diffraction pattern Purpose: Set the zero position for the diffraction shift. Importance: CONVENIENCE for easy resetting of the diffraction shift to the screen center. Method: Center the diffraction pattern using Multifunction X,Y. Procedure The alignment procedure consists of a single step. Description The diffraction shift has two components, an alignment value and a variable 'user' value. If properly aligned, the alignment value will have the diffraction pattern centered on the screen. It then is only necessary to reset the 'user' value to zero to have the pattern back at the screen center.

6.12 Align camera lengths Purpose: Determine the shifts necessary for aligning all camera lengths, so that the diffraction pattern remains in the center when the camera length is changed. Importance: CONVENIENCE for having the diffraction patterns centered when the camera length is changed. Method: Center the diffraction pattern for the reference camera length (~500 mm), then align all camera lengths to the reference camera length. Focus all camera lengths.


Procedure The alignment procedure consists of three steps: • A preparation step in which the image is focused. • A step in which the reference camera length (~500 mm) is focused and centered. • A step in which all other camera lengths are focused and centered. Notes: • The magnification system (projector lenses) is normalized when the magnification is changed to

make the diffraction pattern position better reproducible. • For each camera length the focus setting is stored during this alignment.

6.13 Align camera length Mh Purpose: Determine the shift necessary for aligning the reference camera length when coming from Mh magnifications, so that the diffraction pattern remains in the center. Importance: CONVENIENCE for having the diffraction patterns centered when the camera length is changed. Method: Center the diffraction pattern for the reference camera length (~500 mm), then align all camera lengths to the reference camera length. Focus all camera lengths. Procedure The alignment procedure consists of two steps : • A preparation step in which the Mh image is focused. • A step in which the reference camera length (~500 mm) is focused and centered. Notes: • In principle, always go into diffraction from an SA magnification, not from Mh magnification for proper

diffracting conditions. • For the Mh magnification camera lengths, only a single setting is needed, comparable to the SA

diffraction alignment. The differences between individual camera lengths is handled by the same settings as done in the SA camera length alignment. The need for a separate value is due to the separation of the diffraction alignment for Mi on the one hand (used for the cross-over shift) and SA and Mh.

6.14 Image shift calibration HM Purpose: Calibrate the image shift to physically meaningful values. Importance: ESSENTIAL for meaningful image measurement results. Method: Move the focused beam to the 40 mm circle (the microscope uses the image shift to do this) and adjust the displayed value of the image shift using Multifunction Y. The focused beam is thus used as a reference marker. Procedure The alignment procedure consists of four steps: • In the first step the beam is accurately centered on the screen. • In the second step, the beam is shifted with Multifunction X to the 40 mm circle. Then the displayed

value for the image shift is adjusted to the correct value with the Multifunction Y. • The third and fourth steps repeat the first and second steps but now for the Y direction of the image

shift.


Description The image shift calibration provides the conversion factor for the image shift used in measuring and for the beam shift - image shift.

6.15 Diffraction shift calibration HM Purpose: Calibrate the diffraction shift to physically meaningful values. Importance: ESSENTIAL for meaningful diffraction measurement results. Method: Move the diffraction pattern and adjust the displayed value of the diffraction shift using Multifunction Y. Procedure The alignment procedure consists of five steps: • The first step is a preparation step for the diffraction mode. • In the second step the diffraction pattern must be centered accurately (on the center of the viewing

screen or the tip of the beam stop). • In the third step, the diffraction pattern is shifted with Multifunction X to bring a ring to the center and

the diffraction shift (nm value or angle) is adjusted with Multifunction Y to the correct value. • The fourth step repeats the procedure of the third step for the Y diffraction shift. Description The diffraction shift is converted through the calibration procedure into to physically meaningful units such as Bragg angles and d spacings (in the latter case the Bragg Law formula is used). The diffraction shift can be read off in the flap-out of the Alignment Control Panel and is used in the Measuring Control Panel.

6.16 Beam shift - image shift calibration HM Purpose: Define the beam shift compensation for the image shift. Importance: CONVENIENCE for keeping the beam on the screen when the image is shifted. The beam shift - image shift is used in the Image shift Control Panel and in Low Dose. Method: Move the image off-axis and recenter the beam using Multifunction X,Y. Note: This procedure is complicated by the fact that it is not easy to tell how far the beam-shift/image-shift should be changed. The on-line help file therefore contains a control that indicates the status of the beam-shift/image-shift, beam coils and image coils.

Using this you change the beam shift-image shift until either: • the beam is at the screen edge. • the beam shift-image shift is more than 50% (bar

becomes green) OR one of the coils is more than 80% (bar becomes red).

The microscope will sound a beep when the beam shift-image shift is at its limit but also when one of the coils is at its limit. In the former case you can continue with the calibration, but when a coil is limited very likely you cannot. The control in the help file which is directly linked to the microscope displays the situation.


Procedure The calibration procedure consists of six steps: • In the first step the beam is shifted to the center of the screen. For this purpose the alignment value

is used while the user value is reset to zero. At the same time the image shift is set to zero. • In the second step, the image shift X is changed until the above control indicates a suitable shift, the

microscope beeps (at the limit of the image shift) or the beam moves off the screen (the latter typically happens when the calibration has not been done yet).

• In the third step the beam is recentered using the Multifunction X,Y. If the microscope beeped during the second step, continue, otherwise step back and repeat the second and third steps (so the final calibration is done with the image shift at its limit).

• The fourth and fifth steps repeat the same procedure as the second and third steps, but this time for the Y image shift.

• The final step just ensures that the image-beam shift is reset to zero after the procedure is finished. Description The Tecnai microscope has a function for image shift where the beam position is automatically compensated to keep the beam on the area of interest (what is currently seen on the viewing screen). The beam shift compensation must be calibrated before it will work properly. When the user shifts the image (from the central green ray path to the off-axis, tan ray path), the microscope automatically applies a compensating beam shift.

6.17 Off-axis TV HM image alignment (only if off-axis TV installed) Purpose: Aligning all Mh and SA (as far as attainable) magnifications on the off-axis TV. Importance: CONVENIENCE so that the image is shifted automatically from the center of the viewing screen to the off-axis TV camera . Method: Center a recognizable image on the viewing screen with the specimen stage. Center the image with the Multifunction X,Y knobs on the off-axis TV camera. Repeat for all attainable magnifications.


Note 1: The off-axis TV camera is located about 7 cm from the center of the viewing screen between W and WNW (where N is defined as the direction furthest away from the operator). The image can be shifted to the TV camera by selecting manual mode and the Off-axis TV in the Detector Configuration Control Panel. Note 2: The shift of the image to the off-axis TV uses the image shift coils below the objective lens. The effect of these coils is magnified by the magnification system. The highest magnifications therefore give no problems but some of the lowermost magnifications may be out of reach of the range of these coils. In addition, any other use of these coils (e.g. for the Image-Beam shift) will also affect the range of attainable magnifications. Procedure The alignment procedure consists of four steps : • A preparation step in which an image feature is centered on the viewing screen (with the specimen

stage) in the highest SA magnification. • A step in which the highest SA magnification is centered on the off-axis TV. • A step in which the Mh magnifications are centered on the off-axis TV. • A step in which all other SA magnifications are centered on the off-axis TV (as far as attainable, see

Note 2 above).

6.18 Off-axis TV diffraction alignment (only if off-axis TV installed) Purpose: Aligning all (as far as attainable) camera lengths on the off-axis TV. Importance: CONVENIENCE so that the diffraction pattern is shifted automatically from the center of the viewing screen to the off-axis TV camera . Method: Center the diffraction pattern on the viewing screen. Then center the diffraction pattern with the Multifunction X,Y knobs on the off-axis TV camera. Repeat for all attainable camera lengths. Note 1: The off-axis TV camera is located about 7 cm from the center of the viewing screen between W and WNW (where N is defined as the direction furthest away from the operator). The diffraction pattern can be shifted to the TV camera by selecting manual mode and the Off-axis TV in the Detector Configuration Control Panel. Note 2: The shift of the diffraction pattern to the off-axis TV uses the image shift coils below the objective lens. The effect of these coils is magnified by the magnification system. The highest camera lengths therefore give no problems but some of the lowermost camera lengths may be out of reach of the range of these coils. In addition, any other use of these coils (e.g. for the Image-Beam shift) will also affect the range of attainable camera lengths. Procedure The alignment procedure consists of four steps : • A preparation step in which the image is focused in the SA magnification range. • A preparation step in which the diffraction pattern is centered on the viewing screen. • A step in which the diffraction pattern is centered on the off-axis TV. • A step in which all other camera lengths are centered on the off-axis TV (as far as attainable, see

Note 2 above).


7 Beam LM Procedure

7.1 Preparation Beam LM alignment Purpose: Set up microscope for aligning the upper part of the column (illumination system) in LM. Importance: ESSENTIAL to make sure that the alignment is done for the correct conditions: centered C2 aperture, eucentric height and specimen in focus. Method: C2 aperture centering: • Focus spot and center it on the screen • Turn INTENSITY overfocus (clockwise) • Center aperture until illuminated area is symmetrical around the screen center Eucentric height: with the CompuStage switch on the Alpha wobbler and minimize image movement by changing the Z height.

7.2 Pivot point beam shift LM Purpose: Align beam shift pivot point = make sure that the beam does not tilt when it is shifted. Importance: ESSENTIAL for keeping the beam parallel to the optical axis when shifting. Method: Shifting a beam parallel to itself means that it must always go through the front-focal point (= shift pivot point) of the diffraction lens (the 'objective' lens in LM). This plane is conjugate to the back-focal (LAD diffraction) plane and the alignment of the pivot point can thus be seen in LAD diffraction. The shift 'wobble' done by the microscope should give no beam tilt, so the two central spots in the diffraction pattern should overlap. Procedure The alignment procedure consists of four steps: • Two preparation steps for setting up the image and diffraction pattern, respectively. • Two steps in which the X and Y pivot points are aligned. Notes: • The shift 'wobble' may have one beam position blocked by the specimen. If no second beam is

visible when turning MF-X, then (re)move the specimen. • Diffraction focus: in LAD the objective lens (which is here the focusing lens) is switched to fixed

setting. To focus the diffraction pattern to a spot pattern the Intensity should be used, not the Focus. Only if the pattern cannot be focused with the Intensity should the Focus knob (objective lens) be used. In the latter case, turn the Intensity completely overfocus (clockwise), then focus the LAD pattern with the Focus knob.

7.3 Pivot point beam tilt LM Purpose: Align beam tilt pivot point = make sure that the beam does not shift when it is tilted. Importance: ESSENTIAL for keeping the beam centered during rotation-center alignment and focusing using the wobbler. Method: A tilting beam must remain centered on the specimen (so the tilt pivot point coincides with the specimen). The tilt wobble done by the microscope should give no beam shift, so only one spot should be visible in the image.


Procedure The alignment procedure consists of three steps: • One preparation step for setting up the image. • Two steps in which the X and Y pivot points are aligned.

7.4 LM rotation center Purpose: Make sure that the beam is along the optical axis of the diffraction lens (= the 'objective lens' in LM). Importance: ESSENTIAL for minimizing lens aberrations and image movement during focusing. Method: The microscope 'wobbles' the diffraction lens current, making the image go through focus. Make the sideways movement of the image as small as possible with the rotation center (= tilting the beam to the optical axis). The 'focus wobble' can be made smaller or larger with the Focus Step Size knob. Procedure The alignment procedure consists of two steps: • One preparation step for setting up the image. • A step in which the rotation center is aligned. Note: The rotation center is an alignment that is based on a beam tilt, hence its appearance in the Beam alignment procedure and not the Image alignment procedure.



trackball. When the trackball is moved from left to right, the beam should also move from left to right on the screen. If the beam moves in a different direction, adjust the direction with the Multifunction Y knob.

7.6 Beam shift calibration LM Purpose: Calibrate the beam shift to physically meaningful values. Importance: CONVENIENCE. Method: Move the focused beam to the 40 mm circle and adjust the displayed value of the image shift using Multifunction Y.


Procedure The alignment procedure consists of four steps: • In the first step the beam is accurately centered on the screen. • In the second step, the beam is shifted with Multifunction X to the 40 mm circle. Then the displayed


shift.

7.7 Beam tilt (dark field) calibration LM Purpose: Calibrate the beam tilt (dark field) to physically meaningful values. Importance: ESSENTIAL for meaningful beam tilt values in dark field. Method: Tilt the beam and adjust the displayed value of the beam tilt using Multifunction X,Y. Because few specimens provide the (large) d spacings appropriate for the small beam tilts obtained in LAD, it may not be possible to use a d spacing as calibration. The software therefore suggests to use the camera length value as a reference and simply calculate the angle corresponding to the shift to the 4 cm circle of the viewing screen. Procedure The alignment procedure consists of five steps: • The first step is a preparation step for the diffraction mode. • In the second step the diffraction pattern must be centered accurately (on the center of the viewing




7.8 Spot size-intensity calibration LM Purpose: Make sure that a focused beam remains focused when spot size is changed. Importance: CONVENIENCE for keeping spot focus the same for all spot sizes, ESSENTIAL for proper operation of Intensity Zoom and Intensity Limit. Method: After focusing spot 3, all spots are focused in turn. The deviations in intensity setting from spot focus are stored for all spots. Procedure The alignment procedure consists of two steps: • One preparation step in which the beam is focused for spot size 3. • A step in which all spot sizes are focused. Note: The condenser system (C1 and C2 lenses) is normalized when the spot size is changed to make the spot setting better reproducible.


Description The Intensity (C2 lens) and spot size (C1 lens) settings are not independent. In order to give the same effect for all spot sizes, the Intensity is changed whenever spot size is changed. In addition to the preprogrammed changes, individual instruments differ slightly in their relation between C1 and C2. The spot size-intensity calibration allows adjustment for this individual behavior. For the Intensity Zoom and Intensity Limit functions this procedure defines the Intensity settings at which the beam is focused, which is essential for proper operation of these functions.


8 Image LM Procedure

8.1 Preparation Image LM alignment Purpose: Set up microscope for aligning the lower part of the column (imaging system) in LM. Importance: ESSENTIAL to make sure that the alignment is done for the correct conditions: centered C2 aperture, eucentric height and specimen in focus. Method: C2 aperture centering: • Focus spot and center it on the screen • Turn INTENSITY overfocus (clockwise) • Center aperture until illuminated area is symmetrical around the screen center Eucentric height: with the CompuStage switch on the Alpha wobbler and minimize image movement by changing the Z height.

8.2 Pivot point image shift LM Purpose: Align image shift pivot point = make sure that the LAD diffraction pattern does not shift when the image shift is changed. Importance: ESSENTIAL for the proper functioning of image shifts. Method: Minimize the movement of the diffraction pattern. The microscope 'wobbles' the image shift which should cause no diffraction shift. Procedure The alignment procedure consists of four steps: • Two preparation steps for setting up the image and diffraction pattern, respectively. • Two steps in which the X and Y pivot points are aligned. Note: Diffraction focus: in LAD the objective lens (which is here the focusing lens) is switched to fixed setting. To focus the diffraction pattern to a spot pattern the Intensity should be used, not the Focus. Only if the pattern cannot be focused with the Intensity should the Focus knob (objective lens) be used. In the latter case, turn the Intensity completely overfocus (clockwise), then focus the LAD pattern with the Focus knob. Description The alignment of the pivot points of the image deflection coils is essential to the proper functioning of many other alignments and calibrations: image shifts, diffraction shifts, detector alignments and measurement functions. The image coils pivot points (image shift and diffraction shift) alignment is reasonably insensitive to the operating conditions and normally needs to be done only once. It is important that the image coils pivot points are aligned prior to aligning other parts of the microscope where the image coils are used, otherwise the latter alignments may have to be redone. Alignments where the image coils are used are those where images or diffraction patterns are moved (image shift, diffraction shift/diffraction alignment).

8.3 Pivot point diffraction shift LM Purpose: Align the diffraction shift pivot point = make sure the image does not move when the diffraction shift is changed. Importance: ESSENTIAL for the proper functioning of image and diffraction shifts.


Method: Minimize the movement of the image. The microscope 'wobbles' the LAD diffraction shift which should cause no image shift. Procedure The alignment procedure consists of three steps: • A preparation step for setting up the image. • Two steps in which the X and Y pivot points are aligned. Description The alignment of the pivot points of the image deflection coils is essential to the proper functioning of many other alignments and calibrations: image shifts, diffraction shifts, detector alignments and measurement functions. The image coils pivot points (image shift and diffraction shift) alignment is reasonably insensitive to the operating conditions and normally needs to be done only once. It is important that the image coils pivot points are aligned prior to aligning other parts of the microscope where the image coils are used, otherwise the latter alignments may have to be redone. Alignments where the image coils are used are those where images or diffraction patterns are moved (image shift, diffraction shift/diffraction alignment). For images, see section 6.3.

8.4 LM images Purpose: Aligning the LM-magnification image with the M image and all LM image magnifications with each other. Importance: CONVENIENCE for finding the same image feature centered when crossing from M to LM (or vice versa) and within the LM range. Method: Focus the image (LM magnification) and center the recognizable image feature with MF-X,Y. Procedure The alignment procedure consists of three steps: • A preparation step in which an image feature is centered (with the specimen stage) in the lowest Mi

magnification. • A step in which the whole LM range is aligned with the Mi magnification. • A step in which all LM magnifications are aligned relative to the highest LM magnification. Note: The magnification system (projector lenses) is normalized when the magnification is changed to make the image position better reproducible. Description The alignment uses two different values: • One value determines the shift of the whole LM range relative to the lowermost Mi magnification (and

is thus a shift of the whole LM range) • The other values determine the shifts between individual LM magnifications (defined as the shift

between a particular magnification and the highest LM magnification).

8.5 Align LAD diffraction pattern Purpose: Set the zero position for the LAD diffraction shift. Importance: CONVENIENCE for easy resetting of the diffraction shift to the screen center. Method: Center the diffraction pattern using Multifunction X,Y.


Procedure The alignment procedure consists of a single step. Description The diffraction shift has two components, an alignment value and a variable 'user' value. If properly aligned, the alignment value will have the diffraction pattern centered on the screen. It then is only necessary to reset the 'user' value to zero to have the pattern back at the screen center.

8.6 Image shift calibration LM Purpose: Calibrate the image shift to physically meaningful values. Importance: ESSENTIAL for meaningful image measurement results. Method: Move the focused beam to the 40 mm circle - with the image shift - and adjust the displayed value of the image shift using Multifunction Y. The focused beam is thus used as a reference marker. Procedure The alignment procedure consists of four steps: • In the first step the beam is accurately centered on the screen. • In the second step, the beam is shifted with Multifunction X to the 40 mm circle. Then the displayed

value for the image shift is adjusted to the correct value with the Multifunction Y. • The third and fourth steps repeat the first and second steps but now for the Y direction of the image

shift. Description The image shift calibration provides the conversion factor for the image shift used in measuring and for the beam shift - image shift.

8.7 Diffraction shift calibration LAD Purpose: Calibrate the diffraction shift to physically meaningful values. Importance: ESSENTIAL for meaningful diffraction measurement results. Method: Move the diffraction pattern and adjust the displayed value of the diffraction shift using Multifunction Y. Because few specimens provide the (large) d spacings appropriate for the small diffraction shifts obtained in LAD, it may not be possible to use a d spacing as calibration. The software therefore suggests to use the camera length value as a reference and simply calculate the angle corresponding to the shift to the 4 cm circle of the viewing screen. Procedure The alignment procedure consists of five steps: • The first step is a preparation step for the diffraction mode. • In the second step the diffraction pattern must be centered accurately (on the center of the viewing

screen or the tip of the beam stop). • In the third step, the diffraction pattern is shifted with Multifunction X to bring a ring to the center and

the diffraction shift (nm value or angle) is adjusted with Multifunction Y to the correct value. • The fourth step repeats the procedure of the third step for the Y diffraction shift. Description The diffraction shift is converted through the calibration procedure into to physically meaningful units such as Bragg angles and d spacings (in the latter case the Bragg Law formula is used). The diffraction shift can be read off in the flap-out of the Alignment Control Panel and is used in the Measuring Control Panel.


8.8 Beam shift - image shift calibration LM Purpose: Define the beam shift compensation for the image shift. Importance: CONVENIENCE for keeping the beam on the screen when the image is shifted. The beam shift - image shift is used in the Image shift Control Panel and in Low Dose. Method: Move the image off-axis and recenter the beam using Multifunction X,Y. Note: This procedure is complicated by the fact that it is not easy to tell how far the beam-shift/image-shift should be changed. The on-line help file therefore contains a control that indicates the status of the beam-shift/image-shift, beam coils and image coils.

Using this you change the beam shift-image shift until either: • the beam is at the screen edge. • the beam shift-image shift is more than 50% (bar

becomes green) OR one of the coils is more than 80% (bar becomes red).

The microscope will sound a beep when the beam shift-image shift is at its limit but also when one of the coils is at its limit. In the former case you can continue with the calibration, but when a coil is limited very likely you cannot. The control in the help file which is directly linked to the microscope displays the situation.

Procedure The calibration procedure consists of six steps: • In the first step the beam is shifted to the center of the screen. For this purpose the alignment value





• The final step just ensures that the image-beam shift is reset to zero after the procedure is finished. Description The Tecnai microscope has a function for image shift where the beam position is automatically compensated to keep the beam on the area of interest (what is currently seen on the viewing screen). The beam shift compensation must be calibrated before it will work properly. For a schematic diagram of the beam shift-image shift, see section 6.16.


8.9 Off-axis TV LM image alignment (only if off-axis TV installed) Purpose: Aligning all LM (as far as attainable) magnifications on the off-axis TV. Importance: CONVENIENCE so that the image is shifted automatically from the center of the viewing screen to the off-axis TV camera . Method: Center a recognizable image on the viewing screen with the specimen stage. Center the image with the Multifunction X,Y knobs on the off-axis TV camera. Repeat for all attainable magnifications. Note 1: The off-axis TV camera is located about 7 cm from the center of the viewing screen between W and WNW (where N is defined as the direction furthest away from the operator). The image can be shifted to the TV camera by selecting manual mode and the Off-axis TV in the Detector Configuration Control Panel. Note 2: The shift of the image to the off-axis TV uses the image shift coils below the objective lens. The effect of these coils is magnified by the magnification system. The highest magnifications therefore give no problems but some of the lowermost magnifications may be out of reach of the range of these coils. In addition, any other use of these coils (e.g. for the Image-Beam shift) will also affect the range of attainable magnifications. Procedure The alignment procedure consists of three steps : • A preparation step in which an image feature is centered on the viewing screen (with the specimen

stage) in the highest LM magnification. • A step in which the highest LM magnification is centered on the off-axis TV. • A step in which all other LM magnifications are centered on the off-axis TV (as far as attainable, see

Note 2 above).


9 Beam Nanoprobe procedure

9.1 Preparation Beam Nanoprobe alignment Purpose: Set up microscope for aligning the upper part of the column (condenser system) in Nanoprobe. Importance: ESSENTIAL to make sure that the alignment is done for the correct conditions: centered C2 aperture, eucentric height and specimen in focus. Method: C2 aperture centering: • Focus spot and center it on the screen • Turn INTENSITY overfocus (clockwise) • Center aperture until illuminated area is symmetrical around the screen center Eucentric height: with the CompuStage switch on the Alpha wobbler and minimize image movement by changing the Z height.

9.2 Pivot point beam shift Nanoprobe Purpose: Align beam shift pivot point = make sure that the beam does not tilt when it is shifted. Importance: ESSENTIAL for keeping the beam parallel to the optical axis when shifting. Method: Shifting a beam parallel to itself means that it must always go through the front-focal point (= shift pivot point) of the objective lens. This plane is conjugate to the back-focal (diffraction) plane and the alignment of the pivot point can thus be seen in diffraction. The shift 'wobble' done by the microscope should give no beam tilt, so the two central spots in the diffraction pattern should overlap. Procedure The alignment procedure consists of four steps: • Two preparation steps for setting up the image and diffraction pattern, respectively. • Two steps in which the X and Y pivot points are aligned. Notes: • The shift 'wobble' may have one beam position blocked by the specimen. If no second beam is

visible when turning MF-X, then (re)move the specimen. • Diffraction focus: the Intensity setting is preset to a fixed value that should give a (nearly) parallel

incident beam which in turn should give a spot diffraction pattern. In order to keep the diffraction focus the same as in the microprobe mode (where it can be set more reliably), the pattern should be focused (after it has been focused properly in microprobe) with the Intensity, not Focus.

9.3 Pivot point beam tilt Nanoprobe Purpose: Align beam tilt pivot point = make sure that the beam does not shift when it is tilted. Importance: ESSENTIAL for keeping the beam centered during rotation center alignment, focusing with the wobbler and dark-field operation. Method: A tilting beam must remain centered on the specimen (so the tilt pivot point coincides with the specimen). The tilt wobble done by the microscope should give no beam shift, so only one spot should be visible in the image.


Procedure The alignment procedure consists of three steps: • One preparation step for setting up the image. • Two steps in which the X and Y pivot points are aligned. Note: Unlike the shift pivot point (previous steps), the tilt pivot point is sensitive to objective-lens focus.

9.4 Dynamic conical dark field pivot point Nanoprobe (STEM systems only) Purpose: Align beam tilt pivot point = make sure that the beam does not shift when it is tilted. Importance: ESSENTIAL for keeping the beam centered during dynamic conical dark-field imaging. Method: A tilting beam must remain centered on the specimen (so the tilt pivot point coincides with the specimen). The tilt wobble done by the microscope should give no beam shift, so only one spot should be visible in the image. Procedure The alignment procedure consists of three steps : • One preparation step for setting up the image. • Two steps in which the X and Y pivot points are aligned. Notes: • The AC beam tilt pivot point is sensitive to objective-lens focus. • The AC beam tilt pivot point is used only for Dynamic Conical Dark Field. • Because TIA drives the beam in Dynamic Conical Dark Field, TIA must be running during execution

of this alignment.

9.5 Dynamic conical dark field distortion Nanoprobe (STEM systems only) Purpose: Make sure that the beam tilt describes a circle as seen in diffraction. Importance: ESSENTIAL for proper dynamic conical dark-field imaging. Method: A tilting beam must remain centered on the specimen (so the tilt pivot point coincides with the specimen). The tilt wobble done by the microscope should give no beam shift, so only one spot should be visible in the image. Procedure The alignment procedure consists of four steps : • One preparation step for setting up the image. • One step in which the diffraction pattern is centered. • One step in which the static beam tilt on the AC coils is adjusted until the beam is at the 4 cm circle. • A final step in which the beam scans around and the distortion is adjusted until the movement is

circular.

9.6 Nanoprobe rotation center Purpose: Make sure that the beam is along the optical axis of the objective lens. Importance: ESSENTIAL for minimizing the effects of aberrations on the small spot. Method: The microscope 'wobbles' the objective lens current, making the image and beam go through focus. The 'focus wobble' can be made smaller or larger with the Focus Step Size knob. Because the spot and not the image is important in nanoprobe, optimize the spot by first setting the 'wobble' to the


smallest step (focus step 1), focus the beam (Intensity) and increase the wobble (focus step). Adjust the rotation center until the beam expands and contracts concentrically. Procedure The alignment procedure consists of two steps: • One preparation step for setting up the image. • A step in which the rotation center is aligned.

A misaligned nanoprobe is recognizable from its diffuse tail that is pointing in one direction. In the case of a properly aligned beam, any tail (produced by a too large condenser aperture) should be arranged concentrically around the beam.

Note: The rotation center is an alignment that is based on a beam tilt, hence its appearance in the Beam alignment procedure and not the Image alignment procedure.



trackball. When the trackball is moved from left to right, the beam should also move from left to right on the screen. If the beam moves in a different direction, adjust the direction with the Multifunction Y knob.

9.8 Beam shift calibration Nanoprobe Purpose: Calibrate the beam shift to physically meaningful values. Importance: CONVENIENCE. Method: Move the focused beam to the 40 mm circle and adjust the displayed value of the image shift using Multifunction Y.


Procedure The alignment procedure consists of four steps: • In the first step the beam is accurately centered on the screen. • In the second step, the beam is shifted with Multifunction X to the 40 mm circle. Then the displayed


shift.

9.9 Beam tilt (dark field) calibration Nanoprobe Purpose: Calibrate the beam tilt (dark field) to physically meaningful values. Importance: ESSENTIAL for meaningful beam tilt values in dark field. Method: Tilt the beam and adjust the displayed value of the beam tilt using Multifunction X,Y. Procedure The alignment procedure consists of five steps: • The first step is a preparation step for the diffraction mode. • In the second step the diffraction pattern must be centered accurately (on the center of the viewing




9.10 Dynamic conical dark field beam tilt calibration Nanoprobe (STEM systems only) Purpose: Calibrate the AC beam tilt (dynamic conical dark field) to physically meaningful values. Importance: ESSENTIAL for meaningful beam tilt values in dynamic conical dark field and for ensuring a match between static and dynamic conical dark field. Method: Tilt the beam and adjust the displayed value of the beam tilt using Multifunction X,Y. Procedure The alignment procedure consists of three steps : • The first step is a preparation step for the diffraction mode. • In the second step the diffraction pattern must be centered accurately (on the center of the viewing


bring a ring to the center and the beam tilt value is adjusted with Multifunction Y to the correct value. Description The beam tilt is converted through the calibration procedure into to physically meaningful units. The beam tilt can be read off in the flap-out of the Alignment Control Panel and is used in the Dark Field Control Panel.


9.11 Spot size-intensity calibration Nanoprobe Purpose: Make sure that a focused beam remains focused when spot size is changed. Importance: CONVENIENCE for keeping spot focus the same for all spot sizes, ESSENTIAL for proper operation of Intensity Zoom and Intensity Limit. Method: After focusing spot 3, all spots are focused in turn. The deviations in intensity setting from spot focus are stored for all spots. Procedure The alignment procedure consists of two steps: • One preparation step in which the beam is focused for spot size 3. • A step in which all spot sizes are focused. Note: The condenser system (C1 and C2 lenses) is normalized when the spot size is changed to make the spot setting better reproducible. Description The Intensity (C2 lens) and spot size (C1 lens) settings are not independent. In order to give the same effect for all spot sizes, the Intensity is changed whenever spot size is changed. In addition to the preprogrammed changes, individual instruments differ slightly in their relation between C1 and C2. The spot size-intensity calibration allows adjustment for this individual behaviour. For the Intensity Zoom and Intensity Limit functions this procedure defines the Intensity settings at which the beam is focused, which is essential for proper operation of these functions.


10 Image Nanoprobe Procedure

10.1 SA objective lens preset Nanoprobe Purpose: Setting the eucentric focus preset at the eucentric height. Importance: CONVENIENCE for having the eucentric focus at the eucentric height. Method: Focus the image for the highest SA magnification. Procedure The alignment procedure consists of two steps: • A preparation step in which the SA image is focused at an intermediate magnification. • A step in which the highest SA magnification must be focused. Note: Although the objective lens is at a different setting for a focused nanoprobe image relative to the focus setting for a microprobe image, this does not imply that the microscope imaging system is altered in some way. The difference between the objective-lens settings is caused by the minicondenser lens whose setting has an effect on the magnetic field of the objective lens, so that in nanoprobe a slightly higher current is needed to obtain the same electron-optical effect as in microprobe.

10.2 Mh objective lens preset Nanoprobe Purpose: Finding the difference in focus between the Mh-magnification and SA. Importance: CONVENIENCE for being able to have the image in focus when switching between SA and Mh and vice versa. Method: Focus the image for the lowermost Mh magnification and center the image relative to the highest SA image. Procedure The alignment procedure consists of three steps: • A preparation step in which the image is focused for the highest SA magnification. • A step in which the lowermost Mh magnification is focused. • A step in which all other Mh magnifications are focused.

10.3 Mi objective lens preset Nanoprobe Purpose: Finding the difference in focus between Mi and SA. Importance: CONVENIENCE for having the image in focus when going from SA to Mi and vice versa. Method: Focus the image. Procedure The alignment procedure consists of three steps: • A preparation step in which the lowermost SA magnification is focused. • A step in which the highest Mi magnification is focused. • A step in which all Mi magnifications are focused.

10.4 Align diffraction pattern (Nanoprobe) Purpose: Set the zero position for the diffraction shift. Importance: CONVENIENCE for easy resetting of the diffraction shift to the screen center. Method: Center the diffraction pattern using Multifunction X,Y.


Procedure The alignment procedure consists of a single step. Description The diffraction shift has two components, an alignment value and a variable 'user' value. If properly aligned, the alignment value will have the diffraction pattern centered on the screen. It then is only necessary to reset the 'user' value to zero to have the pattern back at the screen center. The diffraction pattern alignment setting is the only setting that is separate for microprobe and nanoprobe modes. The user diffraction shift as well as the diffraction alignments between the different camera lengths are common for the two modes.

10.5 Beam shift - image shift calibration Nanoprobe Purpose: Define the beam shift compensation for the image shift. Importance: CONVENIENCE for keeping the beam on the screen when the image is shifted. The beam shift - image shift is used in the Image shift Control Panel and in Low Dose. Method: Move the image off-axis and recenter the beam using Multifunction X,Y. Procedure The calibration procedure consists of six steps: • In the first step the beam is shifted to the center of the screen. For this purpose the alignment value





• The final step just ensures that the image-beam shift is reset to zero after the procedure is finished. Description The Tecnai microscope has a function for image shift where the beam position is automatically compensated to keep the beam on the area of interest (what is currently seen on the viewing screen). The beam shift compensation must be calibrated before it will work properly. For a schematic diagram of the beam shift-image shift, see section 6.16.


11 Stigmator Procedure

11.1 Condenser stigmator calibration The microscope contains a condenser stigmator to correct for beam astigmatism. When this stigmator is misaligned, adjustment of the stigmator setting will lead to an apparent beam shift. When the stigmator is aligned correctly, these shifts are compensated by an identical but opposite beam shift - this time using the beam deflection coils. In order to align the stigmator, it is necessary to determine the relation between a change in stigmator setting and the beam shift. This relation is determined in the procedure for aligning the stigmator. The procedure follows these steps: • A preparation step in which the beam is nearly (not completely) focused. • The current through the stigmator is wobbled in the X direction and the operator minimizes beam

movement. • The same is done for the Y direction. • Two highly astigmatic beams (almost lines) should become visible. If the beams do not look like that,

change the INTENSITY until they do. With the condenser stigmator misaligned the two lines will not cross each other in the middle. Adjust the Multifunction knobs until they do.

Condenser stigmator misaligned. Condenser stigmator aligned.

11.2 Objective stigmator calibration The microscope contains an objective stigmator to correct for astigmatism in HM image and LM diffraction. When this stigmator is misaligned, adjustment of the stigmator setting will lead to an apparent HM image (or LM diffraction shift). When the stigmator is aligned correctly, these shifts are compensated by an identical but opposite shift - this time using the image deflection coils. In order to align the stigmator, it is necessary to determine the relation between a change in stigmator setting and the HM image shift. This relation is determined in the procedure for aligning the stigmator.


The procedure follows these steps: • A preparation step in which the HM image is focused. • The current through the stigmator is wobbled in the X direction and the operator minimizes image

movement. • The same is done for the Y direction. The stigmator current wobble will result in two images, which will overlap when the stigmators are aligned correctly. It is advisable to use a specimen with relatively large, easily recognized image detail.

Misaligned objective stigmator. Aligned objective stigmator.

11.3 Diffraction stigmator calibration The microscope contains a diffraction stigmator to correct for astigmatism in HM diffraction and LM image. When this stigmator is misaligned, adjustment of the stigmator setting will lead to an apparent HM diffraction shift / LM image shift. When the stigmator is aligned correctly, these shifts are compensated by an identical but opposite shift - this time using the image deflection coils. In order to align the stigmator, it is necessary to determine the relation between a change in stigmator setting and the LM image shift. This relation is determined in the procedure for aligning the stigmator. The procedure follows these steps: • A preparation step in which the microscope is switched to LM and the image is focused. • The current through the stigmator is wobbled in the X direction and the operator minimizes image

movement. • The same is done for the Y direction. The stigmator current wobble will result in two images, which will overlap when the stigmators are aligned correctly. It is advisable to use a specimen with relatively large, easily recognized image detail.


12 HM-STEM Procedure

12.1 HM-STEM Preparation Purpose: Set up microscope for aligning the column in HM-STEM. Importance: CONVENIENCE as a reminder to set up the proper conditions before starting the actual alignment. For STEM alignment a number of pre-conditions must be met, otherwise no proper alignment can be done: First, the specimen must be at the eucentric height. To do this properly, go to the Nanoprobe mode and press the Eucentric Focus button. The objective lens current is now set to the proper value for Nanoprobe (provided the SA objective preset has been aligned properly). Then bring the specimen into focus with the Z height of the specimen stage. (By doing it this way you will attain a more reproducible setting than by using the alpha wobbler.) Second, HM-STEM relies on a proper setting of several optical parameters: • The proper focusing of the diffraction pattern will be essential for the alignment of the beam shift pivot

points, which in turn is very important for reproducible STEM settings (magnification). The diffraction focusing can only be done properly in the HM Image alignment procedure.

• Since HM-STEM is derived from the Nanoprobe mode, it is essential that the Nanoprobe alignment has been done properly (especially the setting of the SA image focus preset). To avoid problems between Nanoprobe and HM-STEM this setting (which is very important for HM-STEM) can not be set in the HM-STEM alignment procedure.

• Finally, a proper gun alignment (including the spot-size dependent gun shifts) should be present before attempting to align the HM-STEM mode.

Third, Tem Imaging & Analysis (TIA) must be running when for STEM alignment. Fourth, the settings for the Preview view mode must be set so as to give an image size of 512x512 pixels and a frame time of approximately 6 seconds. Finally, the Bright-Field (BF) detector must be selected (and all other detectors deselected) in the STEM Detector Control Panel.

12.2 HM-STEM Objective / Intensity preset Purpose: Set up Objective-lens and Intensity settings for focused beam in HM-STEM. Importance: ESSENTIAL for proper settings for HM-STEM (Objective lens and Intensity settings). Method: First focus the image at the eucentric height, then focus the beam. Important: • The specimen must be at the eucentric height. To do this properly, go to the Nanoprobe mode and

press the Eucentric Focus button. The objective lens current is now set to the proper value for Nanoprobe (provided the SA objective preset has been aligned properly). Then bring the specimen into focus with the Z height of the specimen stage. (By doing it this way you will attain a more reproducible setting than by using the alpha wobbler.)

• During STEM alignment TIA (Tem Imaging & Analysis) must be running.


Procedure The alignment procedure consists of four steps : • A first step (using the Nanoprobe mode settings) in which the specimen is brought to focus (the

eucentric height, which should have been aligned in the Nanoprobe image alignment previously) by adjusting the Z height.

• A second step in which the image is focused (setting the objective-lens preset for HM-STEM) and next the beam is focused (setting the intensity preset). If necessary stigmate the image (if the image is astigmatic, the beam will also appear astigmatic but this is due to the image, not to the shape of the beam itself, and if you "stigmate" the beam with an astigmatic image, you will actually make the beam (and thus the STEM image) astigmatic. Note: Since we want to have the same objective-lens setting in Nanoprobe and HM-STEM, the alternative to setting it by looking at the image is to make sure you can observe the objective-lens excitation (%; e.g. by dragging it into a status display panel or popping up the System status) and making the value in step two the same as in step one.

• A third step in which the microscope is switched to diffraction and the diffraction pattern is centered. • A fourth similar to step three but now for all spot sizes (except 3 which has been done already). Description In principle there is no a priori requirement in scanning to define the objective-lens setting as the same as that in TEM imaging. The only requirement for STEM imaging is that the beam can be focused on the specimen, which can be done through a range of objective-lens/intensity setting combinations. In practice it is, however, not very convenient to use an objective-lens setting that differs appreciably from that in TEM imaging for various reasons: • Some of the settings of Nanoprobe and STEM imaging are shared (they use the same setting;

change one in Nanoprobe and the change will work through in STEM, and the other way around). An example is the alignment of the diffraction pattern. Some of these settings are quite sensitive to objective-lens current so it would be necessary to re-align the Nanoprobe and STEM upon switching from one to the other. It would be possible to decouple these settings (make them different for Nanoprobe and STEM) but that would increase the complexity of the alignments.

• If Nanoprobe and STEM have the same objective-lens setting, one can inspect the beam (e.g. for stigmation) in STEM by stopping the scan, moving the beam to the center, and switching to (TEM) imaging (out of diffraction). The beam is now visible while the TEM image is also in focus. If the objective-lens setting was different, the beam would look defocused in TEM imaging (but it is focused on the specimen if the STEM image is in focus). Changing the focus (objective-lens setting) to obtain a focused beam is possible, but this leads to an objective-lens setting different from that actually used in STEM which in turn leads to changes in astigmatism, rotation center, etc., so it is never possible to be sure that the beam is properly set when changing back to STEM imaging.

12.3 HM-STEM Beam tilt pivot points Purpose: Align beam tilt pivot point = make sure that the beam does not shift when it is tilted. Importance: ESSENTIAL for keeping the beam centered during rotation center alignment. Method: A tilting beam must remain centered on the specimen (so the tilt pivot point coincides with the specimen). The tilt wobble done by the microscope should give no beam shift, so only one spot should be visible in the image.


Important: • The specimen must be at the eucentric height. To do this properly, go to the Nanoprobe mode and


• During STEM alignment TIA (Tem Imaging & Analysis) must be running. Procedure The alignment procedure consists of three steps: • One preparation step for setting up the image. • Two steps in which the X and Y pivot points are aligned. Note: The tilt pivot point set in this subprocedure is the same as the Nanoprobe beam-tilt pivot point. The alignment is repeated here to ensure that the rotation center alignment in STEM can be done properly.

12.4 HM-STEM Rotation center Purpose: Make sure that the beam is along the optical axis of the objective lens. Importance: ESSENTIAL for minimizing lens aberrations and image movement during focusing. Method: The microscope 'wobbles' the objective lens current, making the image go through focus. Make the sideways movement of the image as small as possible with the rotation center (= tilting the beam to the optical axis). The 'focus wobble' can be made smaller or larger with the FOCUS STEP SIZE knob. Important: • The specimen must be at the eucentric height. To do this properly, go to the Nanoprobe mode and


• During STEM alignment TIA (Tem Imaging & Analysis) must be running. Procedure The alignment procedure consists of five steps: • A preparation step in which the beam is focused. The image must be focused by setting the

specimen to the proper eucentric height (by now the eucentric focus for STEM should have been defined).

• A second step in which the beam is (temporarily - for this alignment procedure) defocused. • A third step in which the image movement is minimized. • A fourth step in which the beam is centered and focused. • A fifth step in which the condenser aperture is aligned properly. Note: If the aperture required considerable re-alignment, one should step back through the procedure and repeat from step 2 onwards.


12.5 HM-STEM Beam-shift pivot points Purpose: Align beam shift pivot point = make sure that the beam does not tilt when it is shifted in HM-STEM. Importance: ESSENTIAL for for keeping the beam parallel to the optical axis when shifting so that: • The STEM magnification calibration remains accurate. • The STEM image is not distorted. • The diffraction pattern does not move off the detector during scanning. • The image is in focus from one edge to the other. Method: Shifting a beam parallel to itself means that it must always go through the front-focal point (= shift pivot point) of the objective lens. This plane is conjugate to the back-focal (diffraction) plane and the alignment of the pivot point can thus be seen in diffraction. The shift 'wobble' done by the microscope should give no beam tilt, so the two central disks in the diffraction pattern should overlap. Important: • The specimen must be at the eucentric height. To do this properly, go to the Nanoprobe mode and


• During STEM alignment TIA (Tem Imaging & Analysis) must be running. Procedure The alignment procedure consists of five steps: • A preparation step in normal SA (Microprobe mode) in which the image is focused. • A second step in SA diffraction (Microprobe mode) which the diffraction pattern is focused and

centered. Here the pattern is the TEM pattern which is a spot pattern. • A third step in which the diffraction focus in STEM (Nanoprobe mode) is centered (NOT focused!).

Now it is the STEM (convergent-beam) pattern which has disks, not spots. The correct focus for the pattern is defined in the previous step.

• Two steps in which the X and Y pivot points are aligned ( the Multifunction X knob sets the pivot points, the Multifunction Y the perpendicular correction).

Perform this alignment carefully. Pivot-point misalignment can have considerable effect on the STEM magnification calibration, and perpendicular correction misalignment can lead to distortion in the STEM image.

12.6 HM-STEM Align diffraction pattern Purpose: Make sure the diffraction pattern is centered properly on the viewing screen and all camera lengths are properly focused. Importance: CONVENIENT for making sure the detector alignment is correct. Method: Center the diffraction pattern on the screen. Since the position of the pattern is affected by a number of factors such as condenser-aperture position and rotation center, while the shift to the detector is reproducible (not affected by hysteresis), the only requirement later is to make sure the diffraction pattern is centered at the screen center (with the off-axis shift not active) and then switch the off-axis shift on in order to have the pattern properly centered on the BF-DF detector.


Important: • The specimen must be at the eucentric height. To do this properly, go to the Nanoprobe mode and


• During STEM alignment TIA (Tem Imaging & Analysis) must be running. Procedure The alignment procedure consists of four steps: • In the first step the normal TEM is focused and the beam is centered. • In the second step the normal TEM diffraction pattern is focused properly (focus the diffraction

pattern to a spot pattern on the basis of a preset intensity setting). • In the third step, the microscope is switched to STEM and the STEM diffraction pattern (now a disk

pattern) is centered. • In the fourth step, each camera length is centered on the screen and focused. Notes: • This alignment affects the same diffraction alignment parameter as the diffraction alignment for

nanoprobe. The focusing of the individual camera lengths is the same as in the HM Image camera lengths procedure. These alignments are repeated in this HM-STEM alignment subprocedure for convenience.

• The focusing of the camera lengths is repeated here because it can be done more accurately in STEM. Once we know that the beam-shift pivot points are correct, we know that any movement of the diffraction pattern must be due to a diffraction focus that is away from the back-focal plane. Focusing can thus be done by minimizing movement.

• For the movement of the diffraction pattern in STEM, it is important to make a distinction between any movement seen during the 'normal' scan and during the flyback (the rapid beam movement back to the beginning of the next scan line). During the flyback the beam often cannot keep up due to the inertia in the optics and the diffraction pattern may make an irregular swing. During the flyback the diffraction pattern typically has much less intensity than during the normal scan, producing an irregular line of lower intensity. The flyback should be ignored when it comes to minimizing movement.

12.7 HM-STEM Detector alignment Purpose: Make sure the diffraction pattern is centered properly on the STEM Bright-Field/Dark-Field detector. Importance: ESSENTIAL for obtaining correct STEM Bright-Field and Dark-Field images. Method: First center the diffraction pattern on the screen, then define the off-axis shift necessary to have the pattern centered on the bright-field/dark-field detectors. Since the latter shift is reproducible (not affected by hysteresis), the only requirement later is to make sure the diffraction pattern is centered in the screen center (with the off-axis shift not active) and then switch the off-axis shift on in order to have the pattern properly centered again. Important: • The specimen must be at the eucentric height. To do this properly, go to the Nanoprobe mode and




Procedure The alignment procedure consists of three steps: • In the first step the diffraction pattern (for a camera length of ~500mm) is centered on the viewing

screen with the screen down. • In the second step the off-axis shift is activated and the same camera length is centered on the

STEM detectors, first by moving the pattern to the approximate position with the screen down, then with the screen up by looking at the detector signal.

• In the final step, a number of camera lengths is centered on the detector. Which camera lengths are to be aligned is somewhat instrument-dependent (see below).

Centering on the detector Use a specimen that is thin (or remove the specimen altogether by retracting the specimen holder a bit), because in thick specimens the dark-field signal is too close in intensity to the bright-field signal. Move the diffraction pattern roughly to the place where the detectors are located. When the diffraction pattern is shifted across the detectors, the following behavior is seen: • On the Bright-Field (BF) detector, the signal follows a simple top-hat function(see picture below): low

(when the central disk is not on the BF detector) - high (on the BF detector) - low (off again). To align, first go for maximum intensity in the BF signal. The reduce the X shift until the signal drops off. Then increase the X shift again while 'counting' knob turns until the signal has gone through its maximum and drops off again. Turn back by half of the number of turns counted. Repeat for Y.

• On the Dark-Field (DF) detector, the signal follows a double top-hat function: low - high - low - high -low (initially off the detector, the on one side of the ring-shaped detector, then on the BF detector and thus off the DF, on the other side of the ring, and finally off the detector again). It is also possible for the pattern to follow a simple top-hat. as for the BF. In the latter case the diffraction pattern does not move across the central part of the detector (as in path 2 in the lowermost picture below) but away from the center so the central beam never hits the BF detector properly.

Schematic diagram of the change in detector signals for the BF (blue) and DF (red) detectors observed when the diffraction pattern is moved along a path similar to 1 below.

Schematic diagram of possible paths of the diffraction pattern across the BF (blue) and DF (red) detectors (seen from the top). Along path 1, the detector-signal changes are as sketched in the picture above. Along path 2 the BF signal never gets to be a true BF, and the pattern must be moved first in the perpendicular direction to get the proper alignment.

Always check that the pattern is centered properly for the whole image (if part of it is dark, the alignment is not correct).


Notes: • It is useful to activate the Scope function on the STEM Imaging Control Panel. This function displays

the detector signal levels on a fixed scale and thereby makes it easier to judge the alignment (the STEM image has its contrast and brightness re-adjusted after an image has been collected, which makes it difficult to judge the signal levels from the detector).

• The near-axis position (where the STEM Bright-Field/Dark-Field detector assembly is located) is at ~30 mm from the screen center to the ENE (slightly away from the screen center to the top right). This physical position is of course the same for all camera lengths but the actual diffraction shifts required to get there depend on the camera length (which magnifies the diffraction shift), so the settings are dependent on the camera length.

• Because hysteresis can have considerable effect on the diffraction-pattern position, you should use the normalization facility whenever the optical conditions have changed (the automatic normalizations take care of this, so it is advised to keep them enabled).

• Some of the lowermost camera lengths are too small to reach the near-axis detector (typically the camera length must be at least 90 mm). There is no point in trying to align these. Simply go on to the next camera length.

• Some of the large camera lengths will overlap the bright-field disk onto the dark-field detector and are therefore of little use. You can skip these camera lengths as well.

• When the camera length changes, the signal on the detectors will change, so it will be necessary to adjust contrast and brightness. For this purpose, the alignment procedure makes it easy to switch to detector control by pressing R2 (toggle between detector alignment and BF detector control).

• Since the size of the Condenser aperture determines the beam convergence angle, it also determines the size of the diffraction disks and thereby what the effective camera lengths are for BF/DF imaging (if the central beam is too large, it will fall on the DF detector). To determine if the beam doesn't have long tails due to the combined effects of spherical aberration and large convergence angles (and thus poorly defined spots), you can observe the focused beam in nanoprobe mode (for the particular condenser aperture). Normally the two smallest condenser apertures are the ones that are suitable for STEM.

• Some camera lengths are difficult to use at low STEM magnifications because the spherical aberration of the diffraction lens makes it impossible to have the diffraction remain sufficient stationary to keep it on the detectors (the pattern described by the central-beam disk looks like a bird flying, with its wings flapping up and down). Either use higher magnifications or other camera lengths in those cases.

12.8 HM-STEM Distortion adjustment Purpose: Adjust the AC deflection coils settings so that the resulting STEM image has no distortions. Importance: ESSENTIAL for proper STEM imaging. Method: Collect an image of a cross-grating and adjust the relative strength of X and Y coils, and their perpendicularity. Important: • The specimen must be at the eucentric height. To do this properly, go to the Nanoprobe mode and




Procedure The alignment procedure consists of five steps: • The first step is a preparation step to center and focus the beam. • In the second step a STEM image must be made of a cross-grating specimen. • In the third step the scan rotation is set so the horizontal lines of the cross-grating are horizontal in

the STEM image. The rotation required should be less than ~15° (see note below). • In the fourth step the distortions are adjusted. Multifunction X controls the trapezoid distortion (the

vertical lines of the grating are not perpendicular to the horizontal lines), while the Multifunction Y adjusts the relative strengths of the horizontal and vertical scans (rectangular distortion).

• A check on the previous alignment with the image rotated by 90°. If necessary, iterate steps three and four (see also notes below). Notes: • It is essential that the pivot point alignment has been done properly before the distortion adjustment

is done. • The cross-grating used must be inserted into the microscope in such a way that the horizontal and

vertical lines in the grating are roughly parallel to the horizontal and vertical in the image when the scan rotation (and the default scan rotation) is close to 0° (within ~15°), otherwise the alignment is very difficult (requires many iterations). Judge whether the orientation of the cross-grating is suitable in the second or third step of the procedure. If the orientation is further away from 0°, remove the specimen holder from the microscope and rotate the specimen until its orientation is suitable for this alignment.

• The distortions of the image may be judged more easily by drawing a square in the image. Click on the Image Selection Tool toolbar button in TIA , click in the top left quadrant of the image and drag the cursor to bottom right while keeping the Shift key on the keyboard pressed down (the latter forces the image selection drawn to remain square). It is then easy to count the number of cross-grating squares in the horizontal and vertical dimensions and making sure that the numbers are the same.

• Description • The deflection coils used in the microscope are not all equal and exactly the same from one

microscope to another. There are therefore alignment procedures that will result in adjustments that compensate for the variation in the coils. For the deflection coils used in TEM operation, the two adjustable parameters are the pivot points and the perpendicular correction. The pivot adjusts for the difference in strength between upper and lower coil (e.g the X coil). The perpendicular correction adjusts for a rotation between the upper and lower coils (e.g. the upper and lower X coils) by adding a small deflection to the other lower coil (in that case the lower Y coil). For use in TEM this is sufficient.

• In STEM, however, additional adjustments are necessary, otherwise image distortions (stretch of one direction relative to the other and angular distortion) appear. These adjustments correct for the difference in strength between the X and Y coils and for any deviation from 90° between the X and Y coils. In principle this can be done both for the upper and lower coils separately but in practice this is too difficult to align and a single adjustment is sufficient.

12.9 HM-STEM Default rotation Purpose: Define the rotation correction for STEM imaging. Importance: CONVENIENCE for having the STEM image (at 0° scan rotation) in the same orientation as the TEM image (on the viewing screen). Method: Observe the scan frame on the viewing screen and rotate it until it runs as described below.


Procedure The alignment procedure consists of two steps: • The first step is a preparation step to center and focus the beam on the viewing screen. • In the second step the beam is scanned so the scan frame is visible on the viewing screen. Change

the rotation correction setting until the scan line (the fast scan direction) goes from left to right (W to E) on the screen and the scan frame (the slow scan direction) goes from top to bottom.

Note: The movement of the beam during scanning can be described as follows: • Move the beam to the starting position of the scan image (top left). • Move the beam along a line (the horizontal direction in the scan image). • At the end of the line, jump back to the beginning of the line (the flyback) and change the beam

position one line down. • Repeat this until you reach the bottom of the scan image at the end of the last line. • Go back to the first step. The horizontal direction in scanning is referred to as the 'line', while the vertical direction is called the 'frame'. Since the frame is built up line by line, the scan in the direction of the frame is much slower than that along the lines.


13 LM-STEM procedure

13.1 LM-STEM Preparation Purpose: Set up microscope for aligning the column in LM-STEM. Importance: CONVENIENCE as a reminder to set up the proper conditions before starting the actual alignment. For STEM alignment a number of pre-conditions must be met, otherwise no proper alignment can be done: • First of all, LM-STEM relies on a proper setting of several optical parameters for which the Gun, LM

Beam and LM Image as well as HM-TEM Image alignment must have been done. • Second, Tem Imaging & Analysis (TIA) must be running when for STEM alignment. • Third, the settings for the Preview view mode must be set so as to give an image size of 512x512

pixels and a frame time of approximately 6 seconds and the Search view mode to an image size of 256x256 and a frame time of approximately 3 seconds.

• Finally, the Bright-Field (BF) detector must be selected (and all other detectors deselected) in the STEM Detector Control Panel.

Note: The diffraction focus (LAD) is LM-STEM is kept as set in LAD (proceed to LM, then to diffraction). In most cases this should be kept at the default setting. Unless there is a reason to deviate from this, it is important to make sure the setting is properly done. Go to LM, then LAD and press the Eucentric focus button to reset the LAD focus to the default value.

13.2 LM-STEM Intensity preset Purpose: Set up Intensity setting for focused beam in LM-STEM. Importance: ESSENTIAL for proper setting for LM-STEM (Intensity settings). Method: First focus the image, then focus the beam. Important : During STEM alignment TIA (Tem Imaging & Analysis) must be running. Procedure The alignment procedure consists of three steps : • A first step in which the image is focused and the beam is centered. • A second step in which the beam is focused accurately (sets the Intensity setting). • A third step in which the focusing of the beam is repeated for all spots (except 3 which has already

been done in the previous step).

13.3 LM-STEM Beam-shift pivot points Purpose: Align beam shift pivot point = make sure that the beam does not tilt when it is shifted in LM-STEM. Importance: ESSENTIAL for for keeping the beam parallel to the optical axis when shifting so that : • The STEM magnification calibration remains accurate. • The STEM image is not distorted. • The diffraction pattern does not move off the detector during scanning. • The image is in focus from one edge to the other.


Method: Shifting a beam parallel to itself means that it must always go through the front-focal point (= shift pivot point). This plane is conjugate to the back-focal (diffraction) plane and the alignment of the pivot point can thus be seen in diffraction. The shift 'wobble' done by the microscope should give no beam tilt, so the two central disks in the diffraction pattern should overlap. Note: The diffraction focus (LAD) is LM-STEM is kept as set in LAD (proceed to LM, then to diffraction). In most cases this should be kept at the default setting. Unless there is a reason to deviate from this, it is important to make sure the setting is properly done. Go to LM, then LAD and press the Eucentric focus button to reset the LAD focus to the default value. Important: During STEM alignment TIA (Tem Imaging & Analysis) must be running. Procedure The alignment procedure consists of three steps : • A first step in LAD diffraction which the diffraction pattern is centered. • Two steps in which the X and Y pivot points are aligned ( the Multifunction X knob sets the pivot

points, the Multifunction Y the perpendicular correction). Perform this alignment carefully. Pivot-point misalignment can have considerable effect on the STEM magnification calibration, and perpendicular correction misalignment can lead to distortion in the STEM image. Note: Because of the large shifts of the beam (in LM-STEM) it is possible that one of the wobble directions (or even both) is blocked by the specimen (grid). If this is the case, either move the specimen around a bit or retract the holder slightly (about 1 cm).

13.4 LM-STEM Align diffraction pattern Purpose: Make sure the diffraction pattern is centered properly on the viewing screen. Importance: CONVENIENT for making sure the detector alignment is correct. Method: Center the diffraction pattern on the screen. Since the position of the pattern is affected by a number of factors such as condenser-aperture position and rotation center, while the shift to the detector is reproducible (not affected by hysteresis), the only requirement later is to make sure the diffraction pattern is centered at the screen center (with the off-axis shift not active) and then switch the off-axis shift on in order to have the pattern properly centered on the BF-DF detector. Important: During STEM alignment TIA (Tem Imaging & Analysis) must be running. Procedure The alignment procedure consists of one step in which the LAD diffraction pattern is centered on the screen. Note: In LM-STEM only one camera length is used.

13.5 LM-STEM Detector alignment Purpose: Make sure the diffraction pattern is centered properly on the STEM Bright-Field/Dark-Field detector. Importance: ESSENTIAL for obtaining correct STEM Bright-Field and Dark-Field images. Method: First center the diffraction pattern on the screen, then define the off-axis shift necessary to have the pattern centered on the bright-field/dark-field detectors. Since the latter shift is reproducible (not affected by hysteresis), the only requirement later is to make sure the diffraction pattern is centered in


the screen center (with the off-axis shift not active) and then switch the off-axis shift on in order to have the pattern properly centered again. Important: During STEM alignment TIA (Tem Imaging & Analysis) must be running. Procedure The alignment procedure consists of two steps : • In the first step the diffraction pattern is centered on the viewing screen with the screen down. • In the second step the off-axis shift is activated and the same camera length is centered on the

STEM detectors, first by moving the pattern to the approximate position with the screen down, then with the screen up by looking at the detector signal.

Centering on the detector Use a specimen that is thin (or remove the specimen altogether by retracting the specimen holder a bit), because in thick specimens the dark-field signal is too close in intensity to the bright-field signal. Move the diffraction pattern roughly to the place where the detectors are located. When the diffraction pattern is shifted across the detectors, the following behavior is seen: On the Bright-Field (BF) detector, the signal follows a simple top-hat function(see picture below): low (when the central disk is not on the BF detector) - high (on the BF detector) - low (off again). To align, first go for maximum intensity in the BF signal. The reduce the X shift until the signal drops off. Then increase the X shift again while 'counting' knob turns until the signal has gone through its maximum and drops off again. Turn back by half of the number of turns counted. Repeat for Y. On the Dark-Field (DF) detector, the signal follows a double top-hat function : low - high - low - high -low (initially off the detector, the on one side of the ring-shaped detector, then on the BF detector and thus off the DF, on the other side of the ring, and finally off the detector again). It is also possible for the pattern to follow a simple top-hat. as for the BF. In the latter case the diffraction pattern does not move across the central part of the detector (as in path 2 in the lowermost picture below) but away from the center so the central beam never hits the BF detector properly.

Schematic diagram of the change in detector signals for the BF (blue) and DF (red) detectors observed when the diffraction pattern is moved along a path similar to 1 below. Schematic diagram of possible paths of the diffraction pattern across the BF (blue) and DF (red) detectors (seen from the top). Along path 1, the detector-signal changes are as sketched in the picture above. Along path 2 the BF signal never gets to be a true BF, and the pattern must be moved first in the perpendicular direction to get the proper alignment.

Always check that the pattern is centered properly for the whole image (if part of it is dark, the alignment is not correct).


Notes: • It is useful to activate the Scope function on the STEM Imaging Control Panel. This function displays

the detector signal levels on a fixed scale and thereby makes it easier to judge the alignment (the STEM image has its contrast and brightness re-adjusted after an image has been collected, which makes it difficult to judge the signal levels from the detector).

• The near-axis position (where the STEM Bright-Field/Dark-Field detector assembly is located) is at ~30 mm from the screen center to the ENE (slightly away from the screen center to the top right). This physical position is of course the same for all camera lengths but the actual diffraction shifts required to get there depend on the camera length (which magnifies the diffraction shift), so the settings are dependent on the camera length.

• Because hysteresis can have considerable effect on the diffraction-pattern position, you should use the normalization facility whenever the optical conditions have changed (the automatic normalizations take care of this, so it is advised to keep them enabled).

13.6 LM-STEM Distortion adjustment Purpose: Adjust the AC deflection coils settings so that the resulting STEM image has no distortions. Importance: ESSENTIAL for proper STEM imaging. Method: Collect an image of a cross-grating and adjust the relative strength of X and Y coils, and their perpendicularity. Important: • The specimen must be at the eucentric height. To do this properly, go to the Nanoprobe mode and


• During STEM alignment TIA (Tem Imaging & Analysis) must be running. Procedure The alignment procedure consists of five steps: • The first step is a preparation step to center and focus the beam. • In the second step a STEM image must be made of a cross-grating specimen. • In the third step the scan rotation is set so the horizontal lines of the cross-grating are horizontal in

the STEM image. The rotation required should be less than ~15° (see note below). • In the fourth step the distortions are adjusted. Multifunction X controls the trapezoid distortion (the

vertical lines of the grating are not perpendicular to the horizontal lines), while the Multifunction Y adjusts the relative strengths of the horizontal and vertical scans (rectangular distortion).

• A check on the previous alignment with the image rotated by 90°. If necessary, iterate steps three and four (see also notes below). Notes: • It is essential that the pivot point alignment has been done properly before the distortion adjustment

is done. • The cross-grating used must be inserted into the microscope in such a way that the horizontal and

vertical lines in the grating are roughly parallel to the horizontal and vertical in the image when the scan rotation (and the default scan rotation) is close to 0° (within ~15°), otherwise the alignment is very difficult (requires many iterations). Judge whether the orientation of the cross-grating is suitable in the second or third step of the procedure. If the orientation is further away from 0°, remove the specimen holder from the microscope and rotate the specimen until its orientation is suitable for this alignment.


• The distortions of the image may be judged more easily by drawing a square in the image. Click on the Image Selection Tool toolbar button in TIA , click in the top left quadrant of the image and drag the cursor to bottom right while keeping the Shift key on the keyboard pressed down (the latter forces the image selection drawn to remain square). It is then easy to count the number of cross-grating squares in the horizontal and vertical dimensions and making sure that the numbers are the same.

• Description • The deflection coils used in the microscope are not all equal and exactly the same from one

microscope to another. There are therefore alignment procedures that will result in adjustments that compensate for the variation in the coils. For the deflection coils used in TEM operation, the two adjustable parameters are the pivot points and the perpendicular correction. The pivot adjusts for the difference in strength between upper and lower coil (e.g the X coil). The perpendicular correction adjusts for a rotation between the upper and lower coils (e.g. the upper and lower X coils) by adding a small deflection to the other lower coil (in that case the lower Y coil). For use in TEM this is sufficient.

• In STEM, however, additional adjustments are necessary, otherwise image distortions (stretch of one direction relative to the other and angular distortion) appear. These adjustments correct for the difference in strength between the X and Y coils and for any deviation from 90° between the X and Y coils. In principle this can be done both for the upper and lower coils separately but in practice this is too difficult to align and a single adjustment is sufficient.

13.7 LM-STEM Default rotation Purpose: Define the rotation correction for LM-STEM imaging so that the LM-STEM image is displayed with the same orientation as the HM-STEM image. Importance: CONVENIENCE for making it easier to switch from LM-STEM imaging or HM-STEM imaging and vice versa. Method: Observe the movement in the scan image when the stage is moved from left to right and adjust the rotation correction until the image also moves from left to right. Important: During STEM alignment TIA (Tem Imaging & Analysis) must be running. Procedure The alignment procedure consists of three steps : • The first step is a preparation step to center the diffraction pattern. • In the second step an LM-STEM image is made. • In the third step the specimen is moved from left to right with the stage and the rotation correction

adjusted until the features in the image also move from left to right.


14 HM-EFTEM procedure

14.1 HM-EFTEM Preparation Purpose: Set up microscope for aligning the column for EFTEM. Importance: ESSENTIAL to make sure the alignment is done for the correct conditions: centered C2 aperture, eucentric height and specimen in focus. Also find a recognizable image feature for later alignment steps. Notes: • Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over

correction are often quite critical, otherwise there is no image or diffraction pattern visible on the Imaging Filter), the EFTEM alignment procedure contains two parts, one a pre-alignment, where all alignments are done with the screen down, the other the final alignment where all is aligned on the Filter itself. Once the microscope has been aligned properly, the pre-alignment should not be necessary anymore and the final alignment can be used for fine-tuning.

• The EFTEM alignments are in many cases (lower magnifications and camera lengths) sensitive to hysteresis. It is therefore important to use the normalization facility of the microscope to make image and diffraction-pattern positions reproducible.

14.2 HM-EFTEM Image-shift pivot points Purpose: Align image shift pivot point = make sure that the diffraction pattern does not shift when the image shift is changed. Importance: ESSENTIAL for the proper functioning of image shifts and cross-over correction. Method: Minimize the movement of the diffraction pattern. The microscope 'wobbles' the image shift which should cause no diffraction shift. Procedure The alignment procedure consists of six steps : • Two preparation steps for setting up the image in normal and EFTEM modes. • Two steps for setting up the diffraction pattern in normal and EFTEM modes. • Two steps in which the X and Y pivot points are aligned. In EFTEM a misaligned image shift will lead to an apparent diffraction shift when the cross-over correction is changed. Note: Diffraction focus. The Intensity setting is preset to a fixed value that gives a parallel incident beam which in turn should give a spot diffraction pattern. If the pattern is not focused, it should be focused with the diffraction lens (FOCUS), not Intensity. Description The alignment of the pivot points of the image deflection coils is essential to the proper functioning of many other alignments and calibrations: image shifts, diffraction shifts, detector alignments and measurement functions. The image coils pivot points (image shift and diffraction shift) alignment is reasonably insensitive to the operating conditions and normally needs to be done only once. It is important that the image coils pivot points are aligned prior to aligning other parts of the microscope where the image coils are used, otherwise the latter alignments may have to be redone. Alignments where the image coils are used are those where images or diffraction patterns are moved (image shift, diffraction shift/diffraction alignment).


14.3 HM-EFTEM SA Diffraction-shift pivot points Purpose: Align the diffraction shift pivot point = make sure the image does not move when the diffraction shift is changed. Importance: ESSENTIAL for the proper functioning of image and diffraction shifts and the cross-over correction. Method: Minimize the movement of the image. The microscope 'wobbles' the diffraction shift which should cause no image shift. A common consequence of a diffraction shift that has not been aligned is a mismatch between the image detail selected with the selected area aperture and the area actually contributing to the diffraction pattern. When the image coils have not been aligned properly, image and diffraction shift are not uncoupled and the diffraction shift will also cause an image shift, thereby moving the image detail out of the selected area aperture. In EFTEM a misaligned diffraction shift will lead to an apparent image shift when the cross-over correction is changed. Procedure The alignment procedure consists of four steps : • Two preparation step for setting up the image in normal and EFTEM mode. • Two steps in which the X and Y pivot points are aligned. Description The alignment of the pivot points of the image deflection coils is essential to the proper functioning of many other alignments and calibrations: image shifts, diffraction shifts, detector alignments and measurement functions. The image coils pivot points (image shift and diffraction shift) alignment is reasonably insensitive to the operating conditions and normally needs to be done only once. It is important that the image coils pivot points are aligned prior to aligning other parts of the microscope where the image coils are used, otherwise the latter alignments may have to be redone. Alignments where the image coils are used are those where images or diffraction patterns are moved (image shift, diffraction shift/diffraction alignment). For images displaying the effect of the alignment, see section 6.3.

14.4 HM-EFTEM Mh Diffraction-shift pivot points Purpose: Align the diffraction shift pivot point = make sure the image does not move when the diffraction shift is changed. Importance: ESSENTIAL for the proper functioning of image and diffraction shifts and the cross-over correction. Method: Minimize the movement of the image. The microscope 'wobbles' the diffraction shift which should cause no image shift. A common consequence of a diffraction shift that has not been aligned is a mismatch between the image detail selected with the selected area aperture and the area actually contributing to the diffraction pattern. When the image coils have not been aligned properly, image and diffraction shift are not uncoupled and the diffraction shift will also cause an image shift, thereby moving the image detail out of the selected area aperture. In EFTEM a misaligned diffraction shift will lead to an apparent image shift when the cross-over correction is changed.


Procedure The alignment procedure consists of three steps : • A preparation step for setting up the image in EFTEM mode. • Two steps in which the X and Y pivot points are aligned. The preparation here does not contain a step for the normal mode, because it is assumed that that has already been done for the SA pivot points. Description The alignment of the pivot points of the image deflection coils is essential to the proper functioning of many other alignments and calibrations: image shifts, diffraction shifts, detector alignments and measurement functions. The image coils pivot points (image shift and diffraction shift) alignment is reasonably insensitive to the operating conditions and normally needs to be done only once. It is important that the image coils pivot points are aligned prior to aligning other parts of the microscope where the image coils are used, otherwise the latter alignments may have to be redone. Alignments where the image coils are used are those where images or diffraction patterns are moved (image shift, diffraction shift/diffraction alignment). For images displaying the effect of the alignment, see section 6.3.

14.5 HM-EFTEM Mi Diffraction-shift pivot points Purpose: Align the diffraction shift pivot point = make sure the image does not move when the diffraction shift is changed. Importance: ESSENTIAL for the proper functioning of image and diffraction shifts and the cross-over correction. Method: Minimize the movement of the image. The microscope 'wobbles' the diffraction shift which should cause no image shift. A common consequence of a diffraction shift that has not been aligned is a mismatch between the image detail selected with the selected area aperture and the area actually contributing to the diffraction pattern. When the image coils have not been aligned properly, image and diffraction shift are not uncoupled and the diffraction shift will also cause an image shift, thereby moving the image detail out of the selected area aperture. In EFTEM a misaligned diffraction shift will lead to an apparent image shift when the cross-over correction is changed. Procedure The alignment procedure consists of three steps : • A preparation step for setting up the image in EFTEM mode. • Two steps in which the X and Y pivot points are aligned. The preparation here does not contain a step for the normal mode, because it is assumed that that has already been done for the SA pivot points. Description The alignment of the pivot points of the image deflection coils is essential to the proper functioning of many other alignments and calibrations: image shifts, diffraction shifts, detector alignments and measurement functions. The image coils pivot points (image shift and diffraction shift) alignment is reasonably insensitive to the operating conditions and normally needs to be done only once. It is important that the image coils pivot points are aligned prior to aligning other parts of the microscope where the image coils are used, otherwise the latter alignments may have to be redone. Alignments where the image coils are used are those where images or diffraction patterns are moved (image shift, diffraction shift/diffraction alignment).


For images displaying the effect of the alignment, see section 6.3.

14.6 HM-EFTEM Mh Pre-alignment Purpose: Aligning the Mh-magnification images for EFTEM (as seen on the Filter) with the Mh images for the normal mode, and finding the difference in focus. Importance: CONVENIENCE for being able to find the image at high magnifications. Method: Focus the image for the lowermost Mh magnification and center the image relative to the highest SA image. Procedure The alignment procedure consists of four steps : • A preparation step in which an image feature is centered (with the specimen stage) in the highest SA

magnification for the normal mode. • A step in which the highest EFTEM-SA magnification is focused and centered relative to the normal

mode. • A step in which the lowest EFTEM-Mh magnification is focused and aligned relative to the highest

EFTEM-SA magnification. • A step in which all other EFTEM-Mh magnifications are focused and aligned. Note: Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over correction are often quite critical, otherwise there is no image or diffraction pattern visible on the Imaging Filter), the EFTEM alignment procedure contains two parts, one a pre-alignment, where all alignments are done with the screen down, the other the final alignment where all is aligned on the Filter itself. Once the microscope has been aligned properly, the pre-alignment should not be necessary anymore and the final alignment can be used for fine-tuning.

14.7 HM-EFTEM SA Image-shift pre-alignment Purpose: Aligning all EFTEM-SA images with each other. Importance: CONVENIENCE so that the image remains centered when the magnification is changed. Method: Center a recognizable image with the specimen stage. Lower the magnification one step, center the image with the Multifunction X,Y knobs. Repeat for all magnifications. Procedure The alignment procedure consists of two steps : • A preparation step in which an image feature is centered (with the specimen stage) in the highest SA

magnification for the normal mode. • A step in which all EFTEM-SA magnifications are aligned relative to the highest normal SA

magnification. Notes: • Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over


• In case the cross-over correction is strongly misaligned, it is possible to switch to the cross-over correction adjustment and back by pressing the R2 button.


14.8 HM-EFTEM Mi Image-shift pre-alignment Purpose: Aligning the EFTEM Mi magnification images with SA and finding the difference in focus. Importance: CONVENIENCE so that the image remains centered when the magnification is changed. Method: Center a recognizable image with the specimen stage. Lower the magnification one step, center the image with the Multifunction X,Y knobs. Repeat for all magnifications. Procedure The alignment procedure consists of three steps : • A preparation step in which an image feature is centered (with the specimen stage) in the lowest

EFTEM-SA magnification. • A step in which the highest EFTEM-Mi magnification is aligned relative to the lowest EFTEM-SA

magnification. • A step in which all EFTEM-Mi magnifications are aligned to lowest EFTEM-SA magnification. Notes: • Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over



14.9 HM-EFTEM Mh Image shift Purpose: Aligning the Mh-magnification images for EFTEM (as seen on the Filter) with the Mh images for the normal mode, and finding the difference in focus. Importance: CONVENIENCE for being able to find the image at high magnifications. Method: Focus the image for the lowermost Mh magnification and center the image relative to the highest SA image. Procedure The alignment procedure consists of four steps : • A preparation step in which an image feature is centered (with the specimen stage) in the highest SA

magnification for the normal mode. • A step in which the highest EFTEM-SA magnification is focused and centered relative to the normal

mode. • A step in which the lowest EFTEM-Mh magnification is focused and aligned relative to the highest

EFTEM-SA magnification. • A step in which all other EFTEM-Mh magnifications are focused and aligned. Note: Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over correction are often quite critical, otherwise there is no image or diffraction pattern visible on the Imaging Filter), the EFTEM alignment procedure contains two parts, one a pre-alignment, where all alignments are done with the screen down, the other the final alignment where all is aligned on the Filter itself. Once the microscope has been aligned properly, the pre-alignment should not be necessary anymore and the final alignment can be used for fine-tuning.


14.10 HM-EFTEM SA Image shift Purpose: Aligning all EFTEM-SA images with each other. Importance: CONVENIENCE so that the image remains centered when the magnification is changed. Method: Center a recognizable image with the specimen stage. Lower the magnification one step, center the image with the Multifunction X,Y knobs. Repeat for all magnifications. Procedure The alignment procedure consists of three steps : • A preparation step in which an image feature is centered (with the specimen stage) in the highest SA

magnification for the normal mode. • A step in which the highest EFTEM-SA magnification is aligned on the Imaging Filter and focused. • A step in which all EFTEM-SA magnifications are aligned relative to the highest normal SA

magnification. Notes: • Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over



14.11 HM-EFTEM Mi Image shift Purpose: Aligning the EFTEM Mi magnification images with SA and finding the difference in focus. Importance: CONVENIENCE so that the image remains centered when the magnification is changed. Method: Center a recognizable image with the specimen stage. Lower the magnification one step, center the image with the Multifunction X,Y knobs. Repeat for all magnifications. Procedure The alignment procedure consists of three steps : • A preparation step in which an image feature is centered (with the specimen stage) at the lowermost

EFTEM-SA magnification. • A step in which the highest EFTEM-Mi magnification is aligned relative to the lowermost EFTEM-SA

magnification. • A step in which all EFTEM-Mi magnifications are aligned to the lowermost EFTEM-SA magnification. Notes: • Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over


In case the cross-over correction is strongly misaligned, it is possible to switch to the cross-over correction adjustment and back by pressing the R2 button.


14.12 HM-EFTEM SA and Mi Cross-over correction Purpose: Make sure the cross-over correction is set correctly, so the beam passes through the GIF (and especially through the energy slit) in the same way for all magnifications. Importance: ESSENTIAL for having the image visible on the Imaging Filter, especially at the lower magnifications. The benefits of aligning the beam direction to the GIF A change of beam direction to the GIF leads to a small mistuning of the GIF. This can be corrected by re-tuning the GIF parameters Fx, Fy, Sx, and Sy. Gatan's Autofilter software can store and automatically recall separate values for Fx, Fy, Sx, and Sy for each microscope magnification. So, in principle, it is not necessary to make sure that the beam direction is the same for each magnification. However, after a longer period of time, it can be necessary to re-align the GIF parameters. If the beam direction is different for each magnification then the user must re-tune the GIF parameters at every magnification. Although this is very straightforward, it is time-consuming. Furthermore, if not done consistently for all magnifications, then one can end up with one magnification aligned to the situation one month ago and another magnification aligned to the situation of two months ago. When one switches between these two magnifications one can get the impression that the system is very unstable. A change of beam direction in the x-direction in the GIF also leads to an apparent energy shift in the GIF and thus to disappearance of the beam when small slit widths are used. This apparent energy shift can be easily corrected by pressing 'Align ZLP' but this is inconvenient. Therefore, it is advised to try to minimize the changes of beam direction when the magnification is changed. The cross-over alignment aims at minimizing these changes. In favorite cases, it is possible to get the same beam direction for all HM magnifications. Method: Center the image of the beam stop to the same place as at the highest SA magnification. Procedure The alignment procedure consists of five steps : • A preparation step in the highest SA magnification for the normal mode. • A step in which the Tune GIF procedure must be run for the highest SA magnification. If not already

done, the magnification-dependent GIF tunings in Digital Micrograph should be removed (see below). • A step in which the beam stop is centered on the CCD image. • A step in which the cross-over corrections are set for all EFTEM-SA magnifications except the

highest (where the cross-over correction is zero by definition). • A step in which the cross-over corrections are set for all EFTEM-Mi magnifications. Removing magnification-dependent GIF tunings from DigitalMicrograph DigitalMicrograph keeps magnification-dependent GIF tunings which may interfere with proper operation if the cross-over correction is set properly (in effect the DigitalMicrograph settings compensate for misalignments that may no longer exist). Because of this, the magnification-dependent tunings must be removed. In the DigitalMicrograph menu, select File -> Global info -> Tags -> IF Suite -> Autofilter -> Settings, and delete the branch of the current high voltage.


Delete the branch of the current high voltage. Working with the beam-stop contour Because the beam stop is located below the plane of the differential pumping aperture, it appears to move if the cross-over correction is changed. This feature can be used to align the cross-over correction for the different magnifications so that the GIF always "sees" the cross-over in the same place (thereby reducing the need for magnification-dependent GIF tunings as well as correction of the spectrum energy shift if the magnification is changed). The procedure is as follows: • Set the CCD Search settings such that the full CCD area is used (if necessary, use binning to reduce

the effective image size to 512²). • Start CCD Search and center the tip of the beam stop on the GIF CCD. • Use markers to draw lines around the contour of the beam stop (see image below). • Use the contour at other magnifications to recenter the image of the beam stop by changing the

cross-over correction.


Contour (in red) of the beam stop (in black) on the GIF image.

Notes: • Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over


• In case the image shift is strongly misaligned, it is possible to switch to the image shift adjustment and back by pressing the R2 button.

If the required cross-over shift is more than the range of the deflectors The cross-over shift at the highest SA magnification is zero. When the magnification is decreased, the required cross-over shift will generally increase to a maximum at approximately 20kx...50kx, after which the cross-over shift will decrease again. It can happen that for a few magnifications the required cross-over shift is outside the range of the deflectors. In that case, proceed as follows: Try to get the x-direction and y-direction separately as good as possible. However do not set a cross-over shift close to the end of its range if that is still not enough to get the shadow of the needle back at its reference position. You will only limit the available range of the image shift while you still do not have the optimal beam direction. Instead, set the cross-over shift to not more than about half of its range. Proceed with next magnification. When you have stepped through all magnifications, you can decide how to tackle the magnifications for which the required cross-over shift was out of range: If the magnifications which show out-of-range corrections are in a range which are of minor importance for your applications: Leave things as they are. Keep in mind that, when you are at these magnifications, you might have to recenter the ZLP or tune the GIF. If the magnifications which show out-of-range corrections are important for your applications, but the highest magnifications are not important for your applications: Re-do the cross-over alignment, but this time do not start at the highest SA magnification but at the highest magnification which showed out-of-range corrections. Now the highest SA magnifications will show out-of-range corrections. Keep in mind that, when you are at these high magnifications, you might have to recenter the ZLP or tune the GIF. If the magnifications which show out-of-range corrections are important for your applications, and if also the highest magnifications are important for your applications: Run 'tune GIF' at each magnification. This will make sure that the GIF is well-tuned for every magnification. You can still have energy shifts when you switch magnification. Be aware of the fact that, if you re-do 'tune-GIF' after a longer period of time, you have to re-do 'tune-GIF' for each magnification.


14.13 HM-EFTEM Camera-length pre-alignment Purpose: Determine the shifts necessary for aligning all camera lengths, so that the diffraction pattern remains in the center when the camera length is changed. Importance: CONVENIENCE for having the diffraction patterns centered when the camera length is changed. Method: Center the diffraction pattern for the reference camera length, then align all camera lengths to the reference camera length. Procedure The alignment procedure consists of five steps : • A preparation step in which the image for the normal mode is focused. • A step in which the reference camera length for the normal mode (~500mm) is focused and

centered. • A step in which the equivalent EFTEM camera length is focused and centered. • A step in which all other camera lengths are focused and centered. • A step in which for each camera length the cross-over correction is set. Notes: • Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over



• Although the camera length series for EFTEM typically will contain low camera lengths, this does not imply that such camera lengths are usable on all instruments (very much dependent on a number of factors such as the mechanical alignment of the projector lenses). Typical lowermost usable camera lengths are as follows: BioTWIN lens 700mm TWIN lens 320mm S-TWIN lens 200mm U-TWIN lens 260mm

14.14 HM-EFTEM Camera-length alignment Purpose: Determine the shifts necessary for aligning all camera lengths, so that the diffraction pattern remains in the center when the camera length is changed. Importance: CONVENIENCE for having the diffraction patterns centered when the camera length is changed. Method: Center the diffraction pattern for the reference camera length, then align all camera lengths to the reference camera length.


Procedure The alignment procedure consists of five steps : • A preparation step in which the image for the normal mode is focused. • A step in which the reference camera length for the normal mode (~500mm) is focused and

centered. • A step in which the equivalent EFTEM camera length is focused and centered. • A step in which all other camera lengths are focused and centered. • A step in which for each camera length the cross-over correction is set. Notes: • Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over



• Although the camera length series for EFTEM typically will contain low camera lengths, this does not imply that such camera lengths are usable on all instruments (very much dependent on a number of factors such as the mechanical alignment of the projector lenses). Typical lowermost usable camera lengths are as follows: BioTWIN lens 700mm TWIN lens 320mm S-TWIN lens 200mm U-TWIN lens 260mm


15 LM-EFTEM Procedure

15.1 LM-EFTEM Preparation Purpose: Set up microscope for aligning the column for EFTEM. Importance: ESSENTIAL to make sure the alignment is done for the correct conditions: centered C2 aperture, eucentric height and specimen in focus. Also find a recognizable image feature for later alignment steps. Notes: • Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over


• The EFTEM alignments are in many cases (lower magnifications and camera lengths) sensitive to hysteresis. It is therefore important to use the normalization facility of the microscope to make image and diffraction-pattern positions reproducible.

15.2 LM-EFTEM Image-shift pivot points Purpose: Align image shift pivot point = make sure that the diffraction pattern does not shift when the image shift is changed. Importance: ESSENTIAL for the proper functioning of image shifts and cross-over correction. Method: Minimize the movement of the diffraction pattern. The microscope 'wobbles' the image shift which should cause no diffraction shift. Procedure The alignment procedure consists of six steps : • Two preparation steps for setting up the image in normal and EFTEM modes. • Two steps for setting up the diffraction pattern in normal and EFTEM modes. • Two steps in which the X and Y pivot points are aligned. In EFTEM a misaligned image shift will lead to an apparent diffraction shift when the cross-over correction is changed. Description The alignment of the pivot points of the image deflection coils is essential to the proper functioning of many other alignments and calibrations: image shifts, diffraction shifts, detector alignments and measurement functions. The image coils pivot points (image shift and diffraction shift) alignment is reasonably insensitive to the operating conditions and normally needs to be done only once. It is important that the image coils pivot points are aligned prior to aligning other parts of the microscope where the image coils are used, otherwise the latter alignments may have to be redone. Alignments where the image coils are used are those where images or diffraction patterns are moved (image shift, diffraction shift/diffraction alignment).

15.3 LM-EFTEM Diffraction-shift pivot points Purpose: Align the diffraction shift pivot point = make sure the image does not move when the diffraction shift is changed.


Importance: ESSENTIAL for the proper functioning of image and diffraction shifts and the cross-over correction. Method: Minimize the movement of the image. The microscope 'wobbles' the diffraction shift which should cause no image shift. A common consequence of a diffraction shift that has not been aligned is a mismatch between the image detail selected with the selected area aperture and the area actually contributing to the diffraction pattern. When the image coils have not been aligned properly, image and diffraction shift are not uncoupled and the diffraction shift will also cause an image shift, thereby moving the image detail out of the selected area aperture. In EFTEM a misaligned diffraction shift will lead to an apparent image shift when the cross-over correction is changed. Procedure The alignment procedure consists of four steps : • Two preparation step for setting up the image in normal and EFTEM mode. • Two steps in which the X and Y pivot points are aligned. Description The alignment of the pivot points of the image deflection coils is essential to the proper functioning of many other alignments and calibrations: image shifts, diffraction shifts, detector alignments and measurement functions. The image coils pivot points (image shift and diffraction shift) alignment is reasonably insensitive to the operating conditions and normally needs to be done only once. It is important that the image coils pivot points are aligned prior to aligning other parts of the microscope where the image coils are used, otherwise the latter alignments may have to be redone. Alignments where the image coils are used are those where images or diffraction patterns are moved (image shift, diffraction shift/diffraction alignment). For images displaying the effect of the alignment, see section 6.3.

15.4 LM-EFTEM Image-shift pre-alignment Purpose: Aligning all EFTEM-LM images with each other and with the LM images of the normal mode. Importance: CONVENIENCE so that the image remains centered when the magnification is changed. Method: Center a recognizable image with the specimen stage. Lower the magnification one step, center the image with the Multifunction X,Y knobs. Repeat for all magnifications. Procedure The alignment procedure consists of four steps : • A preparation step in which an image feature is centered (with the specimen stage) in the highest LM

magnification for the normal mode. • A step in which the same feature is centered with the MF-X,Y for the highest EFTEM-LM

magnification. • A step in which all EFTEM-LM magnifications are aligned relative to the highest EFTEM-LM

magnification. • A step in which the cross-over correction is set for each EFTEM-LM magnification.


Notes: • The lowermost EFTEM-LM magnifications may not be usable. Simply skip the alignment for these

magnifications. • Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over



15.5 LM-EFTEM Image-shift alignment Purpose: Aligning all EFTEM-LM images with each other and with the LM images of the normal mode. Importance: CONVENIENCE so that the image remains centered when the magnification is changed. Method: Center a recognizable image with the specimen stage. Lower the magnification one step, center the image with the Multifunction X,Y knobs. Repeat for all magnifications. Procedure The alignment procedure consists of four steps : • A preparation step in which an image feature is centered (with the specimen stage) in the highest LM

magnification for the normal mode. • A step in which the same feature is centered with the MF-X,Y for the highest EFTEM-LM

magnification. • A step in which all EFTEM-LM magnifications are aligned relative to the highest EFTEM-LM

magnification. • A step in which the cross-over correction is set for each EFTEM-LM magnification. Notes: • The lowermost EFTEM-LM magnifications may not be usable. Simply skip the alignment for these

magnifications. • Because of the difficulty of aligning the microscope for EFTEM (image/diffraction shift and cross-over




16 EFTEM Nanoprobe Procedure

16.1 SA objective lens preset EFTEM Nanoprobe Purpose: Setting the eucentric focus preset at the eucentric height. Importance: CONVENIENCE for having the eucentric focus at the eucentric height. Method: Focus the image for the highest SA magnification. Procedure The alignment procedure consists of three steps: • A preparation step in which the SA image is focused at an intermediate magnification. • A step in which the highest SA magnification must be focused. • A step in which the highest SA magnification must be focused on the Filter. Note: Although the objective lens is at a different setting for a focused nanoprobe image relative to the focus setting for a microprobe image, this does not imply that the microscope imaging system is altered in some way. The difference between the objective-lens settings is caused by the minicondenser lens whose setting has an effect on the magnetic field of the objective lens, so that in nanoprobe a slightly higher current is needed to obtain the same electron-optical effect as in microprobe.

Tecnai on-line help Column description 1 Tecnai 12 Software version 4.0

Tecnai on-line help manual -- Column description Table of Contents 1 Troubleshooting (cold emission) ......................................................................................................... 2

1.1 Procedure to check for cold emission .......................................................................................... 2 2 Apertures............................................................................................................................................. 3

2.1 Operation ..................................................................................................................................... 3 2.1.1 Sideways retraction............................................................................................................... 3 2.1.2 Aperture selection ................................................................................................................. 3 2.1.3 Aperture centering ................................................................................................................ 4

2.2 Aperture sizes mounted as default............................................................................................... 4 3 CompuStage ....................................................................................................................................... 5

3.1 Specimen holders ........................................................................................................................ 5 3.2 Eucentric height ........................................................................................................................... 5

3.2.1 To set the specimen to the eucentric height ......................................................................... 6 3.3 Safety features ............................................................................................................................. 6

3.3.1 MaxiTilt system ..................................................................................................................... 6 3.3.2 sEntry system ....................................................................................................................... 6

3.4 Red CompuStage light ................................................................................................................. 7 3.5 Homing......................................................................................................................................... 7 3.6 Parking position............................................................................................................................ 7 3.7 Specimen-holder handling ........................................................................................................... 8

3.7.1 Specimen-holder selection.................................................................................................... 8 3.7.2 Handling instructions............................................................................................................. 8 3.7.3 Inserting a specimen into the single-tilt holder...................................................................... 9 3.7.4 Inserting a specimen holder into the microscope................................................................ 10 3.7.5 Removing a specimen holder from the microscope............................................................ 13

3.8 Stage movement ........................................................................................................................ 14 3.8.1 Speed control ...................................................................................................................... 14 3.8.2 Specimen stage movement by Multifunction knob.............................................................. 14

3.9 Stage Axes................................................................................................................................. 15 4 An introduction to vacuum................................................................................................................. 16 5 Cold Trap........................................................................................................................................... 18 6 Viewing screens ................................................................................................................................ 19

6.1 Main screen................................................................................................................................ 19 6.2 Focusing screen......................................................................................................................... 19

7 Plate camera ..................................................................................................................................... 20 7.1 Plate camera mechanism........................................................................................................... 20 7.2 Plate-camera cassette ............................................................................................................... 20

7.2.1 Cassette parts description .................................................................................................. 21 7.3 Inserting sheet film in a holder (dark room only!) ....................................................................... 22 7.4 Loading the sheet-film magazine (dark room only!) ................................................................... 22 7.5 Loading a cassette into the microscope..................................................................................... 23 7.6 Removing a cassette from the microscope ................................................................................ 23 7.7 Removing exposed film from the receiving magazine (dark room only!).................................... 23 7.8 Removing sheet film from a holder (dark room only!) ................................................................ 24 7.9 Troubleshooting ......................................................................................................................... 24

7.9.1 No exposure is being made ................................................................................................ 24 7.9.2 Plate-camera jam................................................................................................................ 25


1 Troubleshooting (cold emission) Note: The possibility exists that the microscope leaks X-rays when significant cold emission (cold emission means that the emission reads non-zero while the high tension is on but the filament is off) is present. The microscope therefore runs a continuous check for cold emission and if the cold emission exceeds 10 mA the high tension will be switched off. If the user interface is open, the user will be warned about the cold emission. Since the cold emission cannot be checked when the filament is on, the check will be delayed for up to 48 hours if the filament is switched on. After that the filament must be switched off to allow a check to be made. Afterwards the filament can be switched on again. Cold emission is not something that happens suddenly and spontaneously, so the check that has been implemented is sufficient to safeguard safety. Cold emission typically occurs when the gun has been contaminated (dust). To solve the problem, the gun must be cleaned. To solve the problem, the gun must be cleaned.

1.1 Procedure to check for cold emission The following process should expose cold field emission in your gun. 1. Turn the high tension on to its maximum value (i.e., 100 kV in a T10). 2. Be sure the heating filament is turned off. 3. Check the meter for emitted current. 4. If 6 μAmps of current is measured for a sustained period of five or more minutes, cold field emission

may be present. 5. To verify, switch off the high tension, open the emission chamber and check for dust, particles, or

surface irregularities on the electrode. If there is dust or other matter present, blow it away with dry nitrogen.

6. Repeat steps one through 4 above. If cold field emission is still present, call FEI service.


2 Apertures The microscope is equipped with three aperture controls, from top to bottom: • Condenser aperture • Objective aperture • Selected-area aperture Note: Aperture mechanisms, like all other mechanical controls on the microscope should move easily and not require excessive force. If the aperture doesn't move easily, do not try to force it as this may damage the mechanism. The sideways movement of the aperture is equipped with end stops to keep the aperture close to a centered position. The end stops are located in holes going in horizontally in the lower half of the grey, circular block with the aperture numbers, one on the left and another on the right. Do not try to force the aperture beyond these end stops. if the aperture cannot be centered properly, use a small (metric) Allen tool to adjust the position of the end stops.

A side-ways retractable, four-stop aperture mechanism.

2.1 Operation

2.1.1 Sideways retraction Apertures so equipped can be retracted sideways out of the beam by a lever mounted at the underside of the aperture mechanism. Rotate the lever to the right to remove the aperture out of the beam, turn it to the left to insert it in the beam. Aperture mechanisms without the lever either cannot be removed from the beam at all (condenser apertures) or require retraction with the aperture selection ring (retraction along the aperture holder axis).

2.1.2 Aperture selection Turn the large ribbed ring (closest to the column) clock-wise or anti-clockwise to select a different aperture. The small pin on the side of the ring points to the aperture number. On the objective aperture, position 7 has the aperture blade retracted completely from the gap between the objective-lens pole pieces. Position 5 is not usable (it is on the edge of the aperture blade). Positions 4 to 1 are the real aperture positions.


2.1.3 Aperture centering The aperture selected can be centered using the axial (smaller ring inside aperture selector ring) and perpendicular (knob on side of ring with aperture numbers) movements.

2.2 Aperture sizes mounted as default Aperture 1 2 3 4 Condenser 2 30 um 50 um 100 um 200 um Objective (BioTWIN) 20 um 40 um 70 um 150 um Objective (TWIN) 10 um 20 um 40 um 100 um Selected Area 10 um 40 um 200 um 800 um


3 CompuStage The CompuStage is a motor-driven goniometer that provides computer-controlled movement of the specimen on five axes (X, Y, Z, α, β). The CompuStage consists of the following elements: • Hardware (the goniometer), including motor drives and position-measuring system. • Control electronics, including a dedicated microprocessor. • CompuStage server software which provides the link between the user interface (software and

hardware controls like the track ball for X-Y motion) and the CompuStage microprocessor. • Hardware controls, consisting of a track ball for X-Y motion and pressure-sensitive up-down switches

for Z, α and β. • User interface software with translates operator input into goniometer actions. The definition of the physical position of the stage axes in the microscope column is covered in a separate section.

3.1 Specimen holders The CompuStage can be equipped with a variety of holders, which are inserted or removed via an airlock. Specimen holders are inserted into ultra-high vacuum and must be kept clean. Special handling instructions for specimen holders should be adhered to.

3.2 Eucentric height The α tilt of the CompuStage is constructed in such a way that it is possible to tilt around it without having large apparent movements of the point of interest on the specimen. This is called eucentric tilting and is achieved by bringing the point of interest to the same height (with the Z axis) as the α tilt axis itself: the eucentric height. The eucentric height is important because it not only provides an easy way of tilting without having to correct specimen position continuously, but it also defines the reference point inside the microscope for all alignments, magnification, camera lengths, and so on. In general one should work at the eucentric height (the only reason for deviating could be that at very high β tilts and specimen positions away from the center, the range of the Z axis may not be sufficient to bring the specimen to the eucentric height).


3.2.1 To set the specimen to the eucentric height Method 1 Eucentric Focus Preset Once the microscope has been aligned (as it normally should be), the eucentric focus preset (obtained by pressing the Eucentric Focus button) sets the objective-lens setting to the correct value for a specimen that would be exactly at the eucentric height. If the specimen is not at the eucentric height, it will appear out of focus. If you now bring the specimen into focus by changing the Z height, not the focus, it will go to the eucentric height. It may help to switch on the wobbler, since the apparent displacement between the two wobbler images makes it easy to see whether the height is changed in the right direction (the displacement between the images becomes smaller). Method 2 The Alpha Wobbler Since the image displacement on tilting is minimised at the eucentric height, the eucentric height can be set by minimising the displacement while the stage is tilting. For this purpose the CompuStage has the Alpha Wobbler function. When this function is activated, the CompuStage is tilted continuously between two preset tilts (typically -15 and +15°). Change the Z height to make the displacement smaller. When the displacement is minimised the specimen is at the eucentric height.

3.3 Safety features For safety the CompuStage is equipped with a number of special features like the MaxiTilt system which allows maximum use of the available space between the pole piece for tilting while guarding against damage of holder, pole pieces and other objects in the pole-piece gap like the objective aperture holder. Another feature is the sEntry system which guards against insertion of holder that are incompatible (too big) for the objective-lens pole-piece configuration of the microscope.

3.3.1 MaxiTilt system Because tilting takes place around the eucentric point, the motion of the specimen holder may require a lot of space (see picture below).

In the restricted space available between the pole pieces of some objective lens types, there may not be enough room to tilt very far. But the pictures above demonstrate that the available tilt range is very much dependent on the position of the stage (mostly the Y and Z axes). The MaxiTilt system of the CompuStage provides a flexible way of keeping the tilt within a safe range, while at the same time maximising the tilt available. The MaxiTilt senses what the maximum tilt is for the current stage position. If this range is exceeded (a situation called a pole hit), the CompuStage will move a little bit back on the axis that was changed last. If there is no axis identifiable, the B or A tilt will move back. The microscope will display an information message that a pole hit has been detected.

3.3.2 sEntry system The CompuStage is equipped with a SafeEntry (or sEntry) system that prevents holders from being inserted into microscopes where the objective-lens pole-piece configuration is not compatible with the particular holder (like thick holders into objective lenses with a gap that is too narrow for the holder to fit).


The sEntry system consists of a key (a pin varying in shape and diameter) on the holder defining the holder dimensions and a lock on the CompuStage defining the pole-piece configuration. The length of the sEntry key is such that if an incompatible holder is inserted, the key (blocked by the lock) prevents the holder from going between the pole pieces. Although older specimen holders designed fo the manual goniometer will fit in the CompuStage (provided their O-ring is exchanged for a - thicker - CompuStage O-ring), these holders are not equipped with a sEntry key and may therefore be unsafe with objective pole-piece configurations with narrow gaps such as the U-TWIN.

3.4 Red CompuStage light The red CompuStage light has a more generic function than previously used on manual goniometers. It simply means that no airlock actions (insertion or removal) should be executed. This will happen of course while the airlock is being pumped when a holder is inserted into the microscope. It can also mean that it is unsafe to extract the holder under the current conditions (when the β tilt is more than 5 degrees or during movement the red light will also be on). In that case, the unsafe situation must be rectified before the holder can be extracted (reset β tilt to zero or wait until movement is finished).

3.5 Homing Before the CompuStage is ready for use (when the microscope has been switched off altogether or after the CompuStage has been disabled), it must be homed, a procedure in which it finds the zero positions. During homing the CompuStage will move each of the four fixed axes (X, Y, Z and α; the β tilt axis is homed separately whenever a double-tilt holder is inserted) to one extreme. Because of the high tilt applied, this procedure must be executed without a specimen holder. In order to make sure that there is no specimen holder present, the microscope has to ask the operator to identify the specimen holder (which in this case should be 'No specimen holder', otherwise the homing cannot proceed). If no user interface is active, there is no way for communicating with the operator. Consequently, the homing procedure after a start-up will only proceed once the user interface has started. Even if the user interface does not show a message to select a specimen holder after a restart of the microscope, but the CompuStage does not move (easily checked by moving the right-hand track ball a bit), it is very likely that the CompuStage has not been homed. Select the Stage² Settings Control Panel and press the Enabled button. The homing procedure will start.

3.6 Parking position Occasionally it is necessary to get the specimen and/or specimen holder out of the way (for example, when no electron beam is visible initially or in some alignment steps if the specimen blocks the beam). Although it is possible to retract the holder fully and turn it slightly (the initial part of removing the specimen holder), it is better not to do this, because it can lead to a potential leak. The preferred method to 'remove' the specimen holder is to retract it about one or two centimeters and stick something elongated (a pen with a diameter of a centimeter or so will do nicely) between the inside cap of the holder and the CompuStage. The 'elongated' item will prevent the holder from moving back in and the distance is sufficient to remove any obstruction from the field of view. Note 1: Always retract the holder slowly to allow the O-ring to keep the seal. Note 2: Manual-goniometer specimen holders with the sapphire at the end have a longer tip than CompuStage holders and must be retracted at least three centimeters before they are clear from the field of view.


3.7 Specimen-holder handling

3.7.1 Specimen-holder selection There are two ways for informing the microscope which specimen holder is being used: • Not automatic: Each time a holder is inserted, the holder must be selected from the list that pops up

in the message area of the Tecnai user interface. • Default: Only for single-tilt holders, the last-used setting is selected automatically. The CompuStage is equipped with the sEntry system. Associated with this system is an up-down switch on the outer panel of the CompuStage. The top part of the switch depends on the type of objective lens (with smaller lens gaps, the hole in the top is made smaller so only specific types of holders will fit).

The shield of the CompuStage showing the position of the holder-selection switch (gray). The switch itself is controlled by a notch in the lower rectangle. The actual shape of the top of the switch varies and on some microscope will block the sEntry key of the specimen holder from being inserted. • In the up position (left-hand picture above), the Default holder selection is enabled. In this case

the microscope will automatically select a single-tilt holder. In case there are more single-tilt holders present on the system (e.g. normal single- tilt and cryo holder), the holder selected is the last one chosen. If there are more single-tilt holders present, then for the first insertion, move the switch down, insert and identify the holder you are using, then move the switch up again. Thereafter the microscope will automatically select the same holder again.

• In the down position, the holder used must be identified by the user. This must always be done for double-tilt holders (the reason this is necessary has to do with the b tilt cable which must be connected before the holder can be inserted into the microscope), so do not use the switch up position with double-tilt holders.

3.7.2 Handling instructions Specimen holders are a bridge between the air pressure outside the column and the ultra-high vacuum inside. Their cleanliness is an important factor in keeping contamination down (specimen holders are the second-most important source of the contamination - specimens themselves are the primary source


nowadays). Caution should therefore applied to handling specimen holders. The following instructions should be adhered to: • Always use clean nylon or similar gloves when handling specimen-holder parts that enter the vacuum

(that is, between the tip of the holder and the sealing O-ring). • Clean the tip of the holder only with special cleaning fluids or with a fresh piece of window-cleaning

(chamois) leather. • Specimen, spacing washers and clamping devices should be manipulated only using pointed

tweezers or the tools provided. Tools like the hex-ring tool or the needle for levering the single-tilt holder clamp should never be touched by hand on the wrong side (in the case of the hex-ring tool the use of gloves is advised because it is easy to pick it up at the wrong end). Clean the washers, tools and tweezers on a regular basis.

• The O-ring on the specimen-holder rod should be checked for possible dirt or excessive quantities of grease although it should not be completely dry. A very light coating of Fomblin grease (supplied with the microscope) is advised. Take care not apply grease to the conical part of the holder (the part between the thicker and thinner sections of the rod). The conical part is the area where the holder is 'seated' in the CompuStage. Any grease there will very likely result in drift in excess of specimen.

• When a specimen holder is not in use, insert it in the protective holder cover supplied or reinsert it into the microscope. The latter keeps the holder thermally equilibrated with the microscope and CompuStage, thereby reducing drift after holder insertion (especially if there is a considerable temperature difference between the room and the microscope column).

3.7.3 Inserting a specimen into the single-tilt holder (Instructions for mounting specimens in other holders are covered in the manual accompanying these holders.) Note: For easy removal of specimens from the holder, a notch is provided on the holder (see the images below). One tip of a pair of tweezers can be inserted in this notch underneath the specimen (once the clamp has been lifted), making it easy to take the specimen out of the holder. • If necessary, remove the cap at the end of the tube of the specimen-holder cover. • Check that the tip of the holder and the clamping device are clean and dry. • Keep one hand against the cap of the holder, making sure it cannot move out of the cover tube. • Fit the tool (stored in one of the holes in the supports of the cover tube) into the hole in front of the

clamp. Then lift the clamp to its fullest extent.


• Place the specimen in the (roughly) circular recess of the specimen-holder tip. If the specimen is on a re-insertable grid, place the 'ear' of the grid in the 'ear' recess.

• Carefully lower the clamp with the tool onto the specimen. Make sure the specimen remains correctly in position.

Caution: The specimen-securing clamp must be lowered carefully, otherwise the specimen and/or clamp can be damaged. • Retract the holder slightly in the cover and turn it upside down. Tap the cap at the end a few times.

Turn the holder back and check that the specimen has not moved (movement is a sign that it isn't clamped properly).

Note: Never mount magnetic specimens (disks) in the single-tilt holder. The clamp is normally not strong enough to prevent the specimen from flying out due to the objective-lens magnetic field and sticking to the objective-lens pole pieces.

3.7.4 Inserting a specimen holder into the microscope Caution: The following instructions apply to all specimen holders and must be followed completely or damage to airlock, specimen holder or specimen stage may result. For microscopes equipped with a turbo-molecular pump: The turbo-molecular pump (which should not remain running under normal microscopy because of the vibrations it causes) takes several minutes (2-3) to reach operational speed. To speed up specimen exchange, it is advised to switch the pump on (use the toolbar button) before extracting the holder. By the time the specimen has been exchanged the turbo-molecular pump will be near or at its operation speed and pumping on the airlock will begin (almost) immediately. Switch the pump off again after the holder has been inserted fully into the microscope. If the turbo-molecular pump is running on when the specimen holder is inserted into the airlock, the pump will first spin up and only after a few minutes start pumping on the airlock. In this situation (holder triggers the pump), the pump will be switched off automatically after pumping on the airlock has finished. Note 1: Read the specimen-holder handling instructions before proceeding. Note 2: The specimen airlock of the CompuStage and the specimen holder consist of fine, high-quality mechanics. If considerable force is needed for any manual actions on the holder or CompuStage, it is a sign of something being wrong. It should never be necessary to exert strong force and doing so may well result in damage to specimen holder or CompuStage.


Note 3: It is not necessary to switch off high tension or filament during specimen-holder insertion, since the gun is separated from the column by the gun valve (V7) which is closed during specimen insertion (even if the user doesn't close the column valves, the microscope will automatically do so when it detects that a specimen holder has been inserted; it is good practice, however, to close the column valves before extracting the specimen holder from the microscope so they will still be closed when a holder is inserted again). It is advised, however, to keep the column valves closed after insertion of the holder (typically for a few minutes) while the specimen-area vacuum recovers - if only to reduce contamination of the specimen. Using the cold trap is advised to trap water vapour quickly (ion-getter pumps have difficulty in pumping water). Note 4: Always carry out the complete insertion procedure. If the specimen is left in a retracted position, vacuum leakage can occur with consequent contamination (or, if left for a long period, loss of vacuum in the airlock and a crash of the column if the specimen holder is then inserted). If you do not want to insert the specimen holder after all (or if the airlock isn't pumped properly, for example because of a leaking O-ring), leave the airlock cycle to finish pumping (the red LED on the CompuStage goes off) before removing the holder from the airlock. Do not extract the holder while the airlock is being pumped. The specimen holder is introduced through a pre-pumped airlock which ensures that air, introduced with the holder, is pumped away before the airlock is opened to the microscope column. Procedure • Hold the specimen holder with the airlock trigger pin parallel to the small slit in the CompuStage front

plate (at roughly four o'clock). Carefully insert the end of the specimen holder into the airlock cylinder and slide the holder in until a stop is reached. At this point the prepumping of the airlock will start as indicated by the red CompuStage light which will be illuminated. If the light does not come on, the trigger has not been positioned correctly. Slowly turn the holder slightly to the left and the right until it will go in a bit further (the airlock trigger pin now falls properly into its groove).

• The Tecnai user interface will display a message asking for identification of the specimen holder. Select the type of holder from the list and press the Enter button.


Note: Unless a particular type of holder is specifically listed (in which case its has control parameters separate from the 'generic' holder types), it falls in one of two categories: single tilt or double tilt. Single tilt applies to any holder that doesn't have a computer-controlled b tilt (including manual-goniometer double-tilt holders!). For double-tilt holders a few different types may exist (typically only the Philips double-tilt holder is listed unless the control parameters for the other double-holders have been installed). • If the holder is a double-tilt holder, insert the b tilt cable (as also instructed by the microscope) and

press the Enter button. Caution: As long as the red CompuStage light is illuminated, it is unsafe to insert the specimen holder further into the microscope. It is also good practice to observe the pressure in the backing line (which is connected to the airlock during airlock pumping) to make sure the airlock is being pumped properly on a microscope with the standard vacuum system (airlock is pumped by the rotary pump PVP). On systems equipped with a turbo-molecular pump, listen to the sound of the pump. If the turbo doesn't run more smoothly with ongoing pumping, the airlock is leaking.


• When the red CompuStage light has been switched off, rotate the specimen about 120 degrees counter-clockwise as far as it will, then allow it slide in further into the microscope.

Caution: Maintain a firm grip on the holder while it is sliding in (it is being sucked in by the column vacuum) to safeguard against any possible damage to CompuStage or holder. • Once the holder is fully in, carefully tap a few times with a finger against the cap of the holder to help

it settle into position and thereby improve stability.

3.7.5 Removing a specimen holder from the microscope Caution: The following instructions apply to all specimen holders and must be followed completely or damage to airlock, specimen holder or specimen stage may result. For microscopes equipped with a turbo-molecular pump: The turbo-molecular pump (which should not remain running under normal microscopy because of the vibrations it causes) takes several minutes (2-3) to reach operational speed. To speed up specimen exchange, it is advised to switch the pump on (use the toolbar button) before extracting the holder. By the time the specimen has been exchanged the turbo-molecular pump will be near or at its operation speed and pumping on the airlock will begin (almost) immediately. Switch the pump off again after the holder has been inserted fully into the microscope. If the turbo-molecular pump is running on when the specimen holder is inserted into the airlock, the pump will first spin up and only after a few minutes start pumping on the airlock. In this situation (holder triggers the pump), the pump will be switched off automatically after pumping on the airlock has finished. Note 1: Read the specimen-holder handling instructions before proceeding. Note 2: The specimen airlock of the CompuStage and the specimen holder consist of fine, high-quality mechanics. If considerable force is needed for any manual actions on the holder or CompuStage, it is a sign of something being wrong. It should never be necessary to exert strong force and doing so may well result in damage to specimen holder or CompuStage. Note 3: It is not necessary to switch off high tension or filament during specimen-holder exchange, since the gun is separated from the column by the gun valve (V7) which is closed during specimen insertion (even if the user doesn't close the column valves, the microscope will automatically do so when it detects that a specimen holder has been inserted; it is good practice, however, to close the column valves before extracting the specimen holder from the microscope so they will still be closed when a holder is inserted again). Note 4: Always carry out the complete removal procedure. If the specimen is left in a retracted position, vacuum leakage can occur with consequent contamination (or, if left for a long period, loss of vacuum in the airlock and a crash of the column if the specimen holder is then inserted).


Procedure • Close the column valves. • Make sure the specimen holder is in a safe position (if necessary reset a and b to zero, or set all

axes to 0). The red CompuStage light should be off. • Pull the holder as far out of the CompuStage as it will go, then rotate it clockwise as far as it will go

(about 120 degrees). • Carefully extract the holder from the airlock (you have to pull against the vacuum remaining in the

airlock). • For double-tilt holders disconnect the cable plug.

3.8 Stage movement The specimen stage movement is controlled by a track ball (normally the right-hand one, but the assignment can be changed by the user) and/or by Multifunction knob. The movement has two modes of operation: • The track ball (discontinuous movement) mode. • The 'joy stick' (continuous movement) mode. In the track ball mode (the default) the stage will move whenever the track ball has been moved, in the direction and with a displacement related to the direction and displacement of the track ball. As soon as the track ball stops moving, the stage will stop moving as well. For normal operation at moderate to high magnifications this is the preferred mode. However, for searching at low magnifications the track ball mode requires that the user keeps moving the track ball continuously. In the 'joy stick' mode the stage will move in the direction indicated by the movement of the track ball and with a speed related to the displacement of the track ball, and the stage will keep moving in the indicated direction without requiring further input through the track ball. The direction and speed of movement can be influenced by further control of the track ball. To stop the stage movement in the 'joy stick' mode, press one of the track ball buttons. Switching between the two modes is achieved by pressing the two track ball buttons simultaneously.

3.8.1 Speed control The speed of movement of the stage is related to three parameters : • The speed value setting (1 to 9) as defined by pressing the left- (speed down) and right-hand (speed

up) track ball buttons. • The current magnification. • The displacement of the track ball. Note 1: One exception is at the lowermost speed setting. In this case the CompuStage will make its smallest steps, independent of magnification. At low magnifications this may mean that the stage doesn't seem to move at all. If the latter is the case, click once on the right-hand track-ball button to switch the speed one step up. Note 2: At very low magnifications the speed control may appear to function no longer (speed up doesn't increase the speed of the CompuStage). This means that the CompuStage has reached its maximum speed and can go no faster.

3.8.2 Specimen stage movement by Multifunction knob The specimen stage movement can also be assigned to the Multifunction knobs. In this case the Multifunction knobs duplicate the track ball directions (that is, Multifunction X = track ball X). The knobs are thus not connected directly to a stage axis (technically this is not possible within the software


architecture). For most microscopes this means that the X axis (the direction of the a tilt axis) is approximately connected to the Multifunction Y knob.

3.9 Stage Axes The CompuStage is a goniometer with five axes. Three of these are orthogonal translation movements X, Y and Z. The other two are mutually perpendicular rotation movements, α and β. The α tilt is parallel to the X axis and β tilt parallel to the Y axis. The orientation in space of these axes can only be described at a tilt at 0° (since the other axes are mounted on top of the α tilt, they will change their orientation with it). At a tilt 0° the X and Y axes are horizontal and the Z axis vertical. The X axis runs along the rod of the specimen holder. If you look down on the microscope and define the front of the microscope as south, the X axis thus runs NW-SE, with SE in the + direction and the Y axis NE-SW with NE as the + direction. Up is the + direction of the Z axis. The X axis describes a truly linear motion, the Y and Z are in fact circular motions but with such a wide radius that the motion remains close to linear. The Y and Z motions cause the specimen holder rod to pivot around the conical face where it narrows down, just beyond the O-ring.

Because of this pivoting motion, the end of the holder on the outside of the CompuStage moves in the opposite direction! Below is a schematic 3-D view of the specimen holder tip and the orientation of the stage axes.

Note: The notation differs from that on the earlier CompuStage of the CM microscopes where the sign of the X and Y axes is reversed.


4 An introduction to vacuum Electron microscopes cannot operate in air for a number of reasons. The penetration of electrons through air is typically no more than 1 meter, so after coming on meter from the gun, the whole beam would be lost to collisions of the electrons with the air molecules. It is also not possible to generate the high charge difference between the anode and cathode in the gun because air is not a perfect insulator. Finally, the beam on the specimen while in air would trap all sorts of rubbish (air is full of hydrocarbon molecules) on the specimen, crack them (removing hydrogen, oxygen, etc.) and thus leave a thick carbon contamination layer on the specimen. Each electron microscope therefore has a vacuum system. the degree of sophistication of the vacuum system depends on the requirements. Simple imaging of biological thin sections is much less demanding than cryo applications or small-probe analysis in materials science, and a thermionic gun can operate under much worse vacuum than a Field Emission Gun. The most basic vacuum system consists of a vessel connected to a pump that removes the air. The vacuum system of an electron microscope is considerably more complicated, containing a number of vessels, pumps, valves (to separate different vessels) and gauges (to measure vacuum pressures). From the bottom up we can distinguish four vessels in the vacuum system: • The buffer tank • The projection chamber • The column (specimen area) • The electron gun area These are not pumped by single pump, because there is no pump available that handle the full range in vacuum from air pressure (as present after a vessel has been vented) to ultra-high vacuum (in specimen are or gun). The microscope can in essence be divided in two parts, separated by a very small aperture (200 micrometers), the differential pumping aperture, located between the projection chamber and the column. The lower part basically consists of the projection chamber where we observe the image and where plate camera and TV cameras are located. This is pumped by an oil-diffusion pump. Behind the oil-diffusion pump is a rotary pump (the oil-diffusion pump cannot go from vacuum to air, it needs some other pump to back it up). Since the rotary (or pre-vacuum) pump is noisy, it is not running continuously but only when needed. In order to have continuous backing of the diffusion, there is a buffer tank in between them. The buffer tank is slowly filled by the oil-diffusion pump. When its pressure is becoming high, it is emptied by the rotary pump. The upper part consists of the specimen and gun areas which are pumped by one or more ion-getter pumps. These pumps use no oil and are therefore clean. They also achieve higher vacuum than the oil-diffusion pump. The number of ion-getter pumps may range from one to four. Initial pumping of the column and gun on many systems is done by the rotary and oil-diffusion pumps, except for systems equipped with a turbo-molecular pump. In the latter case the oil-diffusion and rotary pumps never pump on the column and gun areas.


The diagram above shows the vacuum system of the Tecnai F20. At the bottom it starts with the rotary pump (PVP) which is not running here (otherwise its symbol would be black). Above PVP is the buffer tank (cyan color), separated by a valve (V1) from the PVP. Another valve (V2) is also closed. When open this would give the PVP access to the projection chamber (the area above V3, the valve that separates it from the oil-diffusion pump ODP). Pir (Pir 1 and Pir 2) and Pen (3) stand for Pirani and Penning which are vacuum-pressure measuring gauges. Valve 4 (closed) separates the projection chamber from the upper part of the column. Pressures are also measured on the basis of the current running in the IGPs. The large cyan area represents the specimen area (as well as some connecting pipes), pumped by a main ion-getter pump (IGP1) and the liner-tube ion-getter pump (IGP4) (liner tubes are thin pipes that connect the projection chamber to the specimen area and the specimen area to the gun). At the same height, to the right, is the turbo-molecular pump (Turbo) that is used to prepump the specimen and gun areas and also pumps on the specimen-holder airlock (on the CompuStage; through the line that is drawn over the top, towards valves 42 and 8). The gun is pumped by two ion-getter pumps (IGP2 and IGP3).


5 Cold Trap The Cold Trap (also called Liquid-nitrogen cooling device or Cryo Trap) consists of a piece of metal around the specimen environment that is cooled to liquid-nitrogen temperature. The cooling is done by the liquid nitrogen in the dewar on the right-hand side of the column. Gases in the vacuum (predominantly water vapour) condense on the cold surface inside the microscope and thereby the partial pressures of these gases is reduced. Use of the cold trap is particularly effective when a holder is introduced into the microscope, because the water vapour coming in with the holder (either from the residual gases in the airlock or adsorbed on the holder surface) is trapped quickly. To use the cooling device, first remove the dewar vessel (lift it up slightly, then tilt away at the bottom). Fill it with liquid nitrogen and replace it onto the support. Make sure not to spill liquid nitrogen onto the viewing windows of the projection chamber as this may cause the glass to crack.

Effective use of the cold trap depends on three factors: the level of the liquid nitrogen in the dewar, the time the cold trap has been in use (effectively the amount of ice already deposited), and the adjustment of the dewar to ensure that no vibrations from bubbling liquid nitrogen are transmitted to the microscope column. Liquid-nitrogen level If the level of the liquid nitrogen is below the half-full level of the dewar, the temperature of the cold trap inside the microscope may increase and some gases may start to evaporate again from the cold surface. In general keep the dewar between full and 3/4 full except when the cold trap has been cold for a long time already (when the rate of liquid-nitrogen boiling will be much reduced). Cold trap effectiveness over time As more and more ice is building up on the cold trap, the effectiveness of the removal of gases is reduced. Especially in environments where a lot of water is introduced with specimens (e.g. cryo-transfer work), the cold trap should be warmed up at least once a day. Rapid warm up can be done either by filling the dewar with hot water (simply throw out the remaining liquid nitrogen and fill the dewar under a hot tap; the dewar doesn't need to be warmed up first, it can stand the temperature change) or remove the dewar and blow with a hair dryer against the copper braids that hang inside the dewar.

Dewar adjustment The dewar should not come into direct contact with the copper braids (wire bundles), insulating cap or the conductor (the rod running from braids towards the microscope). At the same time the amount of space between the dewar and cap must be minimized to prevent cold air from flowing out easily. If this situation cannot be obtained, then the height of the dewar on the drip tray support can be adjusted by removing the screw in the center of the drip tray and adding or removing washers between the dewar support table and the bottom of the drip tray or between the latter and the support rod below the drip tray.


6 Viewing screens The Tecnai microscope is equipped with two (fluorescent) viewing screens, a main screen and a focusing screen.

6.1 Main screen The main viewing screen has a diameter of 160 millimeters (the size of the phosphor, not including the outer rim) except on 300 kV instruments where it is 140 millimeters. The screen is equipped with markings that identify the various sizes of film as well as two circles that aid in identifying the screen center. The small circle in the center has a diameter of 5 millimeters while the larger circle has a diameter of 40 millimeters. The film size indicators take the shape of 'elbows'. Each elbow has three 'dot' markers. The dot markers on the ends closest to the screen center mark the sheet-film negative size 6.5x9 cm, while the dots on the 'elbow' itself (at the corner) marks the 3 1/4x4 inch size. The markers furthest outside are for a film type that is no longer used.

The main screen is moved up and down using a motor drive mechanism. The motor drive is under software control. One, dedicated button for moving the screen up and down is present on the left-hand side of the projection chamber (about five centimeters behind the lever for the small screen). Additionally it is possible to define any of the user buttons to have the screen lift function.

6.2 Focusing screen The small focusing screen is moved in and out by hand with a lever mechanism on the lower left-hand side of the projection chamber. The focusing screen is used together with the binoculars.


7 Plate camera The plate camera is located behind and inside the projection chamber of the microscope. The plate camera consists of a mechanism and a removable cassette.

7.1 Plate camera mechanism The mechanism consists of a pneumatically driven rod that moves forward from behind the cassette. The rod pushes the central section of the cassette out towards the front. This central section is a transport tray, holding a single sheet film holder. The transport tray slides over guides in the bottom of the projection chamber. When it reaches the front, a pin located in the projection chamber pushes in a hole in the front of the tray. This moves two supports in the tray back, allowing the sheet-film holder to fall down inside the tray (until then it was carried at the top of the tray). The exposure is then made. The rod moves back, drawing the transport tray with it. When the tray is withdrawn fully, the sheet-film holder drops down into the receiving part of the cassette. Note: When an exposure is made, the negative lies open in the projection chamber and can be struck by light coming from the room through the front or side windows of the projection chamber. In order to prevent fogging or outright exposure by light, switch off any room lights before the exposure and keep the covers on the projection chamber windows.

7.2 Plate-camera cassette The cassette for sheet film can hold a maximum of 56 sheet-film holders (item 8 in the picture below). This cassette replaces the one holding 36 glass plates or sheet-film inserts. Note for owners of older Philips microscopes: The plate-camera cassettes look similar to older designs for the CM-series and EM400 series (EM400, EM410, EM420, EM430) microscopes but are different. The older versions could hold only 36 plates. If you have such older cassettes, you should make sure the 56- and 36-plate cassettes should not be mixed as this will give rise to plate jam and other problems (this applies to both the cassette top section which has a different width of slit and to the bottom section which has a different spring). The 56-plate have markings to distinguish them from the older cassettes (a marker plate with the number 56 in relief - item 10 in the picture below - and a fluorescing sticker - item 11). The 56-plate cassette is compatible with older microscopes (EM400, EM410, EM420, EM430, all CM microscopes).


7.2.1 Cassette parts description

The complete cassette consists of: • a top magazine (1) carrying the unexposed film material between plate-stack guides (2). • a transport tray (3) belonging in the center of the cassette. • a bottom magazine (7) to receive the exposed film material. Blanking plates (6) can be used to split the cassette in room-light conditions so that a fresh receiving magazine can be put underneath the magazine with unexposed film material. Slide both blanking plates fully into the slits (5) provided above and below the transport tray (3), with the top blanking plate having its curved grip facing upward and the lower one with its grip facing down. Release the clips (9) from the LOWER, RECEIVING MAGAZINE, remove it and replace it with a fresh receiving magazine. Do not take the cover off the top magazine as this will expose the unexposed film material to light! To prevent the transport tray from dropping out during transportation, it is fixed in position by a securing spring (4).


7.3 Inserting sheet film in a holder (dark room only!) The sheet-film holder is a metal plate with upturned edges that will hold a sheet film into position. The top end of the sheet-film holder has a semi-circular cut-out (the finger slot). To load a sheet film in the holder: • Take the sheet-film holder in one hand with the finger slot at the top. • Take hold of the top of a sheet film with the other hand and with the emulsion side upward (the small

notch in the sheet film should be on the top right-hand side). • Slide the sheet film downward underneath the securing rims until the top of the film fits below the rim

inside the holder. Inserting a sheet film into a sheet-film holder.

7.4 Loading the sheet-film magazine (dark room only!) • Remove the upper magazine cover by releasing the

clips (move the top end outward, then release the bottom part from the grip) and pulling it off.

• Stack the required number of sheet-film holders between the guides, with the emulsion (sheet film) upward. Do not exceed 56 plates per magazine.

• Replace the upper magazine cover and fix the securing clips. Note that the cover fits on in only one way (otherwise the clips are misaligned).

The plate stack between the guides of the magazine for unexposed sheet film.


7.5 Loading a cassette into the microscope • Select the tab containing a Vacuum control panel. • If nitrogen gas is available, check that the valve on the bottle or line is open. • Press the Camera Air button and confirm (press the button with the 'V' sign). • Wait until the projection chamber has reached air pressure. • Lift the plate-camera cover (behind the column). • Remove the magazine inside (if present) and insert blanking plates. Replace the magazine with the

new magazine (make sure the blanking plates have been removed and that the magazine settles properly in place). The magazine fits in in only one way, with the guiding pins of the camera housing through the holes alongside the magazine.

• Check the O-ring of the plate-camera cover for cleanliness and proper position. • Replace the cover and make it fits in properly. • Press Camera Air off. • If necessary, close the nitrogen gas valve. • If necessary, adjust the plate-camera stock value. Note: To prevent fogging of the unexposed film material, keep the blanking plates inserted into the cassette during transportation, and load and unload cassettes under somewhat dimmed room-light conditions.

7.6 Removing a cassette from the microscope • Select the tab containing a Vacuum control panel. • If nitrogen gas is available, check that the valve on the bottle or line is open. • Press the Camera Air button and confirm (press the button with the 'V' sign). • Wait until the projection chamber has reached air pressure. • Lift the plate-camera cover (behind the column). • Remove the magazine inside. Insert blanking plates to prevent fogging of the topmost exposures. • Check the O-ring of the plate-camera cover for cleanliness and proper position. • Replace the cover and make it fits in properly. • Press Camera Air off. • If necessary, close the nitrogen gas valve. • If necessary, adjust the plate-camera stock value to zero. Note: To prevent fogging of the unexposed film material, keep the blanking plates inserted into the cassette during transportation, and load and unload cassettes under somewhat dimmed room-light conditions.

7.7 Removing exposed film from the receiving magazine (dark room only!) • Remove the lower magazine from the cassette by releasing the clips (move the bottom end outward,

then release the top part from the grip). The top magazine for unexposed plates can now be lifted off and put aside.

• If a blanking plate is used, pull it out. • Take the exposed plates out of the receiving magazine. It may help to tilt the magazine a little. The

exposed plates rest on a spring-supported platform that will automatically rise when plates are removed.


7.8 Removing sheet film from a holder (dark room only!) • Take the sheet-film holder in one hand with the finger slot at the top (the semi-circular cut-out). Press

with a finger against the sheet film from the back through the finger slot so that the film comes up. • Take hold of the top edge of the sheet film with the other hand and retract it upward out of the holder.

Pressing the sheet film from the back through the finger slot. Take hold of the top edge and withdraw the film from the holder.

7.9 Troubleshooting

7.9.1 No exposure is being made When the Exposure button has been pressed and still no exposure is being made: • Check that the exposure time in automatic mode has a possible value (not 0.0 seconds) and that the

LED of the Exposure button is on. • Check that the screen moves up when the exposure has been started. If the screen does not move

up, the exposure cycle has (probably) tried to move it up and failed within the given time. Move the screen up by pressing the Screen lift control and press the Exposure button.

• Check that the plate stock is higher than zero.


7.9.2 Plate-camera jam The plate-camera mechanism may jam when it moves a plate in or out. In some case the jam is easy to resolve, in other cases it may be more difficult. Below is a description of the procedures to use to clear the plate jam. Warning: do not attempt this when you are uncertain of what you are doing. If you are not the microscope supervisor, please warn the supervisor and let him/her handle this. If you are the supervisor and doubt whether you can handle this, call service! There can be several reasons for plate jams. The procedures for clearing them differ. 1. A sheet-film holder does not come out properly of the upper magazine. 2. A sheet-film holder does not drop down inside the transport tray. Transport tray moves back properly into cassette The most critical aspect in solving a plate jam is whether the transport tray moves back properly into the cassette or is stuck inside the microscope. If the tray moves back, then close the column valves, vent the plate camera/projection chamber, and remove the cassette from the microscope. Go into the dark room, remove exposed and unexposed film material, and then try to establish why the camera jams while using empty sheet-film holders. When removing the film material, pay attention, so you can reload the empty sheet-film holders into the cassette exactly as they were. Possible reasons for plate jams : • A bent sheet-film holder. • Old (36) and new (56) cassettes have been mixed. The slit through which the sheet-film holder

comes out is wider for the old-type of cassette. Loading new sheet-film holders in this cassette will certainly jam, since two holders will try to come out together. The other way around, the old plate holder will not pass through the slit because it is too narrow.

If you have found the reason for the jam, load a cassette with empty sheet-film holders into the microscope. Switch to manual exposure time (you do not need to have the plate camera/projection chamber at vacuum) and 'shoot' a whole magazine to make sure the plate jam does not occur again. If necessary you can look through the window on the left-hand side of the projection chamber and observe the process (on 300 kV machines lift out the cover to see inside - when replacing it, make sure the O-ring is clean and properly seated). Transport tray does not move back into cassette This case is more difficult to solve. You cannot remove the cassette because the rod that moves the transport tray sticks through the tray. Warning: the procedure described below can be dangerous if not handled properly. When the transport tray is moving, the forces on it are such that you damage your hands when they are in the way. • Close the column valves, switch off the HT and vent the plate camera/projection chamber. • Remove the window or cover plate (300kV instruments) on the left-hand side of the projection

chamber. • Switch the plate camera to manual exposure with the longest possible exposure time, and switch to

HOLD (to prevent it switching back to automatic). • Press Exposure. The rod with the transport tray should now come forward again. • If possible, try to remove the jamming plate(s) by lifting them out.


• When done, press exposure again. The rod and transport tray should now move back properly into the cassette.

• If the rod and transport tray are back properly, remove the cassette from the microscope and in the dark room, remove the film material and then reload with empty sheet-film holders, put it back into the microscope and check. If everything works properly, the re-insert the window/cover plate, making sure the O-ring is clean and in its correct place. Replace the test cassette with a full one and pump the plate camera/projection chamber.

If the rod and tray did not move back properly, repeat the exposure (make sure it is still on manual exposure) and try and see if it is clear. If it still doesn't work or if it fails again, call service. Always remove the particular sheet-film holder(s) and inspect for damage. If damaged, discard (do not attempt to repair; that will probably only lead to repeated jams).

TEM manual Customizing Standard User interface 1

TEM manual -- Customizing Standard User Interface Table of Contents 1 Introduction.......................................................................................................................................... 2 2 How to use it........................................................................................................................................ 2 3 Customizations that are supported...................................................................................................... 2 4 Customizations that are not supported................................................................................................ 4 5 Effect on Existing Users ...................................................................................................................... 4

5.1 Expert Users............................................................................................................................. 4 5.2 Standard Users......................................................................................................................... 4

6 Other points to note............................................................................................................................. 4 7 Installing a new TEM version .............................................................................................................. 5 8 Sample Screen Shots.......................................................................................................................... 5


1 Introduction This document explains new functionality added to the TEM software that allows the supervisor to customize certain aspects of the User Interface for new standard users. This user interface customization is intended for standard users only, i.e. not for users belonging to the TEM Experts group. It is aimed primarily at setting up defaults for new microscope users, however the effect on existing users is also explained in a later section. Section 2 describes the general procedure. Section 3 describes the customizations that are possible, and Section 4 describes the customizations that are not currently supported. Section 5 describes how existing users are affected. Finally, sample screen shots are provided in the last section. Since this functionality is available to microscope supervisors only, the reader is assumed to have some basic knowledge of the TEM User Interface and of Windows (e.g. adding users to groups, creating shortcuts and so on).

2 How to use it The general procedure for customizing the User Interface is as follows. 1. Log on to Windows as supervisor (make sure that the “user” supervisor has administrator rights in

Windows). 2. Starting the user interface in customization mode. From the command prompt this can be done by

typing ‘peoui.exe /config’. Alternatively, you may prefer to create a taskbar shortcut for starting the interface in customization mode. Creating shortcuts is described in the standard Windows help system (e.g. searching for ‘creating shortcuts’ will retrieve topics explaining how to add shortcuts for both individual users and groups of users).

3. Once started the title of the user interface should resemble that depicted in figure 1. The important point is that the title of the window at the top of the screen should contain the text ‘CUSTOMIZING INTERFACE SETTINGS FOR STANDARD USERS (I.E. NON EXPERTS)’. This indicates that the user interface is running in customization mode.

4. Although you are logged in as supervisor, the user interface you now see is the interface that will be presented to new standard users when they use the microscope.

5. Make the User Interface changes you require. This is done in exactly the same manner as when an individual microscope user modifies his own personal interface settings – the only difference is that in customization mode the changes are stored in a special location on the computer to be then picked up by new users that log on subsequently. Some examples are given in the following section.

6. Exit the application in the normal manner.

3 Customizations that are supported Note that the detailed procedure for changing the interface is exactly the same as an individual microscope user adopts to modify his own personal interface settings. The only difference is that in customization mode the changes are stored in a special location on the computer to be then picked up by new users that log on subsequently. The following workset related customizations are supported: 1. Changing a workset’s colors Left click the Color icon of the workset you wish to change. This

displays a dialog box on the right allowing you to change the color of various aspects of the workset’s Control Panels’. If the workset you are changing is also the workset currently being displayed on left hand side of the user interface then you will see your changes immediately. For


example, Figure 2 shows the controls to use for selection of the background color for the Control Panels.

2. Renaming a workset Right click on the name of the workset you wish to change and select ‘Edit label’, you can then change the name. If the tab for the workset you have changed is currently visible on the top left hand side of the user interface then you will see your changes immediately. For example, Figure 3 shows the popup menu function ‘Edit Label’ that allows changing the text of the workset.

3. Deleting an existing workset Right click on the name of the workset you wish to change and select ‘Delete’ to delete the workset. The left hand side of the user interface is again updated accordingly. For example, Figure 3 shows the popup menu function ‘Delete’ that allows deleting a workset.

4. Copying an existing workset Right click on the name of the workset you wish to change and select ‘Copy’. This creates a new workset in the user interface that contains the same Control Panels as the workset you copied from. Of course you can then rename the workset (as explained previously), and edit the Control Panels in it (described shortly).

5. Adding a new workset This is performed by first copying an existing workset, then renaming it, then modifiying the Control Panels it contains.

6. Changing the tab order of worksets Hold the left mouse key down while you drag the workset to its new position relative to the other worksets. For example, Figure 5 shows the position of the ‘Tune’ workset changed from below to above the ‘Camera’ workset.

7. Changing the presentation order of Control Panels within a workset Hold the mouse key down while you drag the Control Panel to its new position relative to the other Control Panels in the workset. For example, Figure 4 shows the Plate Camera Control Panel being put below the Dark Field Control Panel.

8. Adding a Control Panel to a workset Hold the mouse key down while you drag the Control Panel from the right-hand pane of the Workspace Layout to the left-hand pane. For example, Figure 5 shows the ‘Direct Alignment’ Control Panel added to the ‘Tune’ workset.

9. Removing a Control Panel from a workset Right click on the name of the Control Panel and select ‘Delete’ or left click and press the Delete key.

The following Status Panel related customizations are supported: 1. Changing the colors used in the Status Panels Left click the Color icon of one of the worksets.

This displays a dialog box on the right allowing you to change the color of various interface items. 2. Adding an Indicator to a Status Panel Right click somewhere over the Status Panel you wish to

change then select the item you wish to add. For example, Figure 6 shows ‘Intensity Zoom’ added to the first Status Panel.

3. Removing an Indicator from a Status Panel Hold the left mouse key down while you drag the item you wish to remove off the Status Panel. For example, Figure 7 (top) shows the ‘Intensity Zoom’ item removed from the first Status Panel.

4. Changing the presentation order of Indicators in a Status Panel Hold the left mouse key down while you drag the item to a new location on the Status Panel. For example, Figure 7 (bottom) shows the items of the first Status Panel arranged in a different order.


4 Customizations that are not supported All customizations that are not mentioned in the previous section cannot be modified for standard users by the supervisor. More specifically the following change is not supported: Binding Panel related customizations. The Binding Panel is located in the bottom left corner of the User Interface, it shows the settings for the various function keys on the control pads. However, it should be noted that the actual bindings for new users are as set by the supervisor under his/her own account (not while in customisation). This only applies to the actual functions connected to the MF and user buttons, not which knobs/buttons are displayed and how (lay-out) in the binding display itself.

5 Effect on Existing Users

5.1 Expert Users Existing expert users are not affected by the customizations.

5.2 Standard Users Existing standard users are not affected by the customizations if they have made their own changes to the interface (i.e. whilst logged in as themselves). However, it is possible for a user to delete his/her current interface changes and revert to the defaults provided by the supervisor by doing the following.

From the command prompt type ‘peoui.exe /resetInterface’. Alternatively, you can create a taskbar shortcut for starting the interface in customization mode. (Creating shortcuts is described in the standard Windows help system.)

Existing standard users that have not changed the User Interface themselves will automatically see the new changes made by the supervisor. Since they are using the default settings for standard users, then changing the default settings affects them too.

6 Other points to note Some general points are worth mentioning: 1. As mentioned above, configuring the user interface for standard users requires logging in as

supervisor and starting the interface in customization mode. However, the supervisor is not prevented from performing actual microscopy whilst running the interface in customization mode. This is not recommended. In this mode, the supervisor is seeing the interface intended for standard users, which is not the interface the supervisor sees when he/she starts the ui in the normal manner. Therefore, recommended procedure is: a) Start the user interface in customization mode. b) Make the interface changes you wish. c) Exit the user interface.

2. The changes affect new standard users only. New expert users are not affected by the customizations.

3. When adding new Control Panels, note that you can select from the ‘User’, ‘Expert’, and ‘Supervisor categories. See Figure 5 where Control Panels from the Expert category were added; but the ‘User’ and ‘Supervisor’ categories are also equally accessible.

4. Changes to the color settings of the Status Panels (e.g. background color or text color) also change the color settings of the Binding Panel.


7 Installing a new TEM version Should a new version of the TEM software be installed, the user interface should be started by the supervisor to copy the user interface customizations from the previous TEM installation. All that is required is to simply start then exit the user interface, i.e. the interface does not need to be started in customization mode (see section 2). Until this is done, any standard users starting the interface will see the default layout that has been supplied with the TEM version just installed.

8 Sample Screen Shots

Figure 1. TEM User Interface title in Supervisor login in customize mode.

Figure 2. Changing a workset’s colors


Figure 3. Renaming and deleting worksets

Figure 4. Changing workset and Control Panel presentation order


Figure 5. Adding Control Panels to a workset


Figure 6. Adding Status Panels indicators

Figure 7. Removing Status Panel indicators and changing the presentation order of indicators

TEM on-line help Freeware 1 Freeware Software version Tecnai 4.0 / Titan 1.1

TEM on-line help manual -- Freeware Table of Contents 1 Introduction.......................................................................................................................................... 3

1.1 Copyright and liability limitation ................................................................................................ 3 2 TEM Account Log................................................................................................................................ 4

2.1 Introduction............................................................................................................................... 4 2.2 The main window...................................................................................................................... 4 2.3 Overview................................................................................................................................... 5

3 Aperture Positions ............................................................................................................................... 6 4 Big Screen........................................................................................................................................... 7

4.1 Introduction............................................................................................................................... 7 4.2 General principles..................................................................................................................... 7 4.3 Popup menu ............................................................................................................................. 7 4.4 Display...................................................................................................................................... 8 4.5 Optics parameters .................................................................................................................... 8 4.6 Stage parameters ..................................................................................................................... 9 4.7 Various parameters .................................................................................................................. 9 4.8 Window position and size ......................................................................................................... 9 4.9 Help access .............................................................................................................................. 9

5 Exposure Log .................................................................................................................................... 10 6 FEG Registers................................................................................................................................... 11

6.1 Introduction............................................................................................................................. 11 6.2 The settings ............................................................................................................................ 11 6.3 FEG Registers Options........................................................................................................... 14

6.3.1 Settings files........................................................................................................................ 15 7 Image Search .................................................................................................................................... 16

7.1 Image Search Control Panel .................................................................................................. 16 7.1.1 Image shift and maximum deflection .................................................................................. 16 7.1.2 Image search controls......................................................................................................... 16

7.2 Image Search Calibrate.......................................................................................................... 17 7.3 Image Search Options............................................................................................................ 18

8 Preset Magnifications ........................................................................................................................ 19 9 Screen Saver..................................................................................................................................... 21 10 Serial Sections............................................................................................................................... 22

10.1 Introduction............................................................................................................................. 22 10.2 Location .................................................................................................................................. 23 10.3 Section.................................................................................................................................... 23 10.4 Overview................................................................................................................................. 24 10.5 Menu....................................................................................................................................... 25

10.5.1 File menu ............................................................................................................................ 25 10.5.2 Setup menu......................................................................................................................... 25 10.5.3 Section menu ...................................................................................................................... 26 10.5.4 Location menu .................................................................................................................... 27 10.5.5 Display menu ...................................................................................................................... 27 10.5.6 Help menu........................................................................................................................... 28

10.6 Control options ....................................................................................................................... 28 10.7 Reference points .................................................................................................................... 28

10.7.1 Setting reference points for new data ................................................................................. 29 10.7.2 Redoing reference points for old data................................................................................. 29

10.8 Section data display ............................................................................................................... 30


10.9 Select section ......................................................................................................................... 31 10.10 Location list............................................................................................................................. 32 10.11 Select location ........................................................................................................................ 32

11 Total Recall .................................................................................................................................... 33 11.1 Total Recall Control Panel ...................................................................................................... 33 11.2 Total Recall Reference Points ................................................................................................ 34 11.3 Reference points .................................................................................................................... 35

11.3.1 Setting reference points for new data ................................................................................. 36 11.3.2 Redoing reference points for old data................................................................................. 36

11.4 Total Recall File...................................................................................................................... 38 12 Vacuum Logger ............................................................................................................................. 39

12.1 Introduction............................................................................................................................. 39 12.2 General principles................................................................................................................... 39

12.2.1 On-line operation ................................................................................................................ 39 12.2.2 Off-line operation ................................................................................................................ 39

12.3 Program structure................................................................................................................... 39 12.4 How to run the logging software ............................................................................................. 39 12.5 Menu....................................................................................................................................... 40

12.5.1 File menu ............................................................................................................................ 40 12.5.2 Display menu ...................................................................................................................... 40 12.5.3 Log menu ............................................................................................................................ 40 12.5.4 Help menu........................................................................................................................... 40

12.6 Logging parameters................................................................................................................ 41 12.7 File.......................................................................................................................................... 41 12.8 Display.................................................................................................................................... 42 12.9 Print ........................................................................................................................................ 42 12.10 Display parameters................................................................................................................. 43

12.10.1 Display settings dialog..................................................................................................... 43 12.10.2 Log scale ......................................................................................................................... 43


1 Introduction This document is a collection of the documentation of the freeware available for the TEM microscope. Before using the software, please refer to the copyright and liability limitation below.

1.1 Copyright and liability limitation Please note: • TEM Freeware is freeware and may be distributed and used freely. • The copyright of the TEM Freeware lies with Mad Max Software. • The creation of the TEM Freeware by Mad Max Software and distribution of the TEM Freeware by

the same or by FEI Company does not imply any future support of the software and future compatibility with new versions of the TEM software.

• TEM is a registered trademark of FEI Company. • The TEM Freeware is NOT a product of FEI Company. It is consequently NOT covered by any

warranties or support agreements between the user and FEI Company. • Limited warranty: Mad Max Software expressly disclaims any warranty for the TEM Freeware. The

TEM Freeware and any related documentation is provided "as is" without warranty of any kind, either express or implied, including, without limitation, the implied warranties or merchantability, fitness for a particular purpose, or non-infringement. The entire risk arising out of use or performance of the TEM Freeware remains with you.

• No liability for consequential damages. In no event shall Mad Max Software be liable for any damages whatsoever (including, without limitation, damages for loss of business profits, business interruption, loss of business information, or any other pecuniary loss) arising out of the use of or inability to use this TEM Freeware, even if Mad Max Software has been advised of the possibility of such damages.


2 TEM Account Log

2.1 Introduction The TEM Account Log software automatically monitors use of the TEM microscope, starting when you log in to Windows and closing when you log off again. You cannot close the program but only minimize it. The program will close automatically when you log off from Windows.

2.2 The main window

The window contains information about the current microscope session. The system assumes that you will work with one or more project codes (up to 60 characters). The project codes normally used are inserted in the drop-down list. The selection in the list is set automatically to your choice during the previous microscope session. Project codes can be added to the list by typing the code in the edit control to the right of the Add code button. Press the Add code (which becomes enabled when values are entered) to enter the new code in the list. The software checks against duplication of codes already present. To delete project code(s) no longer needed, select the required code in the drop-down list, then press the Delete code button. Because the data are logged only when you log off from Windows, you cannot use more than one project code during a session. If it is necessary to use more than project code, you must log off from Windows after work under the first project code is finished (the data are then logged), then log on again and use the other project code.


2.3 Overview You can get an overview of your microscope usage as logged. The overview is taken from the date given (in the edit controls to the right of the Show overview button). Default setting is the first of the current month. The overview window contains two sets of data

On the left-hand side is a list of the individual entries. These entries are sorted initially on date, but they can be sorted differently by pressing the button at the top of the column that is to be the criterion for the sorting. Press the same button again and the sorting reverses its order (from e.g. a..z to z..a). On the right-hand side is a display of the totals per project code (taken from the date defined in the main window). There is no printing function provided, but the totals can easily be copied over to Notepad. Click with the right-hand mouse button in the right-hand text part and choose Select all, then Copy in the popup menu. Then paste into Notepad.


3 Aperture Positions

The Aperture Positions Control Panel.

The Aperture Positions Control Panel displays the current aperture positions (in micrometers). You can use it get an idea of the positions or check the position values (e.g. the positions will differ slightly from MicroProbe to NanoProbe to LM). The positions are displayed for the apertures according to their nominal aperture mechanism. On most microscopes 1 will be C1, 2 C2, 3 Objective and 4 SA, but a few systems may deviate from this. Notes: • The aperture positions as listed are the positions known for the current aperture (size) for the current

mode. As long as you are centering an aperture (Adjust) the known position has not yet been updated so you will not see any change in the aperture positions. The change will become visible once Adjust is switched off again.

• The aperture positions are read out when you press the Get button. The Get button remains yellow during the time it takes to read out the positions.


4 Big Screen

4.1 Introduction Big Screen is an application that can be used to fill the 'data' space of the TEM User Interface (in case there is no other useful information, like CCD or STEM images, to be found there), displaying a selection of microscope settings with a large, easy-readable font, and with a user-selectable foreground (font) and background color, to reduce ambient light from the monitor from interfering with TEM work at low light-level conditions.

4.2 General principles An essential control for Big Screen is the right-mouse click (click with the right-hand mouse button). This will bring up a popup menu with various controls for Big Screen. The Big Screen window can contain up to three panels, one for Optics parameters, one for Stage parameters and the third for Various parameters. These panels can be resized at will by dragging the splitters between them to other positions. The parameters displayed are adjusted automatically, centered horizontally in their panel and with equal spacing in the vertical direction. Big Screen does not check and see if there is enough space available for displaying all labels. It is up to you to define the size of the panels and size of the font to make sure the labels do not overlap and are visible. In principle Big Screen can be run even if the TEM User Interface is not running. There is, however, little point in doing so, since the Control Pads (with the buttons and knobs) are disabled when the TEM User Interface is closed. If the TEM User Interface is closed, locking the Big Screen window to the 'data' space has no effect (apart from making the window non-sizeable). Big Screen will store and recall user's individual settings.

4.3 Popup menu The popup menu (which appears when the right-hand mouse button is clicked inside the Big Screen window) contains six categories of items that control Big Screen: • Display • Optics parameters • Stage parameters • Various parameters • Window position and size • Help access


4.4 Display The display settings that can be chosen are the colors of foreground (font) and background and the size and type of the font used for displaying the microscope parameters. The colors can be selected by making the color selection bar visible. This bar will be displayed on the left-hand side of the Big Screen window and contains the colors available. Two of the fields will contain labels, 'FG' and 'BG' (foreground/font and background). To change the foreground color, click with the left-hand mouse button in the color field required. To change the background color, click with the right-hand mouse button in the color field required. You can also select the foreground color in the font selection dialog.

The Select font item of the popup menu brings up the Font selection dialog wherein the type of font, size and color can be selected. The font color is the same thing as the foreground color.

4.5 Optics parameters Five optics parameters can be selected for display, magnification (or camera length), microscope mode, spot size, intensity setting and defocus value. If none of these values is selected, the Optics panel will not be visible.


4.6 Stage parameters The stage parameters that can be selected for display are all relevant axes. The b tilt axis is only displayed if a double-tilt holder is present. If stage parameters is not selected, the Stage panel will not be visible.

4.7 Various parameters Three plate-camera exposure parameters can be selected for display, measured exposure time, exposure mode (auto or manual) and manual exposure time. If none of these values is selected, the Various panel will not be visible.

4.8 Window position and size The Big Screen window can be locked to the 'data' space of the TEM User Interface (the empty space left over by the user interface in its various display modes). If the display of the TEM User Interface is changed and the Big Screen window is locked, the size of the Big Screen window is adjusted automatically. When locked, the Big Screen window cannot be resized (though it can be moved around).

4.9 Help access By selecting the Show help item of the popup menu (or pressing the F1 function key) the help file is displayed.


5 Exposure Log The Exposure Log Control Panel allows definition of microscope settings that will be stored to file when an exposure is made.

The Exposure Log is a text file with a series of microscope settings (dependent on definition by the user), separated by tabs. Such files can easily be read in in spreadsheet programs like Microsoft Excel, which will translate each tab into a new column (thus the settings are ordered into columns). At the beginning of each session, the Exposure Log will write a line with 'Exposure log' followed by the date and time. After that line follows a second line with a header, describing each item of a column. When the options are changed, the header will be written again (otherwise the columns will not match up with the header anymore). The starting line of the session may be the first line of the file, but data from multiple sessions can be written to the same file (appended automatically). Select File When the Select File button is pressed, a standard Save As dialog is displayed in which a filename can be defined. This filename is stored (for the particular user) at logoff and at next logon automatically selected again. If the file exists, the new data will be appended to the file automatically (so old data do not get overwritten). The button is yellow when a filename has been defined. When the button is still gray, no data will be written to file. Optics On the Optics tab a number of settings related to the optics of the microscope can be selected. Plate On the Plate tab, a number of settings related to the plate camera can be stored such a exposure number, exposure time and film text. Stage On the Stage tab, each axis of the CompuStage can be selected (individually) for storage.


6 FEG Registers

6.1 Introduction The control of the FEG is somewhat complicated because of the wide range of settings under which the gun can be used and the nature of its construction. Two elements that can be varied, the extraction voltage and the gun lens setting, are located above the alignment coils and therefore the alignment of the gun doesn't stay the same when these parameters are changed (the high tension used in turn affects the way the extraction anode and gun lens are used, adding another complication). In some types of application the FEG is only used in one or two specific settings. In these cases (e.g cryo work in Life Sciences), there is little need for frequent changes of the gun settings and the normal method of alignment save and recall in the microscope software will be adequate. In other cases, frequent adjustments are made to gun settings, e.g. changes of extraction voltage and gun lens when switching between normal or high-resolution TEM imaging and small-spot analysis, diffraction or STEM imaging. In the latter case the frequent realignment (or alignment recalls) makes use of the FEG cumbersome, especially since in addition to the alignment recalls several other factors (such as mode, spot size and condenser stigmator settings) are not set along with the alignment. It is in these cases that FEG Registers will be helpful. FEG Registers stores and recalls a number of microscope settings, making it easy to switch between previously defined settings. Each user can store any number of settings under a user-defined label. These settings are saved in a file (one for each user) under the Windows NT user name preceded by usr_. Whenever the user logs in, the settings are loaded from the file. Each of the settings can be selected in the list and set to the microscope. Settings can also be updated and deleted. They can also be saved to and recalled from files with user-defined names. Important note (Titan): Because of the high degree of complexity of the Titan alignments and frequent misalignment (due to user error, not alignment reproducibility problems), the alignment philosophy of the Titan software has been changed. From version 1.0 onwards, users cannot execute a full alignment anymore nor can they save alignment files. In essence users can only make minor modifications (e.g. rotation center) through direct alignments (or alignment procedures for the gun). Because users can no longer save alignment files, the only way to have sets of the alignments saved with easy switching is to use FEG Registers.

6.2 The settings The following settings are stored for each register. Settings Tecnai Titan FEG • Extraction voltage

• Gun lens • Gun tilt • Gun tilt pivot points • Gun shift • Spotsize-dependent gun shift

• Extraction voltage • Gun lens • Gun tilt • Gun tilt pivot points • Gun shift • Gun shift pivot points • Spotsize-dependent gun shift • Gun cross-over • Gun stigmator

Monochromator (on monochromator systems only)

• Potential • Excitation • Monochromator settings (file)

• Potential • Excitation • Monochromator settings (file)


Microscope mode • TEM / STEM • LM, SA, Mh, LAD, D • Microprobe / Nanoprobe • Lorentz • Dark field • Normal / EFTEM

• TEM / STEM • LM, SA, Mh, LAD, D • Microprobe / Nanoprobe • Lorentz • Dark field • Normal / EFTEM

Condenser / Illumination

• Spotsize (active mode only) • Intensity (active mode only,

but ignored in the case of STEM because it is an alignment setting there)

• Beam shift (active mode only) • Rotation center (active mode

only) • Condenser stigmator (active

mode and current channel only)

Note: When motorized apertures are present, the C2 aperture MUST be enabled, otherwise you cannot restore the illumination settings. • C2 Aperture selected and position • Illumination mode (Parallel / Probe) • Condenser lens mode • C2 cross-over mode • Condenser free control mode • Spot number • Intensity • Projection diameter • Illumination diameter (parallel mode) • Convergence angle (probe mode)

Magnification • Magnification (TEM or STEM) • Camera length

• Magnification (TEM or STEM) • Camera length

Direct alignments • Spot-size dependent intensity correction

• Beam shift • Beam tilt pivot points • Rotation center • Diffraction shift • Dynamic conical dark field pivot

points • Descan corrections

Stigmators • Condenser stigmator (the complete list for all spots)

• Three-fold condenser stigmator • Objective stigmator • Diffraction stigmator

Note (Tecnai) : If you want to save settings for different high-tension settings, identify them as such by the label. High tension is neither stored nor recalled automatically. Notes (Titan) : • High tension is stored but not recalled automatically. If the current high tension differs from the stored

one, you will get a warning to that extent. • Currently FEG Registers stores all aperture positions and selections but only restores the C2

aperture. Because it is possible that the apertures have been changed physically, FEG Registers performs the following checks: o If the size of the aperture and the aperture index match, the C2 aperture is selected and its

position will be restored. o If the size of the aperture does not match the size of the current aperture size at the index

(which may mean that the size of the aperture as indicated has been changed on the system, but more likely that the apertures have been changed), FEG Registers will attempt to find an


aperture in the system that has a size matching the stored aperture size. If that is found, that aperture will be selected but the position will not be set to that in the register. If no matching aperture size is found, any aperture that differs in size by less than 5 micrometers will be selected (but again its position will not be set). If no matching aperture within 5 micrometer is found, you cannot restore the FEG Registers beyond the FEG and monochromator setting listed above.

o Note that changing the C2 aperture may take a little time and FEG Registers has to wait until the aperture has changed before it can continue setting the optical settings. Because of this, setting a FEG Register may take more time than previously.

Set When the Set button is pressed the settings from the register currently selected in the list will be set to the microscope. Update When the Update button is pressed, the selected setting is updated to the settings currently active on the microscope. Typical use of this function would be to select a setting and set it back to the microscope. Then modify any microscope settings needed (e.g. the gun alignment) and then update. Update requires confirmation (since the old setting will be overwritten).

Delete When the Delete button is pressed the setting currently selected in the list will be deleted. To guard against accidental deletion, the Delete button will request confirmation. Multiple settings can be selected for deletion (use Shift+Click to select a range or Ctrl+Click to select individual settings). Settings list The settings list gives an overview of all settings of the current user. The settings are automatically stored when the user logs off (it is not necessary to save them to file, settings are added simply by Add and removed by Delete). The settings list initially will display the registers in the sequence they have been defined. It is possible to sort the settings in the list differently by pressing one of the buttons at the top of the list (Lbl, EV, GL, ...). At the first press, the settings are sorted in normal order. When the same button is pressed again the sequence is reversed. The width of the columns can be adjusted by moving the cursor to the boundaries between the buttons and dragging the boundary to left or right. The columns in the list have the following meaning: • Lbl : user-defined label for the setting. • EV : extraction voltage. • GL : gun-lens setting. • Mode : microscope mode • Spot : spotsize • Date (usually out of sight due to lack of space) : the date at which the setting was added. On monochromator systems EV and GL are replaced by Pot (Potential) and Exc (Excitation), respectively. These data are only a subset of the settings stored, but they are the important ones that allow the user to see which setting should be recalled.


A setting is selected for setting to the microscope or updating by clicking the required row. The label of the setting is automatically copied to the settings label edit field to ensure the proper setting is used for updating. Settings label The settings label defines the name of a new register setting (when Add is pressed). If no text is entered, the software will add a label 'Register' with some number attached. Add When the Add button is pressed the settings currently on the microscope are stored in a new register which is added to the list. Flap-out button The flap-out button leads to the Options tab of the FEG Registers Control Panel.

6.3 FEG Registers Options

The control panel for Tecnai The control panel for Titan The Options tab contains a few options concerning which settings are restored to the microscope, and controls for saving the settings to file and loading them from file. Settings options The settings that will be restored to the microscope can be chosen by checking the required check boxes. The categories are listed in the settings table. File Open The Open button brings up the standard Open file dialog from which a file can be selected for loading. See the remark above about overwriting the currently existing settings. File Save The currently existing settings can be saved to file. When the Save button is pressed the settings are saved under a user-defined filename. Note: Under normal circumstances it is not necessary to save settings in a file because a user's settings are saved automatically. Saving files is only useful when a user wishes to have several sets of settings of FEG Registers or for having other users use the settings defined by another user.


File Save As Save as is the same as Save except that another filename can be chosen.

6.3.1 Settings files Settings files can be saved under any file name and in any location with the following restriction: filenames with names that start with usr_ and then a Windows NT username (that is, the name under which users log in) are not allowed. These files will in fact exist for users who have used FEG Registers before and contain their settings. You can open those files and use the settings in there, but you cannot overwrite them. The location of the user files is by default in a folder \”tem”\data\ (where “tem” is either “tecnai” or “titan”) followed by the user name. Note (Titan) : If you save a file in a location other than c:\titan or subfolders, the file will not be found by the update function (available to service) that makes sure that C2 aperture positions are updated when the apertures on the microscope are changed (meaning physically removed and replaced by another aperture). Files do not contain a single setting but all settings together. When you load settings from file you automatically remove all currently existing settings!


7 Image Search

7.1 Image Search Control Panel The Image Search Control Panel provides the controls necessary for the image-search method. With this method the image movement with the track ball normally connected to the stage is done with the image shift instead of the CompuStage, giving faster and smoother image movement. Because of the limited range of the image shift, the image search resets the image shift as needed, with a compensating CompuStage shift motion to keep the same field of view before and after the reset.

Note: Image Search only works in the HM magnification range (Mi and SA magnifications). When Image Search is active and the magnification is changed to LM, the Activate button and Reset buttons become disabled until the magnification range goes back to the HM range (it is not possible to reset the HM image shift while the microscope is in LM).

7.1.1 Image shift and maximum deflection There are two important settings during Image Search, the amplitudes of the image shift position and that of the total deflection. The image shift is but one component active on the image shift deflection coils, other components being the alignment values of the image shift (magnification) and diffraction shift. The total value, given by the maximum deflection can never exceed 100%, even if the image shift itself is less than that. The amplitude available for the image shift will depend on the microscope (and to some extent the magnification). Reset the image shift before the total deflection amplitude reaches its maximum.

7.1.2 Image search controls Activate To start using the Image Search, press the Activate button (it will become yellow). The control of the track ball connected to the stage (which track ball is automatically detected by the software) is changed to the Image Search. To revert to the standard situation, press the Activate button again (the button becomes gray again). When the Activate button is disabled, the Image Search calibrations have not been done. Image shift position The amplitude of the image shift is displayed by a progress bar. Maximum deflection The maximum deflection (the sum of all components working on the image shift deflection coils) is displayed by a progress bar.


Reset To Reset the image shift with a compensating stage movement, press the Reset button. The Reset button remains disabled until the image shift has reached more than 20% of the available image shift range (below that the inaccuracies in the compensating stage movement are such that a reset does not makes much sense). Auto Reset The reset can be done manually (by pressing the Reset button) or automatically (when the Auto Reset checkbox is checked). When Auto Reset is active, the software will do a reset when the image shift amplitude reaches the value for the Maximum deflection range given. When the reset is automatic, the reset is preceded by a beep (PC sound signal). Another beep sounds when the procedure is finished. Maximum deflection range The maximum deflection range value determines the image shift amplitude at which an automatic reset will be done. Flap-out The flap-out button leads to the Calibrate and Options tabs of the Image Search Control Panel.

7.2 Image Search Calibrate The Calibrate tab of the Image Search Control Panel provides the controls necessary for the calibration of the image-search method. There are two things to be calibrated, the relation between the image shift and the stage motion, and the direction of the image shift as seen on the screen. The first determines the accuracy of the compensating motion of the image shift with the stage, while the second ensures that the direction of the image motion is linked to the direction of the track ball movement.

Note: The Image Search method makes use of the image shift with compensated beam shift (a standard feature of the TEM software). For this to work properly, the Image/Beam calibration procedure of the Image HM-TEM alignment procedure must have been done. If the beam shifts away from the center of the screen during image shift, this calibration has not been done properly. Start To start a calibration, select a procedure in the selection list and press the Start procedure. Procedure selection The two calibration procedures are selected in a drop-down list. Instructions Instructions during the calibration procedures will be displayed here. OK Pressing the OK button proceeds with the calibration procedure to the next step.


Cancel Pressing the Cancel button cancels the calibration procedure. The microscope returns to its starting position.

7.3 Image Search Options The Options tab of the Image Search Control Panel provides the controls necessary for the setting the options of the image-search method.

CompuStage backlash correction During a reset the compensating stage motion will contain a backlash correction motion (the stage is first driven a certain distance further negative on X and Y before going to the required position). This backlash correction makes the setting of the stage position more reliable and thus the compensation for the image shift more accurate. Typical values for the backlash correction are between 5 and 20 um. Reset under control pad button The Reset function can also be activated by assigning the function of one of the user buttons L1..L3 and R1..R3. Select the button to use in the drop-down list. The function is only active when Image Search is active. Exposure enabled/disabled while active Because image quality is affected by strong off-axis image shifts, exposures should not be taken without resetting the image shift to zero. In order to make sure this happens, you can choose on of two options: • The exposure button is completely disabled while Image Search is active. In this case you have to

de-activate Image Search (which forces a reset) before you take an exposure. • The Exposure button is enabled while Image Search is active. In this case Image Search catches the

pressing of the Exposure button and no immediate plate exposure takes place. Instead Image search forces a reset and then releases the Exposure button for taking a plate exposure. A message to that extent is displayed in the control panel.


8 Preset Magnifications The Preset Magnifications Control Panel provides rapid switching between a number (up to six) of user-defined magnifications through the user buttons (L1..L3, R1..R3) on the TEM Control Pads. When a previously defined setting (consisting of Magnification, Intensity setting and Spot size) are active, a press of the associated user button switches the microscope to those settings. The Preset Magnifications function thus assists in work on the microscope where a fixed set of magnifications is used repeatedly (as e.g. in diagnostic screening).

The operation is as follows: • Press Active if the button is gray to activate Preset Magnifications. • Decide which user button to use and check the check box for that user button. • Press the associated Define button (which becomes yellow). The software asks to select the required

magnification, intensity setting and spot size. • Press the Define button again (button goes back to normal). The settings are now defined. • To go back to these settings, press the user button on the TEM Control Pad. To redefine the settings under a user button: • Press the required user button. • Press the associated Define button (which becomes yellow again). • Change the settings and press the Define button again. Settings are user-specific and stored automatically when the TEM User Interface is closed and recalled when it is re-opened. First-time users get the settings as defined by the Supervisor (Note: only Supervisor, not Service or Factory), if available. Note: Preset Magnifications requires TEM Scripting on the microscope. Active button The active button determines whether the Preset Magnifications are assigned to the user buttons. The function is active when the button is yellow, inactive when it has the normal button color. Automatic When the Automatic check box is checked, the Preset Magnifications function automatically becomes activated when the TEM User Interface is started (if not, it only becomes active once the Active button is pressed). User button check box The check boxes determine whether a particular user button is used. To activate a user button, check the box and define the settings. To deactivate, click on the check box to remove the check. Magnifications For active user buttons the magnifications used are displayed in a list to the right of the check boxes.


Define buttons The Define buttons allow definition of the microscope settings associated with a user button. For the procedure, see above. Instructions The instructions field at the bottom will display instructions on how to proceed, or a status description or error message.


9 Screen Saver

The TEM Screen Saver can be used as an easy check on the status of an unattended microscope (provided a user is logged on). The TEM Screen Saver (called TEM Vacuum Saver in the Display Properties) acts as a normal screen saver and has three modes of display: 1. There is no connection to the Tem server: the display states Not connected to TEM server and

draws red octagons. 2. There is a connection to the Tem server and the column valves are closed: the display states

Column valves closed and draws green balls. 3. There is a connection to the Tem server and the column valves are open: the display states Column

valves open and draws red octagons. There is no automatic preview in the small “monitor” in the Display Properties, but the Preview button will start a screen saver preview.


10 Serial Sections

10.1 Introduction The serial section software provides an automatic way of retrieving one or more locations within a series of sections by driving the CompuStage to each location as directed by the user. The software is a separate application that you can find in the Start menu under Programs\TEM The serial section software assumes that the specimen contains a number of sections cut from a single block. Each of the sections can be defined and the software can then determine equivalent locations for each section. In this way a three-dimensional impression of the specimen can be obtained, e.g. by recording images for a particular location in each section and combining all these images.

The method used by the software is as follows. A first section must be defined by moving each of the four corners of the section (or specially applied marker points near the corners such as laser-drilled reference holes) with the specimen stage to the screen center (at which point the software will read the stage locations). The four points must always be in a certain sequence. The first two points define the baseline. By definition, this is the bottom of the cutting block which must be as perpendicular as possible to the cutting direction. The third point is one (the best defined) of the corners at the top of the section. Point four is used only for display purposes and needs to be defined only for the first section (its position is calculated for all other sections).

From these data the software determines a number of variables for each section: • the baseline rotation angle (the slope of the line through points 1 and 2) • the baseline stretch factor (the ratio of the distances between points 1 and 2 in any section and that

in the first section). Although not critical for the performance of the software, under normal circumstances the baseline stretch should be very close to 1.0

• the perpendicular stretch factor (the ratio of the projection of point 3 onto the baseline in any section and the same projection in the first section). In practice this factor can vary quite a bit.

After a first section is defined, locations can be stored or other sections can be defined (locations can be stored in any section and at any stage of using the software). For each subsequent section, the three relevant corners (1 through 3) must defined as for the first section. Locations are stored by centering them on the screen and selecting the relevant function (Location, Insert). Once a sufficient set of data has been defined, the software can be instructed to move from section to section (where it will go to equivalent locations for each section) or from location to location within each section. Location-section combinations can be marked as visited to allow the operator to keep track of what has been done (an unmarked location is shown as a red number, a marked one as a black number on a green background). The software can display an overview of the visits status. Marking as visited


can be automatic when the program is instructed to go to a location for a particular section or by active selection by the operator. The choice between automatic or user marking is done under Control options. Stored locations are either defined in the first section or, when the operator stores a location while on another section, the stored location X,Y values are calculated back to the equivalent location in the first section. The location recalculation method works as follows: 1. determine the length of the projection of the location onto the baseline along the perpendicular and

the distance of the projection point on the baseline from point 1. 2. determine for the current section the point on the baseline, adjusted for the baseline rotation and

baseline stretch. 3. project the location from the point of the baseline along the perpendicular, adjusted for the

perpendicular stretch. Note: The method used for calculating the point assumes that any change in location position is wholly determined by a baseline stretch and a perpendicular stretch. The implication is that the cutting direction must be nearly or wholly perpendicular to the baseline. If the stretching due to the cutting deviates significantly from the perpendicular, the software will not work properly. This methodology has been adopted on purpose because there is in the three or four corner points insufficient accuracy to determine a variable cutting direction.

10.2 Location A Stored location is any location at which the operator presses the Insert key or selects Insert from Location menu. If use labels (see Control Options) is active, a dialog will appear asking the operator to enter the label (a string with a maximum of 120 characters) for the location. The number of stored locations that the program can handle is limited only by the amount of memory available on the computer. However, the program will not mark locations visited for more than 64 locations (per section).

10.3 Section A Section represents a piece of tissue on the specimen. Each section is defined by three corner points, the first two defining the baseline (the bottom of the block where the knife hits first), the third one of the upper corners (the best defined of the two).

The fourth corner is set only for the first section and is used only to allow display of the section shape on the screen.

If a section is represented by a single X,Y value (e.g. under Show section or Goto section), that value is the center of the section, defined as the X and Y values that lie halfway between the lowest and highest values for all four corners.


10.4 Overview It can be difficult to keep track of which location in which section has been inspected or recorded. The software therefore marks location-section combinations as visited (either automatically or by operator selection).

Sections are plotted vertically, locations horizontally. Green section-location combinations have been marked as visited. If the left-hand mouse button is double-clicked on any rectangle representing a section-location combination, the program will bring up a dialog giving the information about the location indicated. If the button Go there is pressed, the program will move the specimen stage to that section-location combination (and mark the location as visited if automatic marking is switched on). If the Print button is pressed in the Status overview dialog, the overview is printed. Note that the print-out may spread over several pages if many locations have been stored. The maximum number of locations for the program keeps track is having been visited is 64, the maximum number of sections is 200.


10.5 Menu The menu contains six main items, File, Setup, Section, Location, Display and Help. Depending on the current status of the program, some of the items may be grayed (inactive).

10.5.1 File menu The File Menu provides the operations that are concerned with Data and Settings files as well as Printing. New specimen Resets all series settings (section, location definitions, status) as currently defined

and makes ready for a new specimen.

Open Opens an existing data file allowing continuing working with the specimen. To allow accurate restoring of stage coordinates use reference points.

Save Saves the currently defined series settings into a data file.

Save As Same as Save but allowing change of file name.

Control options Leads to control options dialog.

Default settings Resets settings to default values for program.

Print Screen Prints the graphical display.

Print List Prints a complete list of all sections and all locations per section.

Printer setup Leads to the Printer setup dialog (e.g. for selecting Landscape printing).

Exit Exits the program.

10.5.2 Setup menu In the Setup Menu, the operator defines reference points. It is also possible to move the stage to 0,0 before or during work on a specimen. Reference points Starts procedure for storing reference points (new file) or finding back old points

(stored data on old file).

Show rf. pt. data After reference points from an old file have been found again, the program can display the relation between old and new reference points, such as rotation angle and relative shifts.

Stage to center Moves the specimen stage to 0,0.


10.5.3 Section menu In the Section Menu, the functions concerning the sections are found. Insert Starts the procedure for defining a new section.

Delete Presents a list of sections (as serial numbers plus the section center) from which one

or more sections can be selected for deletion. If a section is marked for deletion accidentally, click again on the section and the highlight will be removed. If only one section is defined as yet, that first section definition will be deleted.

Delete All Deletes all sections currently defined.

Show section Brings up an overview of all data concerning the current section (corner positions, rotation angle, baseline and perpendicular stretch factors). Pressing the Go there button instructs the program to move the stage to the section selected (either to the currently active location or to the section center; the latter if no location has been stored yet).

Goto section Present a list of sections (as serial numbers plus the section center). If one of these sections is selected and OK is pressed, the stage is moved to that section (either to the currently active location or to the section center; the latter if no location has been stored yet).

Next section Move the stage to the next section (either to the currently active location or to the section center; the latter if no location has been stored yet). The menu item is grayed if no next section exists.

Previous section Move the stage to the previous section (either to the currently active location or to the section center; the latter if no location has been stored yet). The menu item is grayed if no previous section exists.

Status overview Displays an overview of section-location combinations visited.

Mark location Marks a location as visited. The menu item is enabled only if User mark has been selected under Control options.


10.5.4 Location menu The Location menu contains a number of functions that are related to the locations stored. Note: A hidden function is present in the program if hyperlabels are (see under Control options): Clicking with the left-hand mouse button on a stored location will bring up a dialog with data (X, Y, label) of the stored location if such a location is near. Pressing the button Goto there will move the stage to the location. Insert Inserts the current stage location in the stored list.

Delete Presents a list of locations (as serial numbers plus the label) from which one or more

locations can be selected for deletion. If a location is marked for deletion accidentally, click again on the location and the highlight will be removed.

Delete All Deletes all stored locations.

Display list Displays a list with all stored locations (X, Y, label) for the current section and allows printing of the list.

Show number Shows a list of stored locations (with number and label) for the current section, allowing selection of one location for which the data (X,Y, label) will be shown. A button Goto allows moving the stage to the stored location.

Goto number Shows a list of stored locations (with number and label) for the current section, allowing selection of one location where the stage will be moved.

10.5.5 Display menu The Display Menu allows the operator to select the method of displaying the section and location data on the monitor. The active selections are checked. Current section Displays the currently active section. The numbers of the current section and current

location are displayed top left. Locations are shown as red (not visited) or black on green numbers (visited). A scale bar is shown at bottom right. If hyperlabels are used (see Control options), it is possible to display the data of each location (X,Y position and label) by clicking on it with the left-hand mouse button. If the button Goto is pressed, the stage is moved to the location.

Overview Displays an overview of all sections.

Whole specimen Displays the sections in relation to the whole specimen. For current section:

Baseline Displays the current section with the baseline horizontally on the monitor.

Screen orientation

Displays the current section in its orientation as on the TEM microscope's viewing screen for the currently active magnification.

Stage X,Y axes Shows the current section with the stage X axis horizontal and the Y axis vertical.


10.5.6 Help menu Show Brings up the Help file. Pressing the F1 function key is equivalent.

About Brings up the program About box with version and copyright information.

10.6 Control options Control options (under Setup menu) allow the operator to choose how to use the Serial Sections software. The options are: • Whether the grid used is a so-called relocatable grid (a grid with a special "ear" on one side that

allows it to be fitted into the single-tilt specimen holder of the CompuStage in only one way). • Whether to use labels (a user-defined string) for stored locations. • Whether to use hyperlabels. Hyperlabels are "hyperlinked" labels of the locations in a section. The

labels will flash blue when the cursor moves over them and when clicked will bring up a dialog with information about the location with a button that allows a direct Goto to the location.

• Whether to mark locations as visited automatically when the program is instructed to move the stage there (By visit) or whether the marking only takes places when the user selects Mark location in the Section menu.

• The colors of the various items on the display: background, foreground (outline of sections and text at top left), sections, locations and current stage location. The definition of these colors is through a standard color definition dialog.

The display colors can also be changed by clicking on the display with the right-hand mouse button, giving the popup menu where the relevant color can be chosen.

10.7 Reference points Reference points are locations on the specimen that are easily recognized and can be used as reference when the specimen is removed from the specimen holder and later put back in again. By finding the reference points again, the program can recalculate the old locations to new ones.


Reference points should be located close to but not at the periphery of the specimen. Locations further from the center increase the accuracy of relocation but if a reference point later lies outside the reach of the stage, it is not possible to recalculate the old locations to new ones. Because specimens are often shifted up to 0.2 mm when re-inserted into the specimen holder, keep the reference points less than 800 um away from the center. Setting the reference points can be done at any stage of working with the Serial Section software (but before the data are saved into a file, otherwise the reference points are not included in the file). When an old file containing reference points is opened, the software will ask to redo the reference points immediately. If the reference points are not redone at this stage, the software will display the stored locations in their original configuration. It is then no longer possible to redo the reference points (unless by reading in the file again).

10.7.1 Setting reference points for new data During the procedure wherein new reference points are defined, the operator is asked to drive the stage to 2 or 3 points that can be recognized. It is suggested to avoid points that lie on the opposite side of the center as this will make it much more difficult to decide later whether the specimen has been reloaded in the same way or upside down. If at least two points are defined lying at an angle of about 120°, then it will be easier to decide. Three points make recognition of an upside-down position of the specimen even easier. If no third reference point is to be set, then simply press OK

while keeping the specimen stage at the same location as for point 2. The third point will then be ignored.

10.7.2 Redoing reference points for old data If an old file containing reference points is reopened, the software will suggest to go through the procedure for relocating the old reference points. The software will show the distribution of the old reference points and list the label of each point in turn. The reference point locations will be updated with the new ones, once a reference point has been found again.

During this procedure the software can assist in finding back the old reference points through the Assist function. The Assist function will list a number of possibilities depending on which reference points must be found again: • move the specimen stage to the old location for point 1 • move the specimen stage in a circle, around 0,0 for point 1, around 1 for point 2, etc. • calculate alternative locations for points 2 and 3 on the assumption that the specimen is upside down After finding reference point 1, the software will calculate what the most likely new location is for reference point 2 and ask if the stage must be driven there. The same is done for point 3 (if a third reference point had been set originally). For each reference point found back as well as the whole set of the reference points the software will go through a number of consistency checks (distances between old points and new points comparable, angles from the center of gravity of the old three-point triangle and old points and the center of gravity of the new three-point triangle and new points comparable). The software can handle rotation, shift and mounting upside down of the specimen. The software also calculates a 'stretch' parameter that is related to the percentage difference in dimensions between old


and new reference points. The software cannot however accommodate severe distortions due to bending of the specimen, so some care should be taken to ensure the integrity of the specimen. The various values can be displayed under Show ref. pt. data.

10.8 Section data display The Section data display dialogs shows the data for the section selected.

These data consist of the X,Y positions of the four corners, the rotation angle of the baseline, and the perpendicular and baseline stretch values.


10.9 Select section For selecting a section (a single section for go to or one or more sections for deleting) the software brings up the Select section or Delete section dialog. The two dialogs are similar in operation, the main differences being the text of the main button (OK or Delete) and the possibility of multiple section selection (use Ctrl+Click or Shift+Click; Ctrl+Click on an already selected - that is, highlighted - section, will remove the selection again).


10.10 Location list The Display list item of the Location menu displays a list with all stored locations (X, Y, label) for the current section and allows printing of the list.

10.11 Select location For selecting a location (a single location for go to or one or more locations for deleting) the software brings up the Select location or Delete location dialog. The two dialogs are similar in operation, the main differences being the text of the main button (OK or Delete) and the possibility of multiple location selection (use Ctrl+Click or Shift+Click; Ctrl+Click on an already selected - that is, highlighted - location, will remove the selection again).


11 Total Recall

11.1 Total Recall Control Panel

The Total Recall Control Panel provides the means for storing specimen locations in such a way that later the specimen can be re-inserted into the microscope and the locations easily found again. This is achieved through the use of two or three reference points, which should be locations on the specimen that are easily recognised. When the reference points are found again upon re-insertion of the specimen holder, the software recalculates the original specimen locations, taking proper account of specimen rotation, shift and mirror (the latter when the specimen is reloaded upside down). A prime application for the Total Recall software is dual-axis tomography, where two separate tilt series are collected of the image area, with a rotation of about 90° of the specimen in between. The combination of these two series reduces the missing wedge to a (smaller) missing pyramid (and thus reduces the consequent aretefacts). However, you can use the software in any other way you see fit. Label At the top of the control panel is an edit control wherein you can define the label used for the next location added. When you enter no label, the software will automatically use the label "Location " plus a serial number. Add Press Add to add the current stage position to the location list. Location list The location list contains all user-defined locations, shown by label and X,Y location. The stage locations are displayed as rounded off micrometers values, but the software stores more accurate values of the stage location. Goto If one of the locations in the list is highlighted (by clicking on it with the mouse), the Goto button is enabled. Pressing the Goto button will have the software drive the stage to the indicated location. Goto's are subject to the normal uncertainties in stage position (~500 nm for X and Y, no backlash correction is done because the original storing of the locations is also done without backlash correction so any such correction later on would be without benefit).


Delete If one of the locations in the list is highlighted (by clicking on it with the mouse), the Delete button is enabled. Pressing the Delete button removes the selected location from the list. Flap-out button The flap-out button leads to the Reference points and File tabs of the Total Recall Control Panel.

11.2 Total Recall Reference Points The Total Recall Reference Points tab provides the means for defining and inspecting reference points.

Define On a new specimen, the reference points are defined by pressing the Define button. The user will be asked to center two or three reference points on the screen with the stage (if you use but two reference points, keeping points two and three the same, the software will not be able to determine if the specimen has been reloaded upside down). The software will display the X,Y locations and labels defined for the reference points. The manipulation of reference points from an existing file is started automatically when a file with reference points is loaded (for further explanation see below).


Show data If an existing file has been reloaded and the reference points found again, the software will display the X,Y locations and labels defined for the reference points. In addition, it is possible to compare some data on the old and new reference points in a separate dialog that is called up by pressing the Show data button.

The data listed are the old and new reference point positions, whether the specimen is upright (same way as before) or upside down, the rotation angles calculated for the average and each individual reference point set (old to new), the shift of the specimen, and the stretch between sets of reference points (in principle all stretch factors should be very close to 1.0). Reference points The bottom part of the panel displays the X,Y locations and labels of the current reference points.

11.3 Reference points Reference points are locations on the specimen that are easily recognized and can be used as reference when the specimen is removed from the specimen holder and later put back in again. By finding the reference points again, the program can recalculate the old locations to new ones. Reference points should be located close to but not at the periphery of the specimen. Locations further from the center increase the accuracy of relocation but if a reference point later lies outside the reach of the stage, then it is not possible to recalculate the old locations to new ones. Because specimens are often shifted up to 0.2 mm when re-inserted into the specimen holder, keep the reference points less than 800 um away from the center. Setting the reference points can be done at any stage of working with the software (but before the data are saved into a file, otherwise the reference points are not included in the file). When an old file containing reference points is opened, the software will ask to redo the reference points immediately. If the reference points are not redone at this stage, the software will display the stored locations in their


original configuration. It is then no longer possible to redo the reference points (unless by reading in the file again).

11.3.1 Setting reference points for new data During the procedure wherein new reference points are defined, the operator is asked to drive the stage to 2 or 3 points that can be recognized. It is suggested to avoid points that lie on the opposite side of the center as this will make it much more difficult to decide later whether the specimen has been reloaded in the same way or upside down. If at least two points are defined lying at an angle of about 120°, then it will be easier to decide. Three points make recognition of an upside-down position of the

specimen even easier. If no third reference point is to be set, then simply press OK while keeping the specimen stage at the same location as for point 2. The third point will then be ignored.

11.3.2 Redoing reference points for old data If an old file containing reference points is reopened, the software will suggest to go through the procedure for relocating the old reference points. The software will show the distribution of the old reference points and list the label of each point in turn. The reference point locations will be updated with the new ones, once a reference point has been found again.

As a first step the software lists the old location for reference point 1 and you can have the software drive the stage there (in case you expect the new point to be close to the old one).

During this procedure the software can assist in finding back the old reference points through the Assist function. The Assist function will list a number of possibilities depending on which reference points must be found again: • move the specimen stage to the old location for point 1 • move the specimen stage in a circle, around 0,0 for point 1, around 1 for point 2, etc. • calculate alternative locations for points 2 and 3 on the assumption that the specimen is upside down


After finding reference point 1, the software will calculate what the most likely new location is for reference point 2 and ask if the stage must be driven there. The same is done for point 3 (if a third reference point had been set originally).


For each reference point found back as well as the whole set of the reference points the software will go through a number of consistency checks (distances between old points and new points comparable, angles from the center of gravity of the old three-point triangle and old points and the center of gravity of the new three-point triangle and new points comparable). The software can handle rotation, shift and mounting upside down of the specimen. The software also calculates a 'stretch' parameter that is related to the percentage difference in dimensions between old and new reference points. The software cannot however accommodate severe distortions due to bending of the specimen, so some care should be taken to ensure the integrity of the specimen.

11.4 Total Recall File The Total Recall File tab contains three buttons which bring up standard File dialogs for loading and saving data from and to file. Total Recall files are text files with the extension .prf. You can in principle open these files in Notepad but if you make changes it is possible that Total Recall can no longer read the file. Open When the Open button is pressed, the Open file dialog comes up where you can select a file for loading. When the loading is finished and the file contains reference points, the procedure for finding the reference points again is started.

Save Once a filename has been defined, the Save button is enabled so you can simply save the data under the same filename (to save under another filename use Save As). Save As When the Save As button is pressed the Save file dialog is brought up, allowing you to define a filename to save the data.


12 Vacuum Logger

12.1 Introduction Vacuum Logger is a program for the TEM microscope that allows logging of vacuum pressures and the high tension condition (disabled, off, on). The logging data are written to a file and, if selected by the user, displayed graphically on the screen. The graph can be printed. Logging files can be read by the program (on-line on the microscope as well as off-line) and displayed graphically. All data logged are in Pascal. The program allows a maximum number of 10000 measurement cycles.

12.2 General principles There are two modes of operation for Vacuum Logger, on-line and off-line. When starting, Vacuum Logger will attempt to connect to the TEM microscope software. If it succeeds, it will continue with on-line operation. If it fails to connect to the TEM software, it will continue with off-line operation. In the latter case, controls and menu items used for on-line operation will be removed or disabled.

12.2.1 On-line operation With on-line operation you can: • Log vacuum system pressures, high-tension status, and vacuum and gun events. • Obtain a graphical display of selected parameters against time elapsed. • Read Vacuum Logger files and display their contents graphically. • Print the graphical display.

12.2.2 Off-line operation With off-line operation you can: • Read Vacuum Logger files and display their contents graphically. • Print the graphical display.

12.3 Program structure The program contains three elements for controlling the logging and graphical display, the program menu, a panel directly underneath the menu for selection of logging parameters such as logging interval and start/stop date and time, and a panel at the bottom for controlling which elements are displayed graphically. The graphical display itself covers the area between the panels at the top and the bottom. Vacuum Logger will store and recall user's individual settings.

12.4 How to run the logging software Program Vacuum Logger is an application, not a service. This implies that you have to be logged in (to Windows NT) to run the software and that the program will be closed (and thus log no further) when you log out from Windows NT.


12.5 Menu The menu contains four main items, File, Display, Log and Help.

12.5.1 File menu New Defines a new name for the log file. For starting logging this is not strictly necessary

as Start always brings up the Save file dialog wherein the filename is defined. If New file has been done, the Save file dialog will not be displayed when Start is pressed.

Open Opens an existing log file and displays the data graphically. Selecting Open always stops any logging currently being done.

Print Prints the graphical display.

Printer setup Leads to the Printer setup dialog (e.g. for selecting Landscape printing).

Exit Exits the program.

12.5.2 Display menu Settings Brings up the Display settings dialog, which allows selection of all display

parameters.

Statusbar Shows or hides the Statusbar wherein display parameters can be defined for individual pressures or high tension.

Lock to data space

The Vacuum Logger window can be locked to the 'data' space of the TEM User Interface (the empty space left over by the user interface in its various display modes). If the display of the TEM User Interface is changed and the Vacuum Logger window is locked, the size of the Vacuum Logger window is adjusted automatically. When locked, the Vacuum Logger window cannot be resized (though it can be moved around).

12.5.3 Log menu Start/stop Starts or stops logging. If delayed action is on, the actual measurements will start

once the start date and time are past. Otherwise logging starts or stops immediately. You cannot pause logging by start-stop-start, because the second start will require a new filename (or, if you select the previous filename, the previous file is overwritten).

Delayed action When checked, delayed action is on, otherwise it is off. After selecting delayed action, you still have to start logging (but now you start the timer that looks if the start date and time have passed).

12.5.4 Help menu Show Brings up this Help file. Pressing the F1 function key is equivalent.

About Brings up the program About box with version and copyright information.


12.6 Logging parameters Logging can be done at selected intervals (1, 5, 10, 30 seconds, 1, 5, 10, 30, 60 minutes), selected from the drop-down list in the panel underneath the menu.

Logging can be started and stopped manually or in delayed-action mode. In delayed-action mode the panel contains additional controls for defining Start date and time and Stop date and time. The formatting of date and time must be exactly as indicated (a maximum of two characters for the date, then month, then year, all separated by hyphens; a maximum of two characters for the hour, then a colon, then two characters for the minutes).

Note: It is possible that the 1 second interval is not totally stable and may result in the program hanging (since the logging data are saved for each measurement the logging file will still be accessible). Especially for larger series of measurements (>200 cycles) it is advised not to use the 1 second interval. For this reason it is also not possible to display the vacuum pressures for the log interval of 1 second. If it is necessary to log at 1 second intervals, first collect the log and then read in the log file to obtain a display.

12.7 File The logging file is a text file. The file starts with a header, which contains the start date and time and a line displaying the names of all vacuum elements measured. The last item is the high tension status. The individual measurements are separated by tabs, making it easy to read the file into spreadsheet programs like Excel (read in as text file). Below come lines of three different types: • Measurement data. Each measurement is on a separate line, containing the date and time of the

measurement. The Event type (listed in the first column) is type 0. The data, separated by tabs, are in Pascal unit, except for the high tension which is 0 (disabled), 1 (off) or 2 (on).

• Vacuum status events. These are events triggered by the vacuum system itself and received by the program. These events can come in at any time (not at the specified measuring intervals) and are logged under Event type 1.

• High tension events. These are events triggered by the high tension system itself and received by the program. These events can come in at any time (not at the specified measuring intervals) and are logged under Event type 2.

The data logged are always the complete data from the microscope, not just the elements selected for display in the graph.


12.8 Display The central part of the Vacuum Logger window is reserved for a graphical display of the pressures and high-tension status. The horizontal scale is the true time of the log (thus even if the logging interval is changed during the acquisition, the values will still plot properly along the scale). • For a display of a file read from disk, the horizontal scale stretches form the start time to the end

time. • During logging, the horizontal scale will start at the start time, but the end of the scale is adjusted in

steps. Initially the program will choose an end value for the horizontal scale. Once the logging time has exceeded that value, the scale will be expanded (typically by a factor 2). Since the display width doesn't change, the display of the pressures will jump back halfway. In this way the graphs will appear to 'grow' progressively to the right as logging proceeds, with stepwise adjustments when the horizontal scale becomes too short.

Underneath the display are some parameters. In the off-line case the top line lists the start time at far left. The corresponding value on the scale is the absolute left-hand point (not a tick that may or may not be centered on the label). The far right right value is the end time of the log and once again the corresponding value is the far right-hand point of the scale. In the center is listed the interval represented by the tick marks. The tick marks are chosen such that they represent unitary (whole) values. This means that the first tick mark in the figure below coincides with the first unit value (1 hour) after the start time, in this case 2:00:00 PM (so not 1 hour after 1:21:21 PM). At the lower line is indicated the number of measurement cycles read from the log file.

In the on-line case the top line is the same as in the off-line case, showing the start and end times of the scale and the tick mark value. The lower line now shows on the left how many measurement cycles have been done, in the center when the next measurement will take place, and on the right the current system time.

12.9 Print The print from Vacuum Logger will be a close a match to the display with some modifications. • The height and width of the print-out will be matched to the paper size (Portrait or Landscape)

available, while the ratio of height and width will be the same as the display. • The background of the graph will be white. Light colors such as yellow are made somewhat darker

for better printing. It is advised to use a color printer, otherwise it may not be possible to distinguish between multiple lines in the graph.

• The printed graph has a header listing the vacuum elements displayed with their scale value. These labels are in the same colors as the lines and act as legend. If a pressure is displayed with log scale, the label is followed by '(log )' with a number indicating the exponent of the value used for the base.


12.10 Display parameters Pressures and the high-tension status can be displayed graphically in the display of Vacuum Logger. The display parameters are defined in the Statusbar underneath the display window.

The following controls are present in the Statusbar, from left to right: • A drop-down list with the settings that can be displayed. • A checkbox that determines whether a setting is displayed. • A drop-down list box with a range of scaling factors (used as maximum display values), including

Default (set by the program to the maximum value found for the particular setting). All pressures are scaled individually. The high-tension status is scaled from disabled (just above the base line) to off (center) to on (just below the top).

• The value of the maximum of the particular setting. When Default is chosen, this is the maximum value of the display scale.

• A checkbox to select between regular and log scaling. • A drop-down list box with a selection of colors.

12.10.1 Display settings dialog In addition to selection of display parameters in the Statusbar, it is also possible to bring up the Display settings dialog, which contains controls for defining the display parameters for all pressures and high tension together.

12.10.2 Log scale Because of the large range available to the vacuum pressures (from air pressure to values in the range of 10-6 Pascal), it can be difficult to display a pressure graphically such that fine detail as well as the whole range is visible. Because of this, pressures can be displayed on a log scale.


Since a log scale in principle can go to very small numbers, the minimum of the log scale is not 0 (as it is for the regular scale). Instead the minimum of the log scale is the truncated exponent of the smallest value of the particular pressure. For example, if the pressures range from 99.9 to 1.1, the truncated log value of 1.1 = 0 will be minimum of the display scale. While a pressure ranging from 3.33e-2 to 1.2e-5 will have as minimum the truncated log value of 1.2e-5 = -5. The maximum value is as defined by the user (Default or one of the fixed values). There is no fixed recipe for determining whether a log scale is useful or not, because it will depend on the pressure range as well as the absolute values. If the pressure range is within a factor 10, a normal scale is more useful. Similarly, a small pressure range can also lead to compression of the graph at the top of the display, for example when there are a few values just inside a lower decade.

The figures above show an example of the effect of plotting a vacuum pressure on a log scale. The regular scale (on the left) is dominated totally by the 'spike' of high pressure and the fine details of the pressure changes are invisible (unless the scale is modified so the 'spike' goes far off the maximum scale. In the log scale display on the right, the fine details are well visible while the 'spike' is still within the whole vertical range.

Tecnai on-line help Modes 1 Tecnai 12 to Tecnai F30 Software version 4.0

Tecnai on-line help manual -- Modes Table of Contents 1 Standard operating procedures........................................................................................................... 3

1.1 Aperture centering........................................................................................................................ 3 1.1.1 Condenser aperture .............................................................................................................. 3 1.1.2 Contrast aperture .................................................................................................................. 3 1.1.3 Diffraction aperture ............................................................................................................... 4

1.2 Setting the eucentric height.......................................................................................................... 5 1.3 Focusing....................................................................................................................................... 6

1.3.1 Wobbler................................................................................................................................. 6 1.3.2 Focused beam ...................................................................................................................... 8 1.3.3 Contrast-enhancement ......................................................................................................... 8 1.3.4 Diffraction focus .................................................................................................................... 8 1.3.5 Small screen and binoculars................................................................................................. 9

1.4 Correction of astigmatism ............................................................................................................ 9 1.4.1 Condenser stigmation ......................................................................................................... 10 1.4.2 Image stigmation................................................................................................................. 10 1.4.3 Diffraction stigmation .......................................................................................................... 17

2 Calibration procedures ...................................................................................................................... 18 2.1 Magnification calibration............................................................................................................. 18 2.2 Camera-length calibration .......................................................................................................... 18 2.3 Image-diffraction rotation angle.................................................................................................. 19

3 Bright-field imaging............................................................................................................................ 20 3.1 Standard method........................................................................................................................ 20 3.2 Working with magnetic specimens............................................................................................. 20

3.2.1 Counteracting the magnetism of the specimen................................................................... 20 3.2.2 Lorentz microscopy.............................................................................................................21

4 Dark-field imaging ............................................................................................................................. 21 4.1 Axial dark-field imaging .............................................................................................................. 21 4.2 Off-axis imaging ......................................................................................................................... 22

5 Diffraction .......................................................................................................................................... 23 5.1 Focusing in diffraction ................................................................................................................ 23 5.2 The shadow image..................................................................................................................... 24 5.3 Selected-area diffraction ............................................................................................................ 25 5.4 Diffraction without selected-area aperture ................................................................................. 26 5.5 Standard convergent-beam diffraction ....................................................................................... 27

5.5.1 Orienting the crystal ............................................................................................................ 27 5.5.2 Fine-tuning pattern.............................................................................................................. 27

5.6 Large-angle convergent beam (LACBED/Tanaka)..................................................................... 27 6 Analysis ............................................................................................................................................. 29

6.1 EDX analysis.............................................................................................................................. 29 6.1.1 Microprobe versus nanoprobe ............................................................................................ 29 6.1.2 Shadowing angle ................................................................................................................29

6.2 EELS analysis ............................................................................................................................ 30 6.2.1 Determining spectrometer acceptance angle β................................................................... 30

7 STEM ................................................................................................................................................ 32 7.1 STEM principles ......................................................................................................................... 32 7.2 The importance of the pivot points .............................................................................................33 7.3 Detectors.................................................................................................................................... 34


7.3.1 Bright-Field (BF) detector.................................................................................................... 34 7.3.2 Dark-Field (DF) detector ..................................................................................................... 35 7.3.3 High-Angle Annular Dark-Field (HAADF) detector.............................................................. 35 7.3.4 Secondary-Electron (SE) detector ...................................................................................... 35 7.3.5 BackScattered-electron (BS) detector................................................................................. 36 7.3.6 Energy-Dispersive X-ray (EDX) detector ............................................................................ 36 7.3.7 Electron Energy-Loss Spectrometer (EELS)....................................................................... 36

7.4 Detector alignment ..................................................................................................................... 36 7.5 STEM adjustments - an in-depth explanation ............................................................................ 37 7.6 STEM Operation ........................................................................................................................ 37

7.6.1 Tem Imaging & Analysis ..................................................................................................... 37 7.6.2 Resolution and frame size .................................................................................................. 38 7.6.3 Detectors and channels ...................................................................................................... 39 7.6.4 Detector control................................................................................................................... 39 7.6.5 Procedure for manual setting of detector contrast and brightness...................................... 40 7.6.6 Filters .................................................................................................................................. 41

8 EFTEM .............................................................................................................................................. 42 8.1 General introduction to energy filtering ...................................................................................... 42 8.2 The (post-column) Imaging Filter ...............................................................................................42 8.3 EFTEM on the Tecnai microscope............................................................................................. 43 8.4 Important concepts in Tecnai EFTEM........................................................................................ 43

8.4.1 Image, diffraction pattern and spectrum ............................................................................. 43 8.4.2 Cross-over correction..........................................................................................................44 8.4.3 Normalizations .................................................................................................................... 44 8.4.4 SA diffraction....................................................................................................................... 45


1 Standard operating procedures

1.1 Aperture centering

1.1.1 Condenser aperture The condenser aperture is the illuminating-beam limiting aperture in the column. The alignment of the aperture is important for two aspects: 1. For convenience, to make the expanding (defocused) and contracting (focused) beam stay centered

on the screen (thereby making it unnecessary to adjust the beam position continuously). 2. To ensure reproducible illuminating conditions. Misalignment of the condenser aperture leads to a

beam tilt (and thus a change in the rotation center). In principle, it is possible to adjust the rotation center whenever the aperture is changed. In practice, it is much easier to make sure the aperture centering is reproducible - and thus the rotation center stays the same.

Note 1: Because of the latter aspect, it is much more important to align the aperture in a reproducible manner (always following the same procedure; that is, use the same magnification and defocus the beam by the same amount each time) than having a ‘perfectly’ aligned aperture (by whatever criterion). Procedure • Select a suitable magnification (the choice is up to user, but select the same one each time). • Focus the illumination using the Intensity knob and center the beam, as necessary, using the beam

shift (left-hand track ball). • Defocus the illumination clockwise (overfocus) by a set amount (e.g. to the 4 centimeter circle on the

viewing screen). The illuminated area should still be around the center of the screen. If not, align the condenser aperture using the mechanical aperture controls to achieve this condition.

• Repeat the last two steps until the illuminated area remains centered. Note 2: There typically is a difference between the aligned condenser aperture in the microprobe and nanoprobe modes (requiring adjustment when switching between these modes). This mechanical misalignment is a consequence of the difficulty of mechanical alignment of the minicondenser lens. Generally, the misalignment is readily apparent when changing from one mode to another. However, when switching to STEM (in essence a nanoprobe mode) from microprobe, this misalignment may not be apparent. It is advised, therefore, to proceed via nanoprobe (for centring the aperture) when going to STEM.

1.1.2 Contrast aperture The contrast aperture eliminates diffracted beams (and, depending on the aperture size, energy-loss electrons) from the beam, thereby giving contrast in the image. In the high-magnification range, the objective aperture (generally located at the level of the diffraction pattern) is the contrast-forming aperture. In the low-magnification range, the objective lens is (nearly) off and the diffraction pattern is located at the selected-area aperture. The latter thus is the contrast-forming aperture for the low-magnification range (it is, however, rarely used in the low-magnification range). High-magnification range – objective aperture When the objective aperture is used, a number of diffracted electrons is scattered back and may interacts with the specimen. These backscattered electrons counteract the losses of secondary electrons and thereby reduce or eliminate charging of the specimen. The latter can have disastrous effects on poorly conducting specimens like biological ones, where the charging is seen as a ‘blowing’ up (in appearance like a balloon) of the specimen, after which the specimen often blows apart. Keeping the


objective aperture in is one way of preventing the destruction of biological specimens. Another method is to keep part of the illumination on a grid bar. The latter method is dangerous, however, because the specimen with blow up as soon as the beam is no longer on the grid bar (e.g. when the specimen or beam is moved). Neither method can be used for EDX analysis (which requires that the objective aperture is removed; and flooding the EDX detector with X rays from the grid bar also will not be conducive to good analysis) and there a properly conducting specimen is required (carbon coating or put on a conducting carbon film). In less strongly insulating specimen, charging may appear as a shivering or repeated jumping of the specimen. It may be possible to reduce the charging by selecting a smaller beam (lower total current) and/or smaller objective aperture. Once again, a carbon coating may prevent charging altogether. Procedure 1 • Select a suitable magnification (as required by the type of image, e.g. 5kx to 20kx for intermediate-

magnification work or 100kx for high-resolution work). • Set proper illuminating conditions (beam defocused – overfocus, i.e. clockwise with intensity – to

illuminate the whole viewing screen or just beyond the rim of the screen). • Switch to diffraction. • Select a camera length of approximately 500 mm. • Insert the required objective aperture into the beam and center it around the central beam spot using

the mechanical aperture controls. Procedure 2 • Insert the objective aperture. • If no bright-field image is visible at all, it may be necessary to use procedure 1 for rough centering

first. Otherwise, shift the aperture until there is no cut-off of the illuminated area visible (if necessary defocus/focus the beam).

Low-magnification range – selected-area aperture Procedure • Select a suitable magnification (around 500x). • Set proper illuminating conditions (beam defocused – overfocus, i.e. clockwise with intensity – to

illuminate the whole viewing screen or just beyond the rim of the screen). • Switch to diffraction (LAD). • Select a low camera length (typically the fourth of the LAD range). • Insert the largest selected-area aperture into the beam and center it around the central beam spot

using the mechanical aperture controls.

1.1.3 Diffraction aperture Diffraction can be done with area selection by means of an aperture (selected-area diffraction) or simply by illuminating an area with the beam (typically convergent beam diffraction with a focused beam though it is not strictly necessary to focus the beam completely). High-magnification range – selected-area aperture In the high-magnification range, the selected-area (SA) aperture acts as the area-selection tool for diffraction work. In the SA magnification range, the first intermediate image coincides with the level of the SA aperture. This range is therefore the optimum for high-quality selected-area diffraction work. The Mi (between LM and SA) and Mh ranges have their first intermediate images below and above the SA aperture level, respectively, and are therefore not as well suited as starting point for SA diffraction (but SA diffraction is very well possible from these ranges).


Note 1: The Tecnai microscope uses (small) image shifts between different magnification steps to align the different magnifications (the image stays centered when the magnification is changed). Because this alignment is executed with the image shift deflection coils, located between the objective lens and the SA aperture, it is impossible to shift both the image and shadow of the SA aperture at the same time (the shift takes place between them). The consequence of the image alignment is therefore an apparent shift of the SA aperture when the magnification is changed. Always select the appropriate magnification first and then insert and center the SA aperture. Note 2: If the diffraction shift pivot point is not aligned properly, changing the diffraction shift (either intentionally by the user or simply because the camera length is changed and the new camera length has a different alignment) will lead to an image shift as well. Since this image shift takes place above the SA aperture, the specimen area will move out of the aperture and the diffraction pattern will come form a different area than the one intended. It is therefore important to ensure that the pivot points of the image coils (the image and - especially – the diffraction shift pivot points) are aligned properly. Because the diffraction shift pivot point is very sensitive to the objective-lens current, it is also important to make sure that the specimen is at the eucentric height (additionally the eucentric height is important for accurate values of camera length and magnification). Procedure • Obtain an image at a suitable magnification. • Insert the SA aperture and center it on the area of interest (by preference near the screen center)

using the mechanical aperture controls. • If no aperture is visible upon insertion, reduce the magnification or select and center a larger aperture

first before continuing on to the aperture with the required size. Note: If the objective aperture is smaller than the central beam, use either a smaller C2 aperture, defocus the beam with Intensity, or use a larger objective aperture. Low-magnification range – objective aperture Procedure • Obtain an image at a suitable magnification. • Insert the objective aperture and center it on the area of interest (by preference near the screen

center) using the mechanical aperture controls. • If no aperture is visible upon insertion, reduce the magnification or select and center a larger aperture

first before continuing on to the aperture with the required size.

1.2 Setting the eucentric height The eucentric height is important in the microscope. It is not only convenient that the area of interest stays centered when tilting around the α tilt axis (but if you never tilt anyway, this feature may be of little interest), but it also defines a reference value for the objective-lens current. The normal changes made to the objective for focusing have little effect, but stronger changes (changes in focus by several tens of micrometers or more) can have considerable effect on: • The effective magnifications and camera lengths (the objective lens not only focuses the image but

also contributes the largest magnification of any lens in the system; strong changes in objective lens current change this magnification and thereby also the final magnification and camera length).

• Proper alignments.


There are several methods for setting the eucentric height. Procedure 1 : the α wobbler • Activate the a wobbler of the CompuStage, typically using the maximum tilt angle available (15° on

all instruments except U-TWIN instruments where the maximum is 5°). For magnetic specimen is may be advisable to use a smaller angle (because of the effect of the specimen’s magnetism on the objective-lens field).

• Minimize the sideways motion of the image with the Z axis height control. • Switch the α wobbler off. Procedure 2 : the focus wobbler • Press the Eucentric focus button to set the objective lens to the eucentric height preset (this method

presumes the microscope has been aligned properly and this preset has been set). • Press the Wobbler button and minimize the distance between the two apparent images with Z axis

height control. • When done, switch the wobbler off. Procedure 3 : minimize the α tilt displacement • Reset the α tilt to 0° (the Stage Control in the Stage Control Panel flap-out has a function for this). • Find and center an easily recognizable feature in the specimen. • Tilt the stage with the α tilt by a small amount (~5°). • If the feature moved away from center, move it back with the Z axis height control. • Reset the α tilt to 0° again and repeat until the feature doesn’t move when the α tilt is used.

1.3 Focusing There are many ways of focusing the image. Some of the more common ones are described below. One simple tool always available is the Eucentric focus function which works both in image and diffraction. In image the Eucentric focus sets the focus to the value for the eucentric height. Assuming that the specimen is at the eucentric height, this eucentric focus function provides a quick method for getting close to the proper focus. The actual value set is set in the various alignment procedures (HM Image, LM Image, Nanoprobe Image). In diffraction, the Eucentric focus function resets the user-defined focus to zero. When properly aligned (by means of the Camera Length Focus in the HM Image procedure), this effectively means that the focus is set to the back-focal plane (see section 1.3.4).

1.3.1 Wobbler The function of the wobbler is to deflect the beam alternately to either side of the optical axis. When the objective lens is focused exactly on the specimen plane, no change in the image is apparent. However, when the objective lens is focused above or below the specimen plane there is an apparent double image so the wobbler is very useful for emphasizing focusing errors. The direction of the wobbler effect should be selected perpendicular to the direction of the structures to be focused. This is adjusted using the Multifunction-Y knob. The amplitude of the wobbler effect is adjusted using the Multifunction-X knob. This angle should be adapted to the size of the objective lens aperture otherwise loss of intensity will occur when the wobbler is operated (if the beam tilt become too high the transmitted beam is stopped by the objective aperture).


The procedure is as follows: • Insert a specimen, adjust its height and focus the image. • Press Diffraction pushbutton (LED on). • Select a camera length of approximately 600 mm. • Focus the illumination using the Intensity knob. • Ensure that the objective aperture is correctly centered. • Press the Wobbler pushbutton: two spots should appear within the area of the objective aperture. If

not, lower the wobbler amplitude until they do. • Press Diffraction pushbutton (LED off) and focus the image to minimum blur. • When finished, press the Wobbler button once again to switch it off.

Focus without use of the wobbler. Focus after use of the wobbler.

Out of focus - wobbler in chosen direction. Out of focus - wobbler perpendicular to c.


1.3.2 Focused beam When the beam is focused on the specimen (or slightly defocused), the beam should look exactly the same as when it passes through a hole in the specimen. If the beam has a diffuse halo around it, the specimen is not in focus. Focus to minimize the halo (it will merge with the beam when the specimen is in focus). In crystalline specimens a diffraction pattern may also appear superimposed on the image. Once again, focus is attained when the diffraction is minimized. This method is especially useful in the nanoprobe mode or without an objective aperture in. Note: The appearance of multiple images (one from the transmitted beam and other ones from strong diffracted beams) is a consequence of the spherical aberration of the objective lens (and the absence of an aperture to block the diffracted beams). Even at true focus, these multiple images may remain visible. In general this aspect is not troublesome, except in the low-magnification range. When the objective lens is (nearly) off, the diffraction lens is used as the ‘objective’ lens. When used in this way, the diffraction lens has a very long focal length (hence the very strong contrast in the LM image) and also high spherical aberration. Because of the latter, the multiple images may be pronounced, making it very difficult to focus (the images will not merge together, even at focus).

1.3.3 Contrast-enhancement In some cases (especially in biology) it is advisable to set the focus deliberately a certain amount underfocus to enhance contrast. The amount of underfocus set depends on the magnification (at high magnification an underfocus image looks blurry while the same amount of underfocus at lower magnifications may look sharp) and the degree of contrast enhancement required.

1.3.4 Diffraction focus Unlike the image, the diffraction pattern does not have a clear criterion for establishing when it is in focus. Often it is presumed to be 'in focus' when the pattern has spots that are as small as possible. This is not strictly true. The diffraction pattern is in focus when the focus lies at the back-focal plane of the objective lens. Only when the incident beam is parallel does the cross-over lie in the back-focal plane.

With a parallel beam incident on the specimen (grey), the objective lens focuses the electrons into a cross-over whose position coincides with the back-focal plane (and thus the true diffraction focus).

When the beam is convergent (but not wholly focused), there still is a cross-over but it is displaced from the back-focal plane upwards (in the extreme case, a fully focused beam, the cross-over lies at the image plane).


When the beam is divergent, the cross-over is displaced downward from the back-focal plane.

If the diffraction pattern is not focused properly, there are a number of consequences: • the camera length can be wrong • the diffraction will be rotated away from its proper orientation • the pattern may be distorted • alignments such as beam shift pivot points can be wrong • the scanning magnification can be wrong due to misaligned pivot points Due to the absence of a clear criterion, we end up with a chicken-and-egg situation (what was first, the chicken or the egg?). For example, if it can be assumed that the shift pivot points are correct, then it is easy to establish the correct diffraction focus by wobbling a beam shift and minimising diffraction-pattern movement. However, the pivot points can only be aligned correctly if the diffraction pattern is focused properly. In order to resolve this situation, we have determined Intensity settings for the different modes (LM, HM-TEM and Nanoprobe) for a parallel beam. These Intensity settings are preset in the alignment procedures, making it easy to find diffraction focus (the spot-pattern condition). After the alignment have been done (camera length focus), the diffraction focus can also be found by simply pressing the Eucentric focus button (this resets the variable diffraction focus to zero). With this method for establishing diffraction focus, the SA aperture is not (and should not be) used.

1.3.5 Small screen and binoculars The binoculars can be used over the whole magnification range and for many different types of images (e.g. bright field, dark field, diffraction) as an aid to more accurate observation and focusing. They give a magnification of 12x and are used together with the small screen as follows: • Introduce the small screen fully into the beam. • Adjust the distance between the eyepieces for maximum comfort. • Focus the binoculars on the small screen making use of the eyepiece adjustment controls. As an aid

to focusing the binoculars, insert the beam stop. When its shadow is sharp, the binoculars are focused and the stop can be removed.

Note: Focusing must be carried out for each eye separately and the eyes should be focused at infinity, fully relaxed.

1.4 Correction of astigmatism Astigmatism is an aberration which is present in all electromagnetic lenses. It is caused by asymmetry of the lens field which can result from inherent asymmetries or from asymmetrical charges on regions close to the beam, e.g. the specimen.


1.4.1 Condenser stigmation • Make sure that the image is focused and reasonably well stigmated (it is not very critical, but if the

image stigmation is much off, the beam may appear astigmatic due to the image astigmatism). • Remove the specimen and the objective aperture from the beam. • In the microprobe mode (LM, HM) select a workset tab containing the Stigmator Control panel and

press the Condenser stigmator button (green LED on). In Nanoprobe or STEM, the same can be done, but it is also sufficient to press the Stigmator button on the left-hand Control Pad (the condenser stigmator is the default stigmator in these modes).

• Astigmatism is corrected when the focused beam remains as circular as possible when going through beam focus (Intensity). Adjust this using the Multifunction-X and Y control knobs. Alternatively, the filament can be undersaturated until structure is visible in the focused beam. Astigmatism is corrected when this structure is as sharp as possible (adjust Intensity, Multifunction-X and Y).

1.4.2 Image stigmation Three factors can cause image astigmatism: 1. Asymmetry of the objective lens. 2. Dirt on or charging of the objective aperture. 3. The specimen itself. The influence of the specimen on the observed astigmatism can be

considerable, particularly in cases where an insulating specimen collects charge, either as a whole or locally. Magnetic specimens also cause strong astigmatism.

High-magnification range Astigmatism is most easily observed on the screen when viewing Fresnel fringes. These fringes result from diffraction phenomena that occur at sharp edges of a specimen when the objective lens is slightly underfocused or overfocused. When the image is underfocused (objective lens weaker than focus), the Fresnel fringe appears as a bright line round the edge of the detail selected. If the detail is a hole, the line will appear on the inside. When the image is overfocused (objective lens stronger than focus), the Fresnel fringe appears as a dark line but otherwise has the same characteristics as in the underfocused condition. With a perfectly symmetrical objective lens field, the fringes will be of uniform width. With an asymmetrical (astigmatic) objective lens, the fringes will also be asymmetrical and, close to the focus, part of the hole will have a bright fringe and the other part a dark fringe associated with it. For more information about astigmatism in electron lenses, reference is made to the many text books on electron optics, for example: Transmission Electron Microscopy (2ed.), L. Reimer (1989), Springer-Verlag, Berlin. Experimental High Resolution Electron Microscopy (2ed.), J. C. H. Spence, Oxford University Press, New York, Oxford. A typical test specimen for measurement and correction of astigmatism is a very thin carbon support film with small perforations. This film must be of a conducting material, because of the high magnifications and thus high beam intensities that will be used and great care should be taken to ensure that the film adheres firmly to the supporting grid. Very small spherical particles can also be used, but this is not advisable because of the possibility that the projected periphery of the particles may not originate from a single plane. In that case, there will be an inherent change of focus around the particle which cannot be distinguished from astigmatism and will thus be corrected when correcting the astigmatism.


In general, accurate correction of astigmatism can be made only at high magnification and under good image visibility conditions. This implies high beam intensity on the specimen. In order to compensate for objective lens astigmatism applicable to the M and SA magnification range, an electromagnetic astigmatism corrector is built into the microscope below the objective lens. There are two possible methods for correcting the astigmatism. In the first, use is made of the special test specimen described above. In the second, use is made of the fact that, at high magnification, all thin objects have a substructure in the size range 0.3-0.2 nm and use is then made of the point/focal line phenomena. The latter method is preferred because of the influence of the specimen itself, causing astigmatism to vary for different areas of the specimen. The method requires some experience, however, and it is recommended that this experience be gained, where necessary, by comparing the effect of the two methods on the special test specimen. Method 1 This method is illustrated in the figure which shows two focal series with astigmatism uncorrected (left-hand side, figure a to c) and corrected (right-hand side, figure d to f). The procedure is as follows: • Obtain a TEM BF image of the test specimen at high magnification (around 100 000x). • Press the Stigmator pushbutton (LED illuminated). • Select a very small hole. This should be of such a size that it is visible in its entirety through the

binoculars at the highest magnification used. • Adjust the Focus until the entire hole is overfocused yet close enough to focus for the fringe

asymmetry to be visible (black fringe inside the hole, Fig. a). Change of focus (lower excitation of the objective lens) produces the images in Figs. b and c.

• Adjust the Multifunction knobs so that the Fresnel fringe is symmetrical when the objective lens is very slightly overfocused.

• To perform this procedure, start adjustment by turning one of the Multifunction knobs until the setting for minimum astigmatism is obtained (best symmetry for overfocused image). Then adjust the other knob for minimum astigmatism.

• Repeat the preceding step at a higher magnification and with smaller focusing step sizes until adequate correction is obtained (Fig. d). The criterion for this is that no asymmetry of the fringe can be seen at one or two step positions overfocus of the finest step size. Change of focus (lower excitation of the objective lens) gives rise to the images in Figs. e and f.


Image astigmatism correction (Method 1). Magnification 100 000x.


Method 2 This method is shown in the figure below which shows two focal series with astigmatism uncorrected (left-hand side, figure a to c) and corrected (right-hand side, figure d to f). The procedure for correction of the astigmatism on the specimen sub-structure is as follows: • Select and center a small C2 aperture (50 um). • Set the magnification to a high value (about 200 000x). • Set the spot size to step 3 and reduce the Intensity as far as possible to be consistent with good

visibility of the structure (the contrast of the substructure disappears if the beam is focused too much).

• Select a thin part of the specimen showing fine structure. • Select focus step 2 and with the focus knob, vary the objective lens from slightly overfocused to

slightly underfocused (total change in focal length less than 1 um). • Look for the line foci (Figs. a, b and c). Set the Focus knob halfway between the settings for these

two foci (Fig. b). • Press the Stigmator pushbutton (LED illuminated). • Adjust the two Multifunction knobs one at a time to decrease the apparent size of the background

structure and at the same time reduce the line effect (Fig. d, e and f) on changing to over and underfocus.

• Repeat the preceding step (at a higher magnification if desired) until the focal distance between the line foci is as small as required (3 nm or even smaller is possible).

Note: With very thin specimens the substructure disappears from the visual image at focus. This can be used as a very sensitive check on the final correction. The two Multifunction knobs are then used to reduce the contrast of the substructure until it finally disappears at focus.


Image astigmatism correction (Method 2). Magnification 300 000x.


LM magnification range When operating in the LM mode, it is possible that some astigmatism will be observed at the higher magnifications. If it is required to make photographs in this mode, astigmatism in the image can be corrected by one of the following methods. Note: The (default) stigmator used in LM is the diffraction stigmator. Method 1 This method is the same as method 1 for the high-magnification range. • Insert a test specimen (as described before under method 1 for the high-magnification range). • Press the Stigmator pushbutton (LED illuminated). • Insert and center the second-largest SA aperture. • Select the highest LM magnification. At this stage, Fresnel diffraction fringes should be observed

around the inside of the holes in the specimen. • If these fringes are not symmetrical, correct the astigmatism using the Multifunction knobs. • Adjust the Focus until the entire hole is slightly overfocused, yet close enough to focus for the fringe

asymmetry to be visible (black fringe inside hole). • Adjust the Multifunction knobs one at a time so that the Fresnel fringe is symmetrical when the image

is very slightly overfocused. This is achieved by turning the two Multifunction knobs until a setting for minimum astigmatism is obtained.

Method 2 This method is illustrated in the figure below showing: a : An overfocused image with asymmetrical Fresnel fringes indicating astigmatism b, c, d : A through-focus series with wobbler in use with astigmatism uncorrected. e, f, g : A through-focus series with wobbler in use with astigmatism corrected. h : A corrected, focused image, wobbler not in use. • Ensure that a platinum aperture not smaller than 150 mm is mounted in the selected-area aperture

holder and that it is clean. • Insert a specimen and center a suitable detail. • Center the selected-area aperture. • Press the Stigmator pushbutton (LED illuminated). • Set the magnification to the highest LM value. • Press the Wobbler pushbutton. • Focus the image so that the blurred image details (Fig. c) are as nearly coincident as possible. • Adjust the Multifunction knobs until the image details are coincident (Fig. f). • Repeat the last two steps.


Image astigmatism correction for LM mode.


1.4.3 Diffraction stigmation It is possible that some astigmatism will be observed in the diffraction pattern when operating in the diffraction mode. This astigmatism can be corrected by making the crossover image of the diffraction lens symmetrical. In this case the Multifunction knob functions are connected to the diffraction lens stigmator. This correction should be made for the camera length being used. • Obtain a TEM BF image of a specimen in the SA magnification range. • Remove specimen and objective aperture from the beam. • Press D button and select the required camera length. • Select spot size 4. • Turn Intensity knob until a low illumination intensity is obtained on the screen. • Adjust the diffraction focus until the diffraction crossover image is obtained (Fig. a below). • Press the stigmator pushbutton (LED illuminated). • Adjust the Multifunction knobs until a symmetrical 3-pointed image is obtained (Fig. b below). • Press the Eucentric focus button to reset the diffraction focus to its proper value.


2 Calibration procedures In normal operation (i.e. with the specimen in the eucentric position), the accuracy of magnification and camera length will be within ±5% of that indicated on the display. To obtain more accurate values, a calibration curve must be made for the microscope using the normalisation facility for each magnification and camera length value. The normalisation facility takes the excitations of all magnifying lenses through a hysteresis cycle, making lens hysteresis conditions consistent and therefore reproducible. Once such a calibration has been carried out, reproducibility to within 1.5% of the calibrated value is obtained. Normalisation is obtained by operation of the projector normalisation procedure (selectable under the User button functions).

2.1 Magnification calibration The calibration procedure can be carried out over the whole range (LM, Mi, SA, Mh). A suitable method is by taking photographs or CCD images of standards over the following magnification ranges: • 50x to 1500x : Grid with a mesh of the order of 1500. • 1500x to 100 000x : Reliable diffraction grating replica. • >100 000x : It is necessary to determine the ratio of the distances between the same two particles (or

points) on two photographs taken at two different magnification settings. In this way it is possible to extrapolate from an accurately calibrated low magnification to the top of the range. Alternatively, obtain high-resolution micrographs with a known lattice spacing.

Important notes: • Ensure that the grating replica has first been checked by light microscopy. • Ensure that the specimen is in the eucentric position. • Begin the calibrations at a very low magnification to enable the elimination of random faults in the

grating. • Do not use a grid opening in which tears occur. • At the intermediate magnifications, check the spacings of the actual lines used against the same

lines in the lower magnifications, again to eliminate random errors. • Do not attempt an accurate calibration with less than 5 lines and only then if they can be checked as

described in the previous point above. • At higher magnifications, calculate the ratios between steps of the magnification range by

photographing two particles at the two magnifications and comparing their separations, or use high-resolution micrographs of a specimen with a known lattice spacing. If available, an FFT may provide an easier and more accurate method of estimating the magnification (by measuring diffraction spacings) than counting lines in the image.

• Ensure that, at each step of the calibration, the normalisation has been executed.

2.2 Camera-length calibration The calibration procedure can be carried out over the whole range (LAD, D). A suitable method is by taking photographs or CCD images of diffraction patterns of standards over the following camera length ranges: • LAD : Grid with a mesh of the order of 1500. Look for the lattice spots of the grid itself. • D : Diffraction grating replica with gold islands or diffraction standard (such as polycrystalline

aluminium).


2.3 Image-diffraction rotation angle The rotation-free series of the Tecnai microscopes result for the majority of magnification and camera lengths in a fixed rotation angle between image and diffraction (dependent on the type of objective lens). The rotation angle is typically not zero (because that would result in a very restricted set of camera lengths available) but 60 or 90°. There may be a few camera lengths and images that do rotate (once again to make these available), typically at the extremes of magnification- and camera-length ranges (especially for energy-filter series). Calibration of the image-diffraction rotation angle can be done by exposure of images and diffraction patterns. A good test specimen for this is molybdenum trioxide crystals on a carbon film. Molybdenum trioxide is (pseudo)orthorhombic with lattice parameters a 0.397 nm, b 1.385 nm and c 0.370 nm. It forms flat crystals, usually lying on [010] that are commonly elongated with the long edges parallel to [001]. From a double-exposure (or overlay) of image and diffraction pattern the rotation angle can be established. To check for 180° inversion, make the diffraction pattern from a relatively thick crystal near one of the points. In the diffraction pattern diffuse intensity will show an 'image' of this point (with the intensities of the diffraction spots also outlining the point. From the direction of the point, it can then be seen whether there is 180° inversion or not.


3 Bright-field imaging

3.1 Standard method Bright-field imaging is the standard method for the TEM. It simply means making an image with the transmitted beam only. To do so, switch to diffraction and insert and center the objective aperture around the transmitted beam. The aperture selected should be small enough to block all diffracted beams on crystalline specimens. On non-crystalline (biological) specimens, the size of the aperture determines the contrast (smaller apertures give better contrast).

3.2 Working with magnetic specimens The field from magnetic specimens affects the field of the objective lens itself. There are a number of consequences: • The rotation center (beam tilt) is affected by specimen position and tilt. • The beam and image astigmatism is affected by specimen position and tilt. • The beam position may be affected by specimen tilt (often flopping around abruptly when the tilt goes

through 0°). In STEM this may have the result that it is impossible to obtain a normal image (strong distortions).

It should be noted that the microscope is equipped with a objective stigmator with a large enough range to allow astigmatism correction of even quite strongly magnetic specimens. The astigmatism correction comes at a price, however. Because the stigmator is not located at the level where the astigmatism is caused (this would be impossible to do construction-wise), the astigmatism correction results in a distortion of the image (one direction is shortened relative to another). This effect is readily observed in high-resolution images (especially of polycrystalline specimens, where the diffraction rings become elliptical). Note: Never use the single-tilt holder with magnetic specimens. The objective-lens field is strong enough to rip the specimen out from underneath the clip and leave it stuck on a pole piece.

3.2.1 Counteracting the magnetism of the specimen In general, prevention is better than a cure and it always pays in convenience and often image quality to spend extra attention in specimen preparation to minimize the actual amount of magnetic material introduced into the microscope (e.g. grind metal disks to a small thickness before jet-polishing so as to keep the amount of material small and keep the disk symmetric, or cut smaller disks (1 to 1.5 mm) and glue them on copper rings). Because of the strong variability of the rotation center with magnetic specimens, it is often easier to use the dark-field tilt to align the rotation center. The following procedure provides a rapid method that leaves the normal microscope alignment unaffected. • With a normal (non-magnetic) specimen set the rotation center. • Switch to diffraction for a selected camera length (e.g. the one closest to 500mm). • Use the normalisation function and accurately center the diffraction pattern on the screen. • Insert the magnetic specimen. • Find a suitable area to start working. • Switch to diffraction, select the same camera length as before and use the normalisation function. • Switch on the dark-field tilt (press the Dark field button) and use the Multifunction knobs to center the

diffraction pattern on the screen. • The incident beam is now parallel to the rotation center without the magnetic specimen. Insert and

center the objective aperture around the beam and go back to imaging. Stay in dark field! • Whenever significant stage-position changes are made, check the beam tilt in diffraction again.


3.2.2 Lorentz microscopy In the microscope it is also possible to observe the magnetic structure of the specimen. In order to do so (in a microscope not equipped with the special Lorenz lens) execute the following before inserting the specimen into the microscope (otherwise the magnetic structure may be changed or even damaged beyond recovery by the objective-lens field): • Switch the microscope to the LM magnification range. • Switch to diffraction (LAD). • Turn the Focus knob anti-clockwise until it beeps (this sets the objective-lens current to zero). • If necessary, go to the HM-Beam alignment procedure and switch the Minicondenser lens (display its

setting in the status display and turn the lens setting to zero). The Minicondenser lens has a small leak field into the objective-lens gap.

• Insert the specimen holder (not the single-tilt holder!) with the specimen. • Find a suitable area of the specimen and go under- or overfocus. The magnetic structure of the

specimen will now become visible as bright and dark lines. • Make sure you stay in the LM magnification range.

4 Dark-field imaging Dark-field imaging means that the image is made by allowing one (or more) diffracted beams through the objective aperture and blocking the transmitted beam. The advantage of the dark-field image is its inherent high and diffraction-selective contrast.

The origin of the high contrast is shown schematically in the figure to the left. Bright-field as well as dark-field images display changes in intensity across the image. In both cases the total range of intensities is roughly similar. In bright-field images, however, the changes in intensity come on top of a high and unvarying signal – the undiffracted electrons. If one attempts to expose for a longer time to improve the signal, the negative becomes overexposed. In the case of the dark-field the background signal is much lower, leading to a much higher contrast in the image.

Note: With negatives the inherent lower contrast of the bright-field image is inescapable. With slow-scan CCD images it is however possible to subtract the uniform background from the image and stretch the contrast. Two different methods can be used for dark field imaging: 1. Off-axis imaging by aligning the objective aperture around the diffraction spot of interest. 2. Axial dark field imaging by tilting the incident beam so that the diffracted beam passes through the

objective aperture along the microscope axis. The axial dark field method is preferred because of the higher image quality obtained with on-axis imaging and ease of use through simple change-over between bright field and dark field.

4.1 Axial dark-field imaging Before activating dark field, execute the following procedure: • Center the beam. • Press Dark-field while in TEM mode in the SA magnification range. • Set the dark-field tilts to 0.00 by pressing Reset. The corresponding dark field beam shift (a value

relative to the bright-field beam shift) will also be set to zero.


Obtaining a dark field image: • In TEM BF mode, select required field of view in the SA magnification range. • Obtain a diffraction pattern of the chosen area. • Center the diffraction pattern on the screen with the diffraction shift. • Decide from which Bragg reflection (or section of a polycrystalline ring) a dark field image is to be

obtained. • Press the Dark-field button (green LED on). • Use Multifunction X and Y knobs to bring the Bragg reflection opposite to the one selected to the

point where the central beam was originally positioned (this should be the center of the screen). • Key Dark-field to return to Bright Field Diffraction mode. • Introduce an objective aperture and center it accurately around the central spot. • Key Dark-field to return to Dark Field Diffraction mode. • Center the chosen diffraction spot accurately in the objective aperture using the Multifunction X and

Y knobs. The objective aperture must be small enough to isolate the chosen spot from neighbouring diffracted beams.

• Press D button (LED off) and a dark field image of those crystal planes causing the selected diffraction spot will now appear.

• Carry out Magnification, Intensity, beam Shift and Focus adjustments as for a Bright Field image. Switching between Dark and Bright Field images may be achieved simply by successively pressing the Dark-field button. Multiple settings for different dark-field tilts can be stored in the dark-field channels.

4.2 Off-axis imaging Procedure • Switch to diffraction. • Center the objective aperture around the diffracted beam required for dark-field imaging. • Switch back to imaging.


5 Diffraction

5.1 Focusing in diffraction Unlike the image, the diffraction pattern does not have a clear criterion for establishing when it is in focus. Often it is presumed to be 'in focus' when the pattern has spots that are as small as possible. This is not strictly true. The diffraction pattern is in focus when the focus lies at the back-focal plane of the objective lens. Only when the incident beam is parallel does the cross-over lie in the back-focal plane.

With a parallel beam incident on the specimen (grey), the objective lens focuses the electrons into a cross-over whose position coincides with the back-focal plane (and thus the true diffraction focus). When the beam is convergent (but not wholly focused), there still is a cross-over but it is displaced from the back-focal plane upwards (in the extreme case, a fully focused beam, the cross-over lies at the image plane). When the beam is divergent, the cross-over is displaced downward from the back-focal plane.

If the diffraction pattern is not focused properly, there are a number of consequences: • the camera length can be wrong • the diffraction will be rotated away from its proper orientation • the pattern may be distorted • alignments such as beam shift pivot points can be wrong • the scanning magnification can be wrong due to misaligned pivot points Due to the absence of a clear criterion, we end up with a chicken-and-egg situation (what was first, the chicken or the egg?). For example, if it can be assumed that the shift pivot points are correct, then it is easy to establish the correct diffraction focus by wobbling a beam shift and minimising diffraction-pattern movement. However, the pivot points can only be aligned correctly if the diffraction pattern is focused properly.


In order to resolve this situation, we have determined Intensity settings for the different modes (LM, HM-TEM and Nanoprobe) for a parallel beam. These Intensity settings are preset in the alignment procedures, making it easy to find diffraction focus (the spot-pattern condition). After the alignment have been done (camera length focus), the diffraction focus can also be found by simply pressing the Eucentric focus button (this resets the variable diffraction focus to zero). With this method for establishing diffraction focus, the SA aperture is not (and should not be) used.

5.2 The shadow image When the diffraction pattern is focused properly at the back-focal plane, the pattern is either a spot pattern in SAED or a disk pattern in CBED (with a fully focused beam). In this case the diffraction pattern contains no image information at all (the magnification of the image in the diffraction pattern is infinite). It is also possible to defocus the diffraction pattern slightly (see below). When this is done, the (by now expanded) spots or disks do contain image information (the magnification is no longer infinite) and so we have obtained a mixture of diffraction and image information. This is called a shadow image. The shadow image (in this case from SAED diffraction) can be understood from the diagram below. When a parallel illuminates an area of the specimen, all transmitted (bright-field) beams converge in a single cross-over in the back-focal plane of the objective lens. In this cross-over we cannot distinguish between beams coming from the different parts of the specimens, because all beams go through a single point (ideally). However, above or below the back-focal plane, the beams do not go through a single point but - in three dimensions - they form a disk and each point in the disk corresponds to an area of the specimen.

In SAED the shadow image is obtained by changing the diffraction focus (FOCUS). In CBED the shadow image is obtained by defocusing the beam on the specimen (INTENSITY). The shadow image is often used when working with crystals : • During tilting it allows observation of both crystal orientation (from the diffraction pattern) and position

(the shadow image), making it easier to correct (with X-Y stage movement) for apparent image shift during tilting (especially with the non-eucentric b tilt).

• It can be used to create multiple dark-field images (one pattern contains the bright-field disk with the bright-field image and several diffraction disks, each with its own dark-field image).

• It can be used to position, focus and stigmate the focused beam accurately while in diffraction (or STEM).

In the shadow image, there are a couple of effects dependent on the direction of defocusing (under- or overfocus). In going from under- to overfocus the shadow image: • flips by 180°. • inverts the contrast. Because of these effects, one should work consistently (either always underfocus or always overfocus).


5.3 Selected-area diffraction Selected-area diffraction has been the main diffraction technique for a long time. However, since the introduction of instruments capable of illuminating areas of nanometer-size, convergent-beam (or with a somewhat defocused or even parallel beam, aperture-less) diffraction has gained in importance. One reason is the resolution limitation of selected-area diffraction (see the nomogram below). Another reason is the difficulty of judging whether –g and +g beams are of equal intensities (and the orientation is thus in a symmetrical condition), which is especially important for high-resolution imaging.

Nomogram showing the relation between objective-lens Cs, electron wavelength λ, diffraction angle α, d spacing and diffraction error δ (the deviation between the center of the SA aperture and the center of the actual area from which the diffracted beam is coming). The nomogram works as follows. Draw a line connecting the wavelength with the diffraction angle α or d spacing. Draw a second line connecting the Cs with the intersection of the first line and the α scale. The intersection of the extrapolation of the second line with the δ scale gives the diffraction error (for the particular α or d spacing assumed). Note that all scales are logarithmic. The lines are drawn for a d spacing of 0.1 nm and the 200 kV objective lenses. Nevertheless selected-area diffraction has still some important applications, mostly where angular resolution in the diffraction pattern (size of the diffraction spots) is important. An example is the detection of weak superlattice spots which become quickly invisible when their small total intensity is spread out over a disk rather than being concentrated in a small diffraction spot. The complete SA diffraction procedure is as follows: • Obtain a TEM Bright Field image in the SA magnification range. • Select required field of view. • Insert a Selected-Area aperture of appropriate diameter. Note: The smallest aperture diameter is in general not smaller than 10 um, which will give a distortion-free diffraction pattern from an area of the specimen of about 400 nm in diameter. • Remove the objective aperture. • Press the Diffraction button (green LED on). • Select required camera length (Magnification).


• Focus the diffraction pattern. • Adjust Intensity of illumination to a suitable level by turning clockwise to reduce the intensity. • Refocus the diffraction pattern if necessary. Note: Recording the diffraction pattern should be carried out with manual exposure-time selection as automatic exposure readings are not reliable in the diffraction mode. The time is best judged from experience gained from a series of test exposures obtained with fixed illumination conditions (emission, high tension, intensity and spot size) for the different sizes of selected-area apertures. As a rough estimate, one-third of the value of the automatic exposure time may be used.

For recording the diffraction pattern, the beam stop may be used to block the central diffraction spot when this is much more intense than the remainder of the pattern. Remove the beam stop after about 90% of the exposure time has elapsed. Selected area diffraction pattern with beam stop blocking the transmitted beam.

5.4 Diffraction without selected-area aperture Instead of using a selected-area aperture to define the area from which a diffraction pattern is obtained, the beam can be located (either in focus - convergent-beam diffraction - or defocused) on an area of interest. There are several advantages to using the beam as area definition rather than the SA aperture: • The optical conditions are closer to those in imaging (which is especially an advantage in orienting a

crystal for high-resolution imaging. • There is often detail visible in the diffracted beams that makes orienting crystals easier (and more

accurate) than with SA diffraction. • Diffraction patterns can be obtained from small areas without the diffraction error of SA diffraction

(see section 5). • The technique can be applied in microprobe and nanoprobe mode (Sa diffraction is often difficult in

nanoprobe). The differences between the modes are: • Microprobe - smaller convergence angles, larger spots, larger area illuminated by parallel beam. • Nanoprobe - higher convergence angles, smaller spots, smaller area illuminated by parallel

beam. The disadvantages of diffraction without an SA aperture are: • Unless the beam is truly parallel the diffraction pattern contains disks, not spots (unless the

diffraction pattern is defocused as well). • With a real convergent beam specimen damage by electron beam may occur. The procedure for 'aperture-less' diffraction is simple: locate the beam on the area of interest and select the Intensity (beam focus) setting. Then go to diffraction.


5.5 Standard convergent-beam diffraction For 'real' convergent-beam diffraction, the beam is simply focused on the specimen. Typical convergent beam diffraction conditions can be either the microprobe or nanoprobe mode, spot sizes in the smaller spot size range (> spot 5), condenser aperture dependent on the diffraction angle required (which is usually determined by the smallest spacing between the diffracted beams for the crystal orientation of interest). The convergent-beam pattern consists of two types of features: 1. The diffraction disks which essentially are images of the condenser aperture. 2. The diffraction information inside the disks. These two types of features react differently to different types of manipulation: 1. The position of the diffraction disks depends on de tilt angle of the incident beam (a tilt in the image is

a shift in diffraction). The position can be changed by either shifting the condenser aperture or tilting the beam itself (dark field). Changing the position of the disks does not affect the overall position of the diffraction information (which behaves like a background over which the aperture images appear to move).

2. The position of the diffraction information depends on the orientation of the crystal. Tilting the crystal thus moves the pattern of diffraction information but leaves the position of the disk unaffected.

These characteristics can be used for fine-tuning of the convergent-beam pattern.

5.5.1 Orienting the crystal In order to minimize distortions of the beam, any fine-tuning (as described below) should be limited to small angles. It is therefore important to make sure the specimen is as well-oriented as can be achieved with the α and β tilts of the specimen holder. It will be seen that the diffraction disks of the CBED pattern stay in position while tilting and that it is the structure inside the disks that moves. Make the pattern as symmetrical as possible by tilting.

5.5.2 Fine-tuning pattern Once the crystal has been oriented as well as possible with the mechanical stage tilts, there are two methods for fine-tuning the pattern. Since the CBED pattern is a reflection of the relative orientation of the crystal and the beam, tilting the crystal and beam have the same effect (except that tilted beams become distorted due to electron-optical aberrations). Tilting the beam is often easier in the end stages of preparing CBED pattern because finer control is possible. Tilting can be achieved in two ways, one using the dark-field tilt (preferred method since it can be reset very easily), the other by moving the condenser aperture.

5.6 Large-angle convergent beam (LACBED/Tanaka) Convergent beam diffraction patterns (CBED) can provide a wealth of information about the crystallography of a specimen. The amount of information available depends on the size of the disks in the CBED pattern, which is limited by the spacing of the disks, since disks are not allowed to overlap (except in coherent CBED, a technique limited to FEG instruments). Even for structures with fairly normal-sized unit cells the disk size on zone axes with low indices is usually small so that only a limited amount of information is obtainable. In many instances, e.g. for the determination of the presence or absence of an inversion center, more information in larger disks is required. This information can be obtained by using the Large-Angle Convergent Beam (LACBED) or Tanaka technique. With this technique, larger convergence angle (and hence large disks) can be obtained without overlap between adjacent disks. This result is obtained by moving the specimen out of the plane of focus (or moving the


focus out of the specimen plane) while keeping the beam focused. When this is done, a diffraction pattern appears overlaid on the image, with the size of the diffraction pattern proportional to the distance between the specimen and the focus plane. The selected area aperture can then be used to select a single beam, either the transmitted beam or a diffracted beam, which is then the only beam contributing to the diffraction pattern. Procedure • Start in the TEM (microprobe) mode. Center the specimen at the eucentric height. Orient the

specimen as required. • Set up the nanoprobe mode with a condenser aperture of 50 um, with beam centered and focused,

and the image in focus. Select a spot size somewhere in the low end of the range (higher spot numbers). Check that the specimen orientation is still in the proper orientation. If high angles are required, increase the size of the condenser aperture.

• Increase the focus (typically by several micrometers) or alternatively move the specimen up with the Z axis height control (the advantage of moving the specimen is that the electron-optical conditions do not change; the disadvantage that the specimen is moved and is no longer eucentric).

• A diffraction pattern will now be visible around the transmitted beam. • Insert a selected-area aperture (start with the smallest) around the transmitted beam. If the aperture

is still too large so that diffracted beams still pass through the aperture, increase the focus current/raise the specimen further.

• Once the diffraction pattern is large enough to allow only one beam to pass through it, center the aperture around the beam of choice (transmitted or diffracted).

• Switch to diffraction and you have the LACBED pattern. Notes: • The maximum diffraction angle is determined by the size of the condenser aperture, the objective-

lens current (the highest angle is typically obtained for objective-lens settings of 5 to 10 um overfocus) and the SA aperture. It is easy to establish if the condenser or SA aperture is angle-limiting. Simply shift one of the apertures slightly. If the shadow at the edge of the diffraction disk moves with it, that is the limiting aperture. If the condenser aperture is angle-limiting (and higher angles are needed), switch to a larger aperture if possible. If it is the SA aperture, switch to a larger SA aperture but remember to check that the aperture allows only one beam to pass!

• A properly focused beam will typically have a small bright spot in the center, surrounded by a halo (caused by the spherical aberration of the objective lens).

• Optimize the diffraction angle by slightly changing the Intensity setting to get the largest disk size and at the same time not running against the SA aperture.

• If ‘sausage’-shaped dark areas are visible in the pattern, the beam is not properly centered inside the SA aperture.

One problem with LACBED patterns is that they mix diffraction and image information because the interaction area between the specimen and beam is large (the beam is focused but not at the specimen itself). This is the price one pays for having no overlap and yet large disks. Because of the mix of image and diffraction information, LACBED patterns often display distortions (due to specimen bending) and thickness changes across the disk. Another ‘feature’ of LACBED patterns is that they show rubbish on the specimen surface with high contrast (but often only visible once the negatives have been recorded). Make sure the specimen is as clean as you can get it. It is also possible to have simultaneous bright-field (transmitted-beam) and dark-field (diffracted) beams in single pattern. To achieve that do not insert an SA aperture but stay in image mode and defocus the beam (Intensity) until the beams of the diffraction pattern touch each other.


6 Analysis There is a big difference between EDX and EELS analysis. In the case of EDX analysis, the detector is mounted close to the specimen and X-rays can be detected from the whole specimen area, not only the area intended (and hit by a focused beam). In the case of EELS it is much easier to keep 'spurious' signals out from the detector. For EDX it is important therefore to pay attention to the conditions for analysis.

6.1 EDX analysis Because of the location of the detector, EDX analysis is sensitive to 'spurious' signals, that is, signals generated outside the focused electron beam and thus outside the area of interest. These signals can in principle come from a specimen-mounting grid, specimen holder, objective aperture and objective-lens pole pieces. Some of these effects can be partially avoided by paying attention to the analysis conditions, but some spurious signals will always exist (for example from electrons backscattered from the specimen area which then hit other areas of the specimen again).

6.1.1 Microprobe versus nanoprobe The electron optics of the column with regard to the two modes result in a large field of view in the microprobe mode (with the beam able to illuminate a wide area) whereas in the nanoprobe mode the beam cannot be defocused further than several micrometers. Stray electrons (scattered at the edge of apertures) can only occur within the area that can be illuminated by the beam. In nanoprobe any stray electrons are therefore confined to a few micrometers around the electron beam itself. Especially in ion-milled or jet-polished specimens, the stray electrons therefore strike only thin parts of the specimen and thus do not have a significant effect on the EDX spectrum (which in principle should contain only information from the area within the beam itself). In the microprobe mode, the stray electrons can strike the specimen over a much larger area, including thick areas where a single incident electron can generate up to tens of X rays. The nanoprobe mode is therefore better suited for EDX analysis.

6.1.2 Shadowing angle The EDX detector is mounted at a finite angle above the plane of the specimen, typically somewhere between 10 and 20°. This has consequences, not only for absorption (at lower angles the path of the X rays through the specimen is longer) but also for the 'visibility' of the specimen. In fact, the angle listed for detectors is somewhat 'cosmetic' because it refers to the angle through the heart of the detector crystal. In practice this can mean that the lower edge of the detector crystal is only slightly above (or even below) 0°. For good analysis the specimen must therefore be tilted. The amount of tilt required depends to some extent on the specimen position (the Y axis, especially when the value is strongly negative) and specimen itself (Focused Ion Beam - FIB - specimens must be mounted so they face the detector), but mostly it is the detector configuration itself. The angle at which shadowing stops can be determined by collecting EDX spectra at various tilt angles (0, 5, 10, …) and comparing the count rates for these angles (note that there is of course a change in effective specimen thickness as the tilt angle goes away from 0°). Find the tilt angle where the increase in count rate to higher tilt angles levels off and use that (with a margin of an additional 5°) as the minimum tilt angle used for EDX analysis. Note: Never use a normal holder for EDX analysis. Special low-background holders with beryllium shielding around the specimen exist in single-tilt and double-tilt versions. Some of the regular holders (especially the single tilt) also have very 'deep' specimen cups and completely shield the specimen from the EDX detector unless the tilt angle is well above 25°.


6.2 EELS analysis EELS analysis can be done in two modes, image mode and diffraction mode. In image mode, the projection-system cross-over, which is imaged again on the EELS spectrometer as a spectrum is a diffraction pattern (this means that the spectrum is a diffraction pattern at the same time). This mode is therefore called diffraction coupling. Conversely, in diffraction, the cross-over contains an image, and this is called image-coupling. The size of the cross-over (and thus to some extent the resolution of the spectrum) is inversely related to the magnification or camera length. There is a second factor related to the diffraction- or image-coupling. The standard mode of operation for EELS analysis is diffraction (image coupling). The diffraction pattern is simply placed at the entrance aperture of the PEELS or Imaging Filter. Area selection is done with the focused beam. One reason for using diffraction is that one can simply move from one area for analysis to another by shifting the beam; the diffraction pattern will remain stationary and so it is not necessary to center the beam on the EELS entrance aperture. This same principle applies to analysis in STEM. It should be realized, however, that the beam shifts (in one direction; the other direction is parallel to the energy axis of the spectrum) will be visible in the spectrum as a spectrum shift. This effect gets magnified at low camera lengths (as often used for EELS). The effect is readily visible when EELS spectrum acquisition is done in STEM with a continuously scanning beam at low camera length. Under these conditions the apparent energy resolution may be 10 eV or more. For proper operation under such conditions a descan facility is needed (but not yet available on Tecnai).

6.2.1 Determining spectrometer acceptance angle β An important parameter in EELS quantification of the spectrometer acceptance half-angle β. (Another parameter, the beam convergence angle α is also important in the sense that it should be less than β, but most users have less problems with determining α than β). The α angle can be measured simply from a diffraction pattern. The equations can be derived as follows: Bragg's Law 2θB = λ / d where 2θB is the Bragg angle, λ the electron wavelength and d the d spacing. Camera constant D d = L λ where L is the camera length and D the distance between transmitted and diffracted beam in the recorded diffraction pattern. CBED α / 2θB = A / D The latter formula says that the angles in the diffraction pattern (convergence angle and a Bragg angle of a diffracted beam) are proportional to the ratio between the distances measured in the pattern (the radius of the diffraction disk A and the distance from transmitted to diffracted beam D). These formulas can be converted to: α = A 2θB / D (a rewriting of the CBED formula) or α = A / L


The β angle is more complicated because it depends on the operating mode of the microscope. If the microscope is in image mode (diffraction-coupled in EELS terminology), the β angle is determined by the objective aperture. To measure this, record a diffraction pattern with the objective aperture in and substitute the radius of the objective aperture for the radius of the diffraction disk in the formula for the α angle above . In diffraction (image-coupled) the β angle is determined by the effective size of the EELS entrance aperture. If the entrance aperture were at the same level as the viewing screen (or plate camera), one could simply take the radius of the aperture and substitute once again in the formulas for α above. However, the aperture is much further down and so it projects a smaller size as seen from the final cross-over in the microscope (in the differential pumping aperture at the top of the projection chamber). To determine the b angle one thus needs to know the distances from the cross-over to the plate camera (the reference for the camera length value; this distance is 438.5 mm) and from the cross-over to the EELS entrance aperture (748.75 mm; it is the same for PEELS and Imaging Filter). From these data it is easy to construct a table with β acceptance angle values (using a spreadsheet program like Microsoft Excel): • Make a first column listing the camera lengths (viewing screen up!) of the microscope (leave the top

cell A1 empty). When the camera lengths are for the EFTEM lens series, make a factor "EFTEM Factor" with value 21.35 to divide the values as listed (these camera lengths refer to the CCD as the reference position, not the plate camera). For the normal lens series this factor equals 1.

• Fill cells B1, C1, D1 and E1 (for PEELS, omit E1 for Imaging Filter) with the values of the entrance apertures.

• Fill in the table with the formula (resulting in mrads) 1000 * “EFTEM Factor” * (dcross-over to plate / dcross-over to EELS) * (aperture size / 2) / (camera length) where aperture size is the value in the first row and camera length is the value in the first column.

An example is shown below. Proj. X-over distances plate 438.5 mm EELS aperture 748.75 mm Aperture (mm) 1 2 3 5 Cam Length (mm) 14 21.69 43.38 65.07 108.45 27 10.85 21.69 32.54 54.23 35 8.37 16.73 25.10 41.83 44 6.66 13.31 19.97 33.28 62 4.72 9.45 14.17 23.61 84 3.49 6.97 10.46 17.43 135 2.17 4.34 6.51 10.85 180 1.63 3.25 4.88 8.13 265 1.10 2.21 3.31 5.52 450 0.65 1.30 1.95 3.25 630 0.46 0.93 1.39 2.32 910 0.32 0.64 0.97 1.61 1750 0.17 0.33 0.50 0.84 2650 0.11 0.22 0.33 0.55 3500 0.08 0.17 0.25 0.42 3900 0.08 0.15 0.23 0.38


7 STEM In the STEM (or Scanning) mode of the microscope images are acquired by moving a focused beam in a raster across the specimen and collecting a signal at each x,y pixel coordinates. The signals for all pixels together make the STEM image.

7.1 STEM principles When a focused beam is moved across a specimen, the signals generated by the interaction between the electron beam and the specimen will vary according to specimen characteristics such as material type (composition and structure), orientation (diffraction from crystals), and topography. When the beam is scanned across the specimen in a rectangular raster, the change in signals allows one to build up an image of the specimen. The scanning of the beam across the specimen on the Tecnai microscope is achieved through a set of beam deflection coils (called the AC coils) that is separate from the 'static' beam shift coils (or DC coils). The AC beam deflection of the scan is thus superimposed on the DC deflection (which is used to align the beam along the optical axis - the rotation center - and center the beam). The AC deflection coils have their own characteristics and thus their own pivot points, separate from those of the DC coils (the procedure to align the AC coils is similar to the DC alignment, though). STEM magnification works different from TEM magnification. The TEM magnification is the result of the magnification by the objective lens (a constant value) and the - variable - lenses of the projector system (Diffraction, Intermediate, Projector lenses). The STEM magnification is the result of the deflection of the beam. If the beam is deflected far from the optical axis, the scanned area is large and thus the STEM magnification low. When the beam is deflected only a little, the scanned area is small and the STEM magnification high. Some STEM detectors are located close to the specimen (secondary-electron, backscattered electron) and for these the setup of the magnification system is irrelevant (they would still work even if the microscope shutter - below the specimen area - would be closed). For other detectors, like the bright-field and dark-field detectors, the setup of the magnification system is important. The setup of the bright-field/dark-field detectors makes clever use of a characteristic of a beam that is being scanned parallel to the optical axis: the diffraction pattern remains stationary while the beam moves. It is therefore possible to detect the bright-field signal by placing the central (transmitted) beam on a bright-field detector, while an annular (ring-shaped) dark-field detector around the bright-field collects the signal from (some of) the diffracted beams. Which diffracted beams (or rather, which range of diffraction angles) are being collected on the dark-field detector depends on the camera length of the diffraction pattern. At low camera lengths the collected angles are large (and the dark-field detector effectively is a high-angle annular dark-field detector), while at higher camera lengths the angles are smaller. The limitation on camera lengths on the lower side is determined by the off-axis diffraction shift required to move the diffraction pattern to the detector (the bright-field/dark-field detectors are located about 3 cm away from the center of the viewing screen), while on the high side the limitation (in this case only for dark-field) is the size of the diffraction disk of the central beam (since the beam is focused, the diffraction pattern has disks, not spots). Once the central-beam disk becomes too large, it starts to fall on the dark-field detector. There are typically two camera lengths around 90 to 100mm. One of these is rotation-free, the other is not (it is rotated relative to the other camera lengths). The rotated camera length is used for STEM high-angle dark-field imaging with the normal dark-field detector (on the lowermost rotation-free camera lengths the high diffraction angles are shadowed by the differential pumping aperture, so very little high-angle dark-field signal is available).


Currently only the 'Nanoprobe' type of STEM (that is, the microscope is effectively in the nanoprobe mode) is implemented in the Tecnai software, but later Microprobe and LM STEM will be implemented as well.

7.2 The importance of the pivot points In TEM mode, the pivot points are important in ensuring that a pure beam shift or pure beam tilt takes place. However, since generally one does not shift the beam off-axis during TEM operation, the pivot points are not extremely critical. In STEM they are. When the pivot points are aligned properly, the beam will remain parallel to the optical axis while it is scanning within the raster (with the limitation noted below). It will, for a certain STEM magnification, cover a certain area of the specimen. If the pivot points are incorrect, the beam will either fan in a divergent pattern or in a convergent pattern across the specimen.

When the pivot points are set correctly, the scanning beam will remain parallel to the optical axis. All beams will pass through the same cross-over in the back-focal plane of the objective lens. When the pivot points are wrong the beam will either diverge away from the optical axis or converge (picture below). When the beam is diverging, the outermost positions of the beam are further away on the specimen than in the parallel-beam case. Consequently, the beam scans a larger area of the specimen and the scanning magnification will be lower. When the beam is scanning a converging pattern, the outermost beam remain closer to the center than in the parallel-beam case, the scanned area is smaller and thus the scanning magnification is higher. Note that in all three cases the beams pass through a cross-over, but that only in the parallel-beam case does the cross-over lie in the back-focal plane (but it is always possible to 'focus' in diffraction to the cross-over).


One limitation on the degree to which the beam remains parallel to the optical axis is spherical aberration. Beams that travel far off-axis are affected more strongly than beams closer to the axis. The consequence is that the scanning beam at low magnifications will not remain truly parallel to the optical axis but tilts slightly towards the optical axis. The main consequences are a change in effective magnification across the image and some movement of the diffraction pattern. Note : In addition to spherical aberration of the objective lens (which does affect the scan pattern), there can also be an effect of spherical aberration of the diffraction lens for some camera lengths. This effect is noticeable from the stronger movement of the diffraction pattern.

7.3 Detectors Since the beam in STEM is focused on the specimen, the signal is generated only from the area where the beam is actually located. This makes it possible to use a wide range of detectors for STEM. Some of these detectors produce a signal in a similar way to TEM imaging. The Bright-Field (BF) detector collects the same signal as the TEM Bright-Field image : the transmitted beam. Other detectors such as the Secondary-Electron (SE) or Energy-Dispersive X-ray (EDX) detector can only be used with a focused beam because these detectors lack the optics (electron or X-ray) to separate signals from different areas of the specimen when a broad, defocused beam is used. In principle, all detectors can be used to acquire STEM images (even a slow-scan CCD could be used in principle to record and process signals and thereby generate a STEM image). In practice some detectors receive too weak a signal (EDX) or read out too slowly (CCD) to be used for image acquisition.

7.3.1 Bright-Field (BF) detector There are two different types of bright-field/dark-field detectors: non-retractable (in the so-called near-axis position) and retractable (centered). The non-retractable BF detector consists of a scintillator (converting electrons to light) and photo-multiplier (detecting and amplifying the light signal, then converting it to electron current). The scintillator has a diameter of 7 mm. The assembly (together with the dark-field detector) is located in the so-called near-axis position underneath the projection chamber, which is about 3 cm ENE of the center of the viewing screen. The retractable BF detector consists of a solid-state detector with a diameter of 10 mm. WARNING : POTENTIAL DAMAGE ! The retractable BF detector can be damaged by a very high electron dose. If you are using the detector with a very high beam current (e.g. for X-ray mapping), change to a high camera length so the central-beam disk is large and the electron dose per detector area is not too high. Important note on the retractable BF detector. The area covered by the BF detector is larger than the holes in the DF2 and DF4 detectors. Consequently, when you set contrast and brightness with the BF inserted but the DF retracted and then insert one of the DF detectors, the area of the BF detector receiving signal will be much reduced and typically no BF signal is observed any more. Set the contrast and brightness again (AutoCB) and the signal will re-appear.


Schematic drawing of the retractable BF/DF detectors.

7.3.2 Dark-Field (DF) detector The non-retractable DF detector is an annular solid-state detector. Its inner diameter is 7 mm, the outer diameter is 20 mm. It is positioned around the BF detector (and thus also in the near-axis position). The acceptance angles of the DF detector (inner and outer) are determined on the one hand by its dimensions (inner and outer radius) and on the other hand by the camera length chosen (the values are given by the arc tangent of the relevant radius - 3.5 or 10 mm - divided by the camera length - in mm of course). The retractable DF detectors (there are two with different diameters) are annular solid-state detectors with outer diameters of 24 mm and inner diameters of 2 (DF2) and 4 (DF4) millimeters. These detectors are located above the the retractable BF detector.

7.3.3 High-Angle Annular Dark-Field (HAADF) detector The HAADF detector is an annular detector consisting of a scintillator-photomultiplier. This detector is placed in a housing above the projection chamber and is moved in an out. This detector has been optimised for two aspects, in order to allow atomic-resolution STEM imaging (on FEG instruments) : • single-electron sensitivity • high angles The inner acceptance angle can be seen directly from the shadow of the detector on the fluorescent screen (when it is inserted), the outer acceptance angle is five times the inner angle.

7.3.4 Secondary-Electron (SE) detector The SE detector consists of a scintillator (converting electrons to light) and photo-multiplier (detecting and amplifying the light signal, then converting it again to electron current). It is mounted above the specimen and can be used to detect secondary electrons emitted by the top surface of the specimen. The detector itself enters the area around the specimen through a hole in the upper objective-lens pole piece. This is possible only for the TWIN objective lens for 120 and 200kV. For all other lenses or voltages the hole in the pole piece would degrade the quality of the pole piece (and thus the quality of the objective lens) too much and/or the strength of the magnetic field prevents the (low-energy) secondary electrons from reaching the scintillator. In order to attract the secondary electrons an extraction anode is placed above the specimen. The extraction anode can be supplied with voltages in the range -50 to +150 Volts. Where a backscattered-electron (BS) detector is mounted in combination with an SE detector, the extraction anode is integrated with the BS detector. Because of the placement of this detector, alignments affecting the positioning of the diffraction pattern (as for BF/DF) have no relevance (since that effect occurs much further down the column than where the


SE detector is mounted). The pivot point alignment are important, however, because they affect the scan raster itself.

7.3.5 BackScattered-electron (BS) detector The BS detector is a solid-state detector mounted above the specimen (just below the upper pole piece of the objective lens). The detector is not truly annular (ring-shaped) but consists of a plate of total area 16 mm² with a hole in the center - through which the incident beam passes - and one truncated corner (to make room for the EDX detector). For additional details, see also the last two paragraphs under SE detector.

7.3.6 Energy-Dispersive X-ray (EDX) detector The EDX detector is a solid-state detector that detects X rays. X rays entering the detector are converted to a number of electron-hole pairs that is proportional to the energy of the X ray. The EDX detector is located as close as possible to specimen, typically at an angle of 15 to 20 degrees above the horizontal. Because of the proximity of the detector, it is very difficult to exclude 'spurious' X rays, generated outside the area of interest (for example, by electrons scattered by the specimen or at the edge of the condenser aperture). In order to reduce the 'spurious' X rays, it is essential to use a proper low-background specimen holder (regular holders have the additional disadvantage that the holder area around the specimen is 'deep', blocking the EDX detector from 'seeing' the specimen unless a high specimen tilt is applied). The EDX signal can be recorded for each pixel in a STEM raster, allowing collection of X-ray images. Since collection of EDX spectra is slow, these images are generally recorded with a limited number of pixels and in a single acquisition, and processed afterwards.

7.3.7 Electron Energy-Loss Spectrometer (EELS) The EELS spectrometer (either a PEELS or the Imaging Filter in spectroscopy mode, mounted underneath the projection chamber) can be used the record the EELS signal. The EELS signal can be recorded for each pixel in a STEM raster, allowing collection of energy-loss images. Since collection of EELS spectra is relatively slow (in any case much slower than collecting video signal like BF or DF), these images are generally recorded with a limited number of pixels and in a single acquisition, and processed afterwards.

7.4 Detector alignment For proper STEM operation with the BF/DF detectors, the diffraction pattern must be centered correctly on the detectors. In order to make this easier, the microscope has a separate alignment (an additional diffraction shift per camera length) that can be used to switch rapidly between a centered diffraction pattern and a pattern shifted to the detectors. The advantage of the detector alignment is the reproducibility of the off-axis shift. Once the diffraction pattern has been centered on the screen, it is much easier to shift accurately to the detector. Note: The centering of the diffraction pattern on the screen depends on the diffraction shift set as well as on the rotation center. Misalignment of the rotation center (which effectively is a beam tilt) gives a diffraction shift, so check the rotation center as well. In addition, the centering of the diffraction pattern may be sensitive to the magnification-system lens settings. Using projector normalization makes the position much better reproducible.


7.5 STEM adjustments - an in-depth explanation The pivot points are not the only adjustments made to the STEM system. Ideally deflection coils have the following characteristics: • Shifts on the x and y coils keep the beam parallel to the optical axis - implying that the upper-x and

lower-x coils are perfectly matched (and the same for upper-y and lower-y). • The upper and lower coils are perfectly aligned (that is, the is no rotation between the upper and

lower coils). • The x and y coils produce the same amount of shift (the magnetic fields of the coils are equal). • The x and y coils are perfectly perpendicular to each other. In practice small deviations occur for all these parameters. On the Tecnai all of these are compensated by alignments for the AC coils. The pivot points and the perpendicular correction are the most important user adjustments. The pivot points (which determine the ratio between the upper and lower coils, for x and y separately) make sure the beam moves parallel to the optical axis. The perpendicular correction corrects for any rotation between the upper coils and the lower coils by adding a small lower-y shift to the x shift (executed itself by upper and lower-x) and a small lower-x shift to the y shift (executed by the upper and lower y shift) The relative strengths of the coils as well as the perpendicularity are set through separate adjustments on the upper-x and upper-y coils . All these adjustments should result in a STEM image with minimum distortions.

7.6 STEM Operation

7.6.1 Tem Imaging & Analysis The scanning system of the Tecnai microscope consists of two elements: • hardware, such as scanning (beam) coils, inside the microscope, and software to control these. • hardware (the scanning acquisition board) inside the PC, and software to control it. The hardware inside the microscope provides all the controls necessary for scanning, such as magnification, scan rotation and so on, except for two elements. The microscope hardware itself cannot scan the beam and it cannot read the detector signals. These functions are done by the scanning acquisition board. The scanning acquisition board puts out voltages for x and y (called line and frame in STEM), causing the beam to be moved around the specimen. These voltages can cover the full range possible or can be only a subrange. The microscope itself translates these voltages into beam deflections. The amplitude of the beam deflection is defined by the combination of the voltage from the scanning acquisition board (in TIA) and by the scanning magnification (set by the microscope software). This combination has important consequences for the magnification and resolution in an image, as described below. The detector signals are fed into the scanning acquisition board. The Analog-to-Digital (ADC) converters of the board convert the current coming off the detector and convert it into a digital signal. The ADC inputs of the scanning acquisition board are not fed directly from the detectors but instead from three video channels (with each channel directly connected to an ADC converted). The detectors themselves can be switched from one channel to another. This setup exists for two reasons: • it makes it possible to have more detectors than the maximum number of ADC converters available. • the video channels themselves contain hardware that adjust the amplification and offset of the

detector signals to the optimum range for the ADC converters, similar to the detector contrast and brightness (except that the video level adjustment is a factory/service setting because it is fixed - that is, not dependent on beam current, specimen or detector type).


7.6.2 Resolution and frame size There are three factors that affect the STEM image: • Resolution (scan acquisition board) • Frame size (scan acquisition board) • STEM magnification (Tecnai microscope) The first two names are somewhat arbitrary, because there are no unambiguous terms that clearly define these factors. The three factors are not independent of each other and a good understanding of the how and what of them is important. What is STEM magnification? STEM magnification differs from TEM magnification. In the case of the TEM magnification, the lenses of the projection system (Diffraction, Intermediate and Projector 1 and 2) are changed to give different magnifications. The STEM magnification, in contrast, has nothing to do with the projection system, but is the result of a deflection of the beam. When the beam is deflected over a large range, the area covered by the scan raster is large and the STEM magnification is low (large field of view = low STEM magnification). At very high STEM magnifications, the beam is scanned only over a very small area. The scan acquisition board can output voltages within a certain maximum range. It is also possible to have the acquisition cover only part of the maximum range. These ranges can be covered by a user-defined number of pixels. Resolution refers to the number of pixels taken across the maximum scan range. This a resolution of 2048 means that the full range is covered by 2048 pixels (in the x direction). A resolution of 512 means that the same range is covered by 512 pixels, so the distance between the pixels is 4x larger than for the resolution 2048. Frame size refers to the range covered by the acquisition. If the value equals the resolution, the scan covers the maximum range. Any lower value has the scanning take place over a sub-range. Thus, at a resolution of 2048 and a frame size of 512 the scan covers only a quarter of the range (and thus the total area covered is 1/16th because both x and y are affected). STEM magnification affects the maximum range of the scan frame and is thus a factor by which the output signal from the scan acquisition board is multiplied (or rather divided, because a higher STEM magnification reduces the size of the scan frame). Some examples Let us assume a STEM setup with the following parameters: resolution 2048, frame size 2048, STEM magnification 20000x. What happens when we change: • Resolution to 512. The Frame size will automatically go to 512, the STEM magnification stays the

same and the field of view stays the same as well. The only thing that changes is that the image now consists of 512x512 pixels instead of 2048x2048 pixels.

• Frame size to 512. The scan frame now covers only a subrange (512/2048 so one-quarter of the total range available) and the effective magnification in the image has increased by a factor 4 (even though the STEM magnification setting itself remain unchanged).

• STEM magnification to 80000x. The area scanned by the beam is reduced by a factor 4. The other settings are unaffected. Unlike the case where the frame size was changed, it is now still possible to collect an 2048x2048 image at this STEM magnification.

Why is there a duplication in the controls (Frame size and STEM magnification)? The scan acquisition board does not have enough range (theoretical maximum 4096, in practice limited to 2048) in its settings to give a reasonable range of scan magnifications and keep a reasonable number of pixels in the image. The STEM magnification range covers a factor 100 or more. If the frame size were to be used for that


purpose, the image at the highest magnification would only have a maximum of 41x41 pixels (in practice 20x20 pixels due to other limitations). Ultimately, the effective magnification of the image as seen on the monitor or a print also depends on the how the image is displayed or printed. You can, for example, display an image of 16x16 pixels on the monitor such that 1 image pixel is 1 monitor pixel and the image size is about 5x5 mm. You can also display that same image much larger so that 1 image pixel covers 40 monitor pixels and the image size on the monitor is about 200x200 mm. In the latter case the apparent magnification is clearly much larger than in the former. The only absolute criterion for size in the image is therefore the scale bar.

7.6.3 Detectors and channels The STEM detectors are not connected directly to the Analog-to-Digital Converters (ADCs) of the scan acquisition board but through so-called channels. The microscope is equipped with three channels, each of which is connected to one of the four ADCs of the scan acquisition board (not all channels may be connected on the system, e.g. if only two detectors are present typically only channels 1 and 2 are connected). The connection between a detector and a channel is subject to user selection.

7.6.4 Detector control STEM detectors are electronic devices that detect a certain signal, and then amplify and offset the signal. The amplification of the signal is necessary because the original signal is only very small (down to just a few electrons), too small to be detected by the Analog-to-Digital (ADC) converter used to convert the signal from a current to a digital value. In addition, the amplification ensures that the transport of the signal from the detector to the ADC does not add unwanted noise to the signal (or even drowns the signal totally in noise). The offset of the signal is necessary to make sure that the amplified signal remains within the 'boundaries' allowed by the system (so it isn't below the minimum or above the maximum). The amplification and offset are under control of the microscope operator (because they change, dependent on the amount of signal in the image, which in turn depends on the amount of current in the electron beam and the effects of the specimen). Control of amplification is called detector contrast, and the offset is called brightness.

Schematic diagram of the scanning signal and the effects of amplification and offset. The original signal (black bar 1) is amplified. Various degrees of amplification lead to signals 2, 3 and 4, of which only 2 fits into the allowed range, given by the blue bar 6, but the signal is too low. With the offset (green lines), a fixed current is added to the signal (5), bringing it within the allowed boundaries (6).

Contrast and brightness are interdependent in the sense that the brightness comes on top of the contrast. This means that changing the brightness setting has no effect on the contrast (the total range of the signal stays the same; it is only shifted up or down), but changing the contrast does change the


brightness level (in the diagram above, the green lines would move up when the red lines would go to higher angles). A further control over contrast and brightness is present at the level of the video signal. Since we display the image in a digital form, it is possible to subtract or add a constant value from / to the image, or multiple / divide the image by a constant factor. These are the video contrast and brightness levels used for display. There is a significant difference between detector contrast and brightness and video contrast and brightness. The latter only affects how the image is displayed on the monitor and doesn't change the information in the image (by resetting contrast and brightness you can always get back to the original setting). The detector contrast and brightness are absolute. If the detector brightness is set so high that the whole image is white, there is no manipulation of the video contrast and brightness possible that will result in information in the image. In general the optimum setting for detector contrast and brightness is such that the whole range of the video signal fits within the allowed video signal boundaries (0 to 65536, or 16 bit). Usually the 16-bit range in the video levels is sufficient to allow later manipulation of the video contrast and brightness (or any other form of image processing) to bring out any detail needed. Detectors signal levels above or below the maximum and minimum will be uniform white and black and will no longer have any image information in them.

7.6.5 Procedure for manual setting of detector contrast and brightness (As an alternative to using the Scope signal, see section on STEM Imaging Control Panel) • Start TIA and make sure a scanning acquisition display window is active (press Init in the Scanning

Control Panel). • Double-click on the image in the scanning acquisition display window. • In the Image Properties dialog, set the Scale Limits to 0 (Lower) and 65000 (Upper).

• Make sure the TIA control panels are visible and click on the Video tab


• Click on the Lock button to lock the video scale (no more automatic adjustment). Now the video range is set to its maximum and the output signal from the detector can be set to cover the full range from black to white by adjusting detector contrast and brightness.

7.6.6 Filters Ideally images recorded on an electron microscope contain information and no noise (random variation not related to information coming from the specimen). In practice there are a number of sources of noise - which reduces the quality of the image - such as electron shot noise and electronic noise. The shot noise (the square root of the total number of electrons making up the image per pixel) is unavoidable and can only be reduced by increasing the electron dose, thereby making the ratio of the number of electrons and its square root larger and thus the noise less significant. In the case of STEM there are two aspects to electronic noise that can affect the images: • noise on the beam deflection (beam position) • noise on the video signal In the case of noise on the beam deflection, the voltage coming from the Digital-to-Analog Converter (DAC) of the scan acquisition board is imperfectly translated to an accurate scanning beam position due to various reasons. In order to reduce this type of noise, the STEM system can be equiped with scan filters (FEG instruments only). In the case of noise on the video signal, the detected electron signal is imperfectly translated to a digital value for a pixel in the scanning image. In order to reduce this type of noise, the STEM system is equiped with video filters. Both the scan filter and the video filters are low-pass filters, that is, they allow a low-frequency signal to pass through while a high-frequency signal is stopped. Since real information in the scanning image typically comes from low-frequency signals, while most electronic noise has a high frequency, the filters remove the major part of the noise. Inherently, the filters use our knowledge from STEM image acquisition. For example, if we collect a STEM image with 100 milliseconds per pixel, then we know that the beam deflection and the video level will change 10 times per second. If the beam deflection system or video signal changes much more rapidly than that, it must be due to noise. We can then smooth out the rapid variation (noise) to end up with a better STEM image. The scan filter is needed only on FEG systems. It is set in the STEM Imaging Scan (Expert) control panel. For this beam deflection filter there are four settings, Off (very fast scanning), Fast, Medium and Slow (slow scan image acquisition). When the scan filter is in automatic mode, it will be adjusted to the scanning speed. In manual control, which is only available by experts, the use of the wrong setting can lead to excessive noise (Off while doing the final image acquisition slowly) or image distortion especially at the left-hand side where each new line starts (Slow while scanning very rapidly). The filter must therefore be matched to the scan speed used. Only experts can override the automatic mode. The video filter is set in the STEM Detector Selection (Expert) control panel. For the video filter, each channel has a wide range of settings, selectable from a drop-down list. The filters can also be controlled automatically in which case they are matched to the scan speed. In manual control, which is only available to experts, the correct filter setting can be determined by lowering the value until the image starts to have horizontal streaks (signal variations are smeared out over several pixels). Now increase the value again by approximately two steps (or at least until the streaks are completely absent).


8 EFTEM EFTEM (Energy-Filtered TEM) makes use of the fact that (some of) the beam electrons lose energy inside the specimen (this process is called inelastic scattering). Energy losses on the one hand degrade the image (since electrons that have lost energy are focused differently from those that have not lost any energy, strong energy losses result in image blurring, an effect that increases in strength with specimen thickness). On the other hand, the energy losses contain specific information about the specimen (chemical and physical). In EFTEM, the electrons are separated according to their energies, making it possible to filter out the blurring effects or retrieve the chemical or physical information from the specimen.

8.1 General introduction to energy filtering When passing through a specimen, electrons interact with it. Some are scattered without losing energy (elastic scattering, e.g. in the case of crystals Bragg scattering) which implies that the electrons are deflected by a certain scattering angle and no longer form part of the transmitted beam. Other electrons lose energy inside the specimen by interacting for example with electrons in the specimen itself. Often this takes the form of a beam electron ejecting an electron from its orbit around the nucleus of an atom in the specimen. The beam electron loses an amount of energy (at the very least the amount needed to eject the specimen electron) and the atom loses an electron and often reacts by emitting an X ray. Energy losses are always accompanied by scattering through an angle as well. The inelastic scattering angles are generally much smaller than elastic scattering angles and, in contrast with Bragg scattering, are variable. Statistically, the inelastic scattering angles increase with the energy loss. The average scattering angles for low energy losses is low and these electrons effectively remain part of the transmitted beam (they cannot be stopped by the objective aperture). For higher energy losses the larger angles makes it possible to stop these electrons with the objective aperture. When the energy-loss electrons are separated according to their energy, they form an energy-loss spectrum. A typical energy-loss spectrum consists of the zero-loss peak (those electrons that have lost no or negligible energy), and towards higher energy losses a signal that at first increases strongly (within the first 50 volts, usually with a maximum around 25 volts) and then decays towards high energy. The decay is not monotonous but superimposed on this general background are so-called energy loss edges (regions where the signal first rises steeply, then decays again towards higher energy), which are the result of energy losses through interactions with specific atoms (the edges are thus element-specific). The amount of inelastic scattering increases with specimen thickness (in general quite rapidly), dependent on the incident electron energy and on the material the beam is interacting with (in general the heavier the material, the stronger the interaction). An important parameter for EFTEM is the so-called Mean Free Path, which is a dimension that indicates the pathlength inside the specimen wherein all electrons (statistically speaking) will have undergone one inelastic scattering event (since some electrons will have undergone multiple scattering by then, after one mean free path there still remain some unscattered electrons). Typical mean free paths are of the order of 50-100nm for 120kV electrons, 100-200 for 200kV and 150-300 for 300kV. Above one mean free path is is still possible (and often even advantageous) to filter images in order to remove energy-loss electrons. However, the other main application - elemental mapping - becomes much more difficult and often even impossible at such specimen thicknesses. For good elemental mapping ultra-thin specimens are therefore important.

8.2 The (post-column) Imaging Filter For EFTEM, the microscope is equipped with an Imaging Filter, which is mounted underneath the projection chamber (a so-called post-column filter, as opposed to an in-column filter, where the filter itself forms part of the microscope column). The Filter accepts electrons passing through an entrance aperture


of 3 mm diameter in the center of the projection chamber. The electron beam passes through a (sector or prism) magnet that describes part of a circle. The sector magnet separates the electrons according to their energy into an energy spectrum. After the magnet lies a retractable slit and thereafter a series of lenses. The lenses restore the image (which can also be a diffraction pattern) at the entrance aperture so it can be viewed on a TV or recorded on a slow-scan camera.

8.3 EFTEM on the Tecnai microscope The post-column filter has a number of advantages over the in-column filter, but also one strong disadvantage: the image at the viewing screen is magnified by about 15 to 20x when it reaches the slow-scan camera. This implies that the field of view (in the image) or of the diffraction angles (in diffraction) would be seriously limited. At a minimum magnification in HM of about 2000x, the minimum EFTEM image would be about 40000x (EFTEM is possible in LM, but very limited because the objective lens is off). In order to get around this problem, the Tecnai microscope changes its magnification series between normal TEM (or screen down) and EFTEM (screen up). In EFTEM the magnification or camera length at the level of the viewing screen is about 10 to 20x smaller than in normal TEM, resulting in magnifications or camera lengths that are similar in TEM and EFTEM. The lower magnifications and camera lengths are possible because one requirement for normal TEM is relaxed: it is not necessary to illuminate the image across the whole viewing screen. It should be realised, however, that the total magnification of the magnification system (Diffraction, Intermediate and Projector lenses) is often very low and in some cases even less than one (the fixed contribution from the objective lens, in the range 20 to 50x - dependent on the type of objective lens, remains).

8.4 Important concepts in Tecnai EFTEM

8.4.1 Image, diffraction pattern and spectrum The microscope contains a number of image and diffraction planes and these play an important role in EFTEM imaging and diffraction under certain circumstances. The primary image plane is, of course, the specimen plane itself, while the first diffraction plane lies directly underneath at the back-focal plane of the objective lens (the level of the objective aperture and the first diffraction pattern). The second image plane occurs at the level of the SA aperture (called the first intermediate image, at least, for HM imaging). Thereafter the occurrence of the planes depends on the mode of operation: imaging or diffraction.

Schematic diagram of image and diffraction planes from the differential pumping aperture downward. The microscope is in diffraction and an image plane occurs at the level of the pumping aperture and at the level of the energy-loss spectrum. With an Imaging Filter, the diffraction pattern at the level of the viewing screen comes back after the lenses (not shown here) to the right of the slit.


In image mode, one image plane lies at the level of the viewing screen (or plate camera), with a second one at the level of the recording camera of the Imaging Filter (behind the imaging lenses of the Filter itself). In this mode, a diffraction plane occurs at the level of the differential pumping aperture (a 200 mm aperture that separates the projection chamber from the column) and a second at the slit of the Filter. The second diffraction plane thus combines a diffraction pattern with a spectrum. In diffraction mode, all image and diffraction planes are reversed with respect to the image mode. One important aspect about these image and diffraction planes is the inverse relation between magnification or camera length of these planes. Thus, if the magnification in image mode is high, then the size (camera length) of the diffraction pattern in the differential pumping aperture and at the slit of the Filter is small. If the image magnification is low, then the diffraction patterns are large. And, since the magnification series for EFTEM contain extremely small real magnification, the diffraction patterns in the pumping aperture and at the Filter slit can be enormous! The same applies to the images when the microscope is in diffraction with a small camera length. Under these circumstances (low magnifications, small camera lengths) alignments become highly critical and some phenomena may appear: • Astigmatism that can be strong and increases to lower magnifications due to charging of the slit of

the Filter. If the diffraction pattern at the spectrum plane becomes very large, the slit not only cuts off energy-loss electrons but also works like a very small and asymmetric objective aperture. This may lead to increasing image astigmatism towards lower magnifications. To avoid or reduce such effects, use a small objective aperture (thereby removing most of the diffracted beams) and use a somewhat larger slit width.

• The slit becomes visible as a shadow in the image or diffraction pattern. At low magnifications or camera lengths the electrons beams go through high angles relative to each other and, due to spherical aberration, cannot be focused properly at a single plane - the slit - causing the higher-angle beams to be cut off. Increase the size of the slit, if possible.

• Slight beam shifts make the diffraction pattern disappear at low camera lengths. When the image at the differential pumping aperture becomes very large (as happens at low camera lengths), even slight beam shifts may cause the beam to be intercepted by the differential pumping aperture. Use the specimen stage rather than the beam shift for aligning the area of interest for diffraction.

8.4.2 Cross-over correction From the discussion above, it will be clear that for proper EFTEM operation at low magnifications or small camera lengths the alignment of the beam in the differential pumping aperture will be critical (otherwise the whole beam may be blocked and no image or diffraction pattern is obtained at all). For this purpose a so-called cross-over alignment is used (the beam goes through a cross-over in the differential pumping aperture). The cross-over alignment shifts the image or diffraction pattern in the differential pumping aperture. When the microscope is aligned properly, the image or diffraction pattern on the screen does not shift. There is one important aspect to the cross-over correction: in image mode the cross-over correction is the same as a diffraction shift and vice versa (the cross-over correction uses the same image deflection coils as the image/diffraction shift. This means for example that the alignment values of the diffraction shift (the centering of all diffraction pattern together, not the alignment of the individual camera lengths) also affect the cross-over correction. Also if (by accident) a diffraction shift is executed in image mode (such as happens when the Diffr shift is constantly active on the Multifunction knobs), the cross-over correction will be affected.

8.4.3 Normalizations The image or diffraction shifts used for the image/diffraction alignment and the cross-over alignment can be very sensitive to the actual values of the magnetic fields of the lenses in the projection system


(Diffraction, Intermediate and Projector lenses). The lens normalizations bring these lenses to more reproducible settings - and, as a consequence, more reproducible image/diffraction shifts and cross-over corrections. It is therefore advised to allow the microscope to execute a projector-system normalization whenever the magnification or camera length is changed (an alternative to the automatic normalization it is also possible to do the normalization by hand, by assigning projector normalization to one of the Control Pad user buttons).

8.4.4 SA diffraction Because of the cross-over correction (which shifts the image while in diffraction, but the shift occurs before the SA aperture so the 'area of interest' moves relative to the SA aperture), SA diffraction is difficult to execute in EFTEM. In addition, the larger area of selection (relative to CBED) causes problems at low camera lengths (because the image in the differential pumping aperture and spectrum planes is so large) such as diffraction astigmatism that cannot be corrected. The best procedure for SA diffraction in EFTEM is as follows: • Focus the beam on (an uninteresting area of) the specimen. Use no SA aperture. • Switch to EFTEM and select a suitable camera length. Set up everything as needed. • Go back to normal TEM image operation. • Defocus the beam somewhat (but still no SA aperture) and center the area of interest in the beam. • Go again to EFTEM diffraction and observe the shadow image in the central (transmitted beam). • Insert an SA aperture (it should be visible from the shadow it casts in the diffraction shadow image). • Center the SA aperture around the area of interest. • Defocus the beam further until the beams in the diffraction pattern shrink to spots.

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Electron Microscope Services

Instructions

Multi Position Sample Holder

For

FEI Compustage

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FEI Compustage Multi Position Sample Holder Includes:- 1 Multi Sample holder 1 Loading jig with circlip expander 1 Circlip Extraction Tool 016 1 Circlip pusher 1 spare "0" ring 1 spare circlip 1 Instruction manual The specimen sample holder is always shipped and is best stored in the wooden storage box with the acrylic cover tube. If the rod is to be transported by post or courier then a packing piece needs to be placed between the handle and the box end.

Specimen loading / unloading Use the specimen loading jig at all times whilst removing or loading samples. This will prevent undue pressure on the bearing surfaces of the rod and prevent any distortion of the tip.

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Carefully place the rod onto the loading jig with the circlips facing upwards. Hold the rod steady using a gloved hand or lint free tissue between the hand and the rod, this will prevent tarnishing and will maintain the cleanliness of the rod. How to use the circlip extraction tool

Using the circlip extraction tool, place the end into the circlip. Press the circlip extraction tool (Circlip pliers) together to expand the tip and there by grip the internal grove of the circlip.

Grid

Circlip Pliers

Circlip

Sample holder

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Hold the specimen rod onto the stand and carefully pull out the circlip vertically upwards, to remove the circlip from the rod. With the circlip tool over a clean surface or over a piece of lint free tissue release the tension on the circlip extraction tool. This will release the circlip and allow the tool to continue removing further circlips. Place the specimen grids into the specimen rod hole vacated by the circlip and check that it is seated flat into the base. Again using the circlip extraction tool, place the end into the circlip. Press the circlip extraction tool together to expand the tip and there by grip the internal surface of the circlip. Press the circlip into the recess by pressing the expanded circlip tool squarely and vertically down into the circlip recess. Release the tension on the circlip extraction tool. This will release the circlip and allow the tool to be removed from the circlip which should now be held firmly by the rod, and there by securing the specimen grid.

Circlip Grid

Sample holder

Circlip pliers

Pressure here to grip the Circlip Ring

Circlip Pliers

Circlip

Reduce pressure here to release circlip

Sample holder Grid

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Check that the circlip has been pressed home and is in good contact with the grid with the cirlip pusher This will prevent circlip loss while entering the microscope and will prevent specimen drift and charging while under the beam of electrons. Note the use of the pusher tool is only for testing that the circlip is sitting squarly in its correct position. It is not an insertion tool.

If the circlip is not a good fit into the sample holder the circip will need to be expanded using the mandrill on the side of the loading jig. Regally inspect the circlip for signs of damage. (see maintenance section) View of the sample holder when it is inside the microscope

Grid

Sample on 3.05mm Grid

Circlip

Specimen sample holder

Sample holder

Circlip Pusher

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Inserting the rod into the microscope The original manufacturer instructions should be followed. Care should always be taken, never to damage the tip on entry to the microscope. Select standard holder when asked by the Microscope Computer, which type of sample holder? Changing samples while the sample holder is in the microscope Rotate the knob on the side of the handle to change samples while under the electron beam. Number 1 sample is nearest to the handle and number 4 sample is nearest to the tip. Clockwise moves the sample from position 1 to position 2

Changing samples

Rod insertion position Sample position 1

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Cleaning Remove the circlips from the rod and clean the tip of the rod and the circlip using as little mechanical polishing as is necessary to remove the tarnish. Wenol or any other suitable EM cleaning agent can be used. It is recommended that the cleaning agent is removed using 5% Quadraline / distilled water in an ultrasonic bath, followed by distilled water, followed by acetone or methanol. Only the first 30mm of rod end should be immersed into the liquids for cleaning. Other parts of the rod may be wiped with a solvent loaded lint free cloth. Maintenance It may become necessary during use, to apply a small film of vacuum grease to the external 'O' ring of the rod to assist insertion and withdrawal from the EM. We would recommend the use of Fombalin high vacuum grease in very small amounts, as per the Microscope manufacturers instructions. It is recommended after a period of time depending on use that the external 'O' ring on the rod be replaced. This is best achieved by removing the old ring using a wooden cocktail stick. Slide the sharp end of the cocktail stick under the 'O' ring and using a gloved hand roll the 'O' ring out of the grove and off the rod. Clean the 'O' ring grove using a polishing cloth and then wipe over the surface with a small amount of solvent Acetone, Ether, or any suitable EM cleaning solvent. During use the circlips may become loose in the specimen rod due to wear this will be noticed while loading the circlips into the rod. In this situation a circlip expansion mandrel has been provided in the loading jig. Using gloved hands or lint free tissue to hold the circlip push the circlip onto the expansion mandrel which is attached to the Vee block end on the specimen support jig. This will expand the external diameter of the circlip, and allow it to grip the rod internal surface once again.

Sample position 3

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If the circlips, during inspection are noticed to be none round or damaged it is recommended that they be replaced. See replacement parts section. Replacement parts: Circlips pks (3) EMS022/015 Circlip extraction tool EMS 022/016 External “O” ring EMS 022/ Ext. 0 ring

Circlip Mandril which is used to expand circlips

Push circlip up the mandrill

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Warranty E.M.S Specimen rods are guaranteed for a period of 12 months against faulty workmanship or faulty materials. Exclusions are:- When the bearing jewel has been damaged, When the rod has been bent, When the rod has been unassembled. For information about repair or cleaning of EMS or any other Sample holder contact:- Electron Microscope Services 2 Felsted, Markland Hill Bolton. BL1 5EY U.K. Tel/Fax 44- (0) 1204 845428 Or Tel/Fax 44- (0) 1942 876045 [email protected] The manufacturer reserves the right to make small changes to the product which may show as a slight variations in the operation manual.

TEM on-line help Options Version Tecnai 4.0 / Titan 1.1

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TEM on-line help manual -- Options Table of Contents 1 TEM Low Dose and Spotscan............................................................................................................. 5

1.1 Introduction to Low Dose imaging................................................................................................ 5 1.1.1 Beam blanker ........................................................................................................................... 5 1.1.2 Low dose states........................................................................................................................ 5 1.1.3 Low Dose setup........................................................................................................................ 7 1.1.4 An overview of Search, Focus and Exposure optics ................................................................ 8 1.1.5 Working with Low Dose in diffraction........................................................................................ 9 1.1.6 TEM control buttons and knobs.............................................................................................. 10 1.1.7 User levels in Low Dose ......................................................................................................... 10 1.1.8 Spotscan................................................................................................................................. 10

1.2 Low dose Control Panel ............................................................................................................. 11 1.2.1 Expose panel.......................................................................................................................... 13 1.2.2 Focus panel ............................................................................................................................ 15 1.2.3 Series panel............................................................................................................................ 16 1.2.4 Double panel .......................................................................................................................... 17

1.3 Low Dose Spotscan tab ............................................................................................................. 18 1.4 Low dose Settings tab................................................................................................................ 21 1.5 Low dose Calibrate tab .............................................................................................................. 23 1.6 Low dose Options tab ................................................................................................................ 26 1.7 Low dose TV / CCD tab ............................................................................................................. 28 1.8 Low Dose Settings ..................................................................................................................... 30 1.9 Low Dose calibrations ................................................................................................................ 31

2 TEM Photomontage .......................................................................................................................... 32 2.1 Introduction ................................................................................................................................ 32 2.2 Getting started............................................................................................................................ 32 2.3 Photomontage Control Panel .....................................................................................................32 2.4 Photomontage display................................................................................................................ 34 2.5 Recording on plate negatives..................................................................................................... 34

2.5.1 The display ............................................................................................................................. 35 2.5.2 The menu ............................................................................................................................... 36

2.5.2.1 File menu ..................................................................................................................... 36 2.5.2.2 Medium menu .............................................................................................................. 36 2.5.2.3 Calibrate menu ............................................................................................................ 36 2.5.2.4 Setup menu ................................................................................................................. 37 2.5.2.5 Montage menu............................................................................................................. 37 2.5.2.6 Exposure menu............................................................................................................ 37 2.5.2.7 Help menu ................................................................................................................... 37

2.5.3 Control options ....................................................................................................................... 38 2.5.4 Setting up for a montage ........................................................................................................ 40 2.5.5 Define boundary points........................................................................................................... 40 2.5.6 Set magnification .................................................................................................................... 41 2.5.7 Calibrate rotation .................................................................................................................... 41 2.5.8 Modifying a montage .............................................................................................................. 41 2.5.9 Automatic exposure................................................................................................................ 42 2.5.10 Manual exposure .................................................................................................................... 43

2.6 Recording on CCD and automatic montage .............................................................................. 44


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2.6.1 The display ............................................................................................................................. 45 2.6.2 The menu ............................................................................................................................... 46

2.6.2.1 File menu ..................................................................................................................... 46 2.6.2.2 Medium menu .............................................................................................................. 47 2.6.2.3 Calibrate menu ............................................................................................................ 47 2.6.2.4 Magnification calibration .............................................................................................. 48 2.6.2.5 Image-shift calibration.................................................................................................. 48 2.6.2.6 Stage calibration .......................................................................................................... 48 2.6.2.7 Setup menu ................................................................................................................. 49 2.6.2.8 Exposure menu............................................................................................................ 49 2.6.2.9 Mount menu................................................................................................................. 49 2.6.2.10 Help menu ................................................................................................................... 49

2.6.3 Control options ....................................................................................................................... 50 2.6.4 Setting up for a montage ........................................................................................................ 51 2.6.5 Acquire image......................................................................................................................... 52 2.6.6 Define area ............................................................................................................................. 52 2.6.7 Set magnification .................................................................................................................... 53 2.6.8 Automatic exposure................................................................................................................ 54 2.6.9 Manual exposure .................................................................................................................... 54 2.6.10 Mounting the exposures into the montage ............................................................................. 55

2.7 File formats ................................................................................................................................ 56 2.7.1 Binary data type...................................................................................................................... 56 2.7.2 Extended-header format.........................................................................................................56 2.7.3 MRC file format....................................................................................................................... 58

2.7.3.1 The primary header. .................................................................................................... 58 2.7.3.2 The extended header................................................................................................... 59

3 TEM Grid Scanning ........................................................................................................................... 61 3.1 Introduction ................................................................................................................................ 61 3.2 Getting started............................................................................................................................ 61 3.3 Grid scanning Control panel....................................................................................................... 61 3.4 Grid scanning display................................................................................................................. 63 3.5 The display................................................................................................................................. 63 3.6 The menu ................................................................................................................................... 64

3.6.1 File menu................................................................................................................................ 64 3.6.2 Setup menu ............................................................................................................................ 65 3.6.3 Hole menu .............................................................................................................................. 65 3.6.4 Location menu ........................................................................................................................ 65 3.6.5 Help menu .............................................................................................................................. 65

3.7 Control options ........................................................................................................................... 66 3.8 Calibrate grid.............................................................................................................................. 68 3.9 Reference points ........................................................................................................................ 68 3.10 Show ref.pt. data ........................................................................................................................ 70 3.11 New spiral .................................................................................................................................. 70

4 TEM Smart Tilt .................................................................................................................................. 71 4.1 Getting started............................................................................................................................ 71 4.2 Smart Tilt Control Panel ............................................................................................................. 71 4.3 Smart Tilt Options ...................................................................................................................... 73 4.4 Smart Tilt display........................................................................................................................ 74 4.5 The display................................................................................................................................. 74 4.6 The menu ................................................................................................................................... 75

4.6.1 File menu................................................................................................................................ 75 4.6.2 Calibrate menu ....................................................................................................................... 75 4.6.3 Tilt menu................................................................................................................................. 76


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4.6.4 Focus menu............................................................................................................................ 76 4.6.5 Help menu .............................................................................................................................. 77

4.7 Camera length calibration .......................................................................................................... 77 4.8 Alpha and beta tilt calibration ..................................................................................................... 77 4.9 Full (zone axis) tilt ...................................................................................................................... 78 4.10 Bragg tilt ..................................................................................................................................... 79 4.11 Focussed diffraction and shadow image.................................................................................... 80

5 TEM Compucentricity ........................................................................................................................ 81 5.1 Introduction ................................................................................................................................ 81 5.2 Getting started............................................................................................................................ 82 5.3 Compucentricity Control Panel................................................................................................... 82 5.4 Compucentricity Options ............................................................................................................ 83 5.5 Compucentricity Data................................................................................................................. 83 5.6 Compucentricity Calibrate .......................................................................................................... 84 5.7 Compucentricity Cal. data .......................................................................................................... 86

6 TEM k-Space Control ........................................................................................................................ 87 6.1 Introduction ................................................................................................................................ 87 6.2 Getting started............................................................................................................................ 87 6.3 General introduction................................................................................................................... 87

6.3.1 Definition of the crystal structure ............................................................................................87 6.3.2 Determination of the orientation of the crystal ........................................................................ 88 6.3.3 Changing the orientation ........................................................................................................ 88

6.4 Display elements........................................................................................................................ 88 6.4.1 Lab grid................................................................................................................................... 88 6.4.2 Stage grid ............................................................................................................................... 89 6.4.3 Zone axes............................................................................................................................... 89 6.4.4 Kikuchi map ............................................................................................................................ 90 6.4.5 Diffraction pattern ................................................................................................................... 93 6.4.6 Lattice ..................................................................................................................................... 93 6.4.7 Markers................................................................................................................................... 94

6.5 Two-zone indexing ..................................................................................................................... 95 6.5.1 Two-zone indexing dialog.......................................................................................................95 6.5.2 Zone axis calculator dialog ..................................................................................................... 97 6.5.3 Match constraints dialog......................................................................................................... 98

6.6 Two-zone indexing procedure in steps ...................................................................................... 99 6.7 Manual indexing ....................................................................................................................... 103 6.8 Special keyboard keys ............................................................................................................. 104 6.9 k-Space Control Control Panel................................................................................................. 105 6.10 k-Space Control Crystal ........................................................................................................... 108 6.11 k-Space Control Info ................................................................................................................ 110 6.12 k-Space Control Display........................................................................................................... 111 6.13 k-Space Control View............................................................................................................... 113

7 TEM Free Lens Control ................................................................................................................... 114 7.1 Introduction .............................................................................................................................. 114 7.2 Free Lens Control Panel .......................................................................................................... 115 7.3 Free Lens Registers................................................................................................................. 116

8 Magnification Calibration ................................................................................................................. 118 8.1 Magnification Calibration Control Panel ................................................................................... 118

8.1.1 The HM magnification calibration procedure ........................................................................ 119 8.1.2 The Mi plus SA magnification calibration procedure............................................................. 120 8.1.3 The HR-TEM magnification calibration procedure................................................................ 120 8.1.4 The LM magnification calibration procedure......................................................................... 120 8.1.5 The D camera length calibration procedure ......................................................................... 120


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8.1.6 The HM-STEM magnification calibration procedure............................................................. 121 8.1.7 The LM STEM magnification calibration procedure.............................................................. 121 8.1.8 Measurement on cross-grating............................................................................................. 122 8.1.9 Measurement with image shift .............................................................................................. 123 8.1.10 Measurement on lattice image ............................................................................................. 123 8.1.11 Diffraction-ring analysis ........................................................................................................ 124 8.1.12 Measurement with diffraction shift ........................................................................................ 126

8.2 Magnification Calibration Report Control Panel ....................................................................... 126 8.2.1 Tabular format report ............................................................................................................ 128 8.2.2 History format report ............................................................................................................. 130

8.3 Magnification Calibration File Control Panel ............................................................................ 131 8.4 Magnification Calibration Options Control Panel...................................................................... 131

9 TEM AutoGun (FP5460/20)............................................................................................................. 133 9.1 AutoGun procedures ................................................................................................................ 133

9.1.1 Auto Conditoning .................................................................................................................. 133 9.1.2 Auto Align ............................................................................................................................. 133

9.2 AutoGun (User) – (Thermionic) ................................................................................................ 135 9.3 AutoGun (Expert) – (Thermionic) ............................................................................................. 137 9.4 AutoGun Control (Expert) – (Thermionic)................................................................................. 139 9.5 AutoGun (Supervisor) – (Thermionic) ...................................................................................... 140 9.6 AutoGun Control (Supervisor) – (Thermionic).......................................................................... 142 AutoGun (User) – (FEG) ..................................................................................................................... 144 9.7 AutoGun (Expert) – (FEG) .......................................................................................................146 9.8 AutoGun Control (Expert) – (FEG)........................................................................................... 148 9.9 AutoGun (Supervisor) – (FEG)................................................................................................. 149 9.10 AutoGun Control (Supervisor) – (FEG) .................................................................................... 151

10 AutoAdjust ................................................................................................................................... 152 10.1 AutoAdjust (User) Control Panel .............................................................................................. 152 10.2 AutoAdjust (Expert) Control Panel ........................................................................................... 154 10.3 AutoAdjust Calibrate (Expert) Control Panel ............................................................................ 156

10.3.1 Calibrations........................................................................................................................... 156 11 TrueImage™ Focus Series Acquisition ....................................................................................... 159

11.1 Introduction .............................................................................................................................. 159 11.2 Automated Experiments Control Panel .................................................................................... 159 11.3 TrueImage™ Focus Series Acquisition Settings...................................................................... 160 11.4 Settings .................................................................................................................................... 161

11.4.1 Acquisition parameters .........................................................................................................161 11.4.2 Reconstruction parameters .................................................................................................. 163

11.5 File formats .............................................................................................................................. 164 11.5.1 Binary data type.................................................................................................................... 164 11.5.2 Extended-header format....................................................................................................... 164 11.5.3 MRC file format..................................................................................................................... 165

11.5.3.1 The primary header. .................................................................................................. 165 11.5.3.2 The extended header................................................................................................. 167


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1 TEM Low Dose and Spotscan

1.1 Introduction to Low Dose imaging Radiation-sensitive materials get damaged when viewed in the electron microscope. In some cases the damage is tolerable, as it is too small to affect the validity of the information extracted from the electron microscope. In other cases, the damage itself is the ultimate limitation to the information sought (not the microscope-related parameters such as contrast or resolution), and it is, therefore, necessary to keep the total radiation on the specimen at a minimum level. For example, the atomic structure of proteins and nucleid acids falls apart significantly after receiving a dose of about 10 to 20 e-/Ǻ², even at liquid nitrogen temperature. As a consequence, the current density on the detector cannot be increased arbitrarily and images of these materials are intrinsically noisy. Since the allowed exposures are already severely limited by this condition, any pre-exposure of the area of interest is undesirable. Low dose strategies, therefore, are designed to minimize such pre-exposure to a level that is negligible compared to the tolerance of the specimen. The Low Dose software provides a number of dedicated tools to accomplish that. Low Dose works on film as well as on CCD cameras. Please note that the CCD camera must support use of the beam blanking (pre-specimen "alternate shutter") for shuttering to be effective in Low Dose. To use Low Dose: • Open the Workspace layout (bottom-right selection list) and drag the Low Dose Control Panel into a

workset. • Go to the tab of the workset and press the Low Dose button.

1.1.1 Beam blanker In order to prevent accidental exposure of the specimen by electrons the Low Dose software uses a beam blanker. This blanker deflects the beam in the electron gun by a gun tilt (by an amount that can be calibrated by the user). The beam blanker is used to blank the beam during state transitions (see below) and it acts as the actual exposure shutter for the Low Dose exposure on plate (for the CCD exposures the "alternate shutter" of the CCD is used and the beam blanker is only used under circumstances where the CCD does not close the shutter - when the fluorescent screen is down). The beam blanker is also under user control, so the user can blank the beam for as long as needed.

1.1.2 Low dose states Low dose contain three states that have a certain degree of optical independence : • Search • Focus • Exposure The general idea is to use the Search state to search the specimen under very low dose and high-contrast conditions for suitable areas. There are two ways of doing this: • Using a low magnification at a high defocus. • Using defocused diffraction. In either case one must choose a high spot number to ensure that the electron dose at the specimen is at most a few hundredths of an e-/Ǻ²s. Once a suitable area has been identified, Low Dose is switched to the Focus state. This state should be set to a magnification that is well-suited for focusing and astigmatism correction. The actual area of interest for recording should be avoided, so the Focus state uses an off-axis image shift (with a corresponding beam shift) to allow performing focus and stigmator


6

adjustments on an adjacent area. The location of that area is defined through distance and rotation controls. Two separate positions can be set, e.g. when one of the positions falls on a grid bar or for focusing a tilted specimens.

Schematic diagram of the operation of the off-axis shift of the Focus state. The user controls the image shift below the specimen, thereby bringing an off-axis area (tan ray path) into view. The microscope automatically compensates the image shift with a beam shift to keep the illumination centered on the area currently in view (otherwise the illumination would move away from the center and remain on the area that should not be damaged). The Exposure state has the conditions appropriate for the actual exposure. Under normal circumstances the Exposure state is switched to only initially during setup (defining the Low Dose settings) or after an exposure as a check.

Condenser and aperture

Beam shift

Minicondenserlens

Objectivelens

Image shift

SA plane

Objectiveaperture

Image deflectioncoils

Objective-imaging

lens

Specimen

Objective-condenser

lens

Beam deflectioncoils


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1.1.3 Low Dose setup Unless use is made of previously established conditions, the normal way of operation of the Low Dose function consists of : 1. Preparation

a) Switch to the Exposure state (press Exposure button). Select the Settings tab in the flap-out and press Exposure Reset. Set conditions like magnification, spot size, intensity. Center the beam. Read off the exposure time and enter it in the Expose panel. Use the electron dose rate read-out to verify that the dose on the specimen per exposure will be below the tolerance limit.

b) Switch to the Focus state (press Focus button). Select the Settings tab in the flap-out and press Focus Reset. Set conditions like magnification, spot size, intensity. Set the required off-axis shift (with Multifunction X,Y or the trackbars in the Focus panel). Center the beam. If necessary repeat for the second Focus substate (except for magnification, spot size and intensity which are the same for the two substates). Make sure that the illumination of the focus areas does not overlap with the area of interest (Exposure state area).

c) Switch to the Search state (press Search button). (If the microscope is equipped with an off-axis TV that must be used in the Search state, use the TV toggle to switch to the off-axis detector position.) Select the Settings tab in the flap-out and press Search Reset. Set conditions like magnification, spot size, intensity. Switch to Exposure state and center a recognizable image feature with the goniometer, then switch back to the Search state and center the same feature using the Multifunction X,Y knobs. Verify that the electron dose rate on the specimen is negligible (i.e. ≤ 0.01 e-/Ǻ²s). On Titan you can use the Dose rate display value in the TEM user interface information area.

d) Switch to Exposure, Focus and Search again and check the conditions (especially centering of the beam).

e) If necessary, recenter the beam and repeat the whole procedure. f) To check for overlap between the focus-state illumination and the exposure area, leave the

illumination on a suitable specimen (one that will show damage) for a while in both focus and exposure states, then check on the imprints left by the beam at low magnification (Search state). The imprint of the Focus illumination which should be off-center) should not overlap with that of the Exposure state.

2. Operation

a) Switch to the Search state and locate a number of good specimen areas. Store these using the Stage control panel function.

b) (If the microscope is equipped with an off-axis TV that must be used in the Search state, use the TV toggle to switch to the off-axis detector position.)

c) Press the Blank button (blank the beam). d) Move to the first good area (or the best) and wait a short time to allow the goniometer to settle. e) Press the Blank button again (unblank the beam). f) If necessary, center the area accurately with the goniometer. g) Switch to Focus and focus the image. h) (If the microscope is equipped with an on-axis CCD that must be used in the Focus state, use the

TV toggle to switch to the on-axis detector position.)


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i) If necessary, press the Blank button (blank the beam). j) Press the Expose button (or the Exposure button on the TEM Control Pad) to make the

exposure. k) (If the microscope is equipped with an off-axis TV that must be used in the Search state, the

detector shift is set to on-axis automatically before the exposure and reset to its original position afterwards.)

l) After the exposure is finished, go to the Exposure state and check the conditions (unless another low-dose exposure must be made at the same position).

1.1.4 An overview of Search, Focus and Exposure optics The settings of Search, Focus and Exposure achieve a certain amount of decoupling between the states. In general the Search state is almost totally independent of Focus and Exposure, while some settings of the Focus state are coupled to the Exposure state. Spotsize The spotsize (C1 lens) is independent for all three states.

Intensity The intensity (C2 lens) is independent for all three states.

Illumination modes The illumination modes are independent between Search on the one hand and

Focus and Exposure on the other (Focus and Exposure are thus the same by definition). This means e.g. that Search may be in Microprobe and Focus/Exposure in Nanoprobe.

Imaging modes The imaging modes are independent between Search on the one hand and Focus and Exposure on the other (Focus and Exposure are thus the same by definition). This means e.g. that Search may be in diffraction or LM and Focus/Exposure in HM imaging (Microprobe).

Focus The focus settings are independent between Search on the one hand and Focus and Exposure on the other (Focus and Exposure are thus the same by definition). Separate settings are stored by the software for : • HM Image focus • LM Image focus • D (HM) diffraction • LAD (LM) diffraction Focus differences between magnifications used for the Focus and Exposure states can be either compensated (properly) by using the Parfocal Magnification series alignment. As an alternative, the focus difference can be measured and entered as a single-exposure series and series switched on. The software will the automatically apply the focus change (but only during the actual recording of the exposure, not when the state is changed from Focus to Exposure).

Beam shift The Search and Focus states react to beam shifts in Exposure states and in their own states. The beam shift is built up as follows. First of all any 'User' beam shift at the start of using Low Dose is ignored (this means that the right point to start is to define the Low Dose settings with the beam well aligned by means of the 'Align Beam Shift' alignment). The Exposure state stores its own settings for the beam shift, made during Low Dose operation. The Focus and Search states each have their own independent beam shifts on top of the Exposure beam shift. The Focus beam shift comes on top of the compensation for the image shift.


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Image shift The Search state has an independent image shift relative to the Exposure state to allow accurate alignment of the Search image to that of the Exposure image to ensure recording of the proper area when coming from a Search state at much lower magnification. The Search state image shift can be set only by using the Multifunction knobs. The Focus state image shift is a totally different setting because it uses the image shift with compensated beam shift.

Diffraction shift The Search state has a setting for the diffraction shift that is added to any shift in Focus and Exposure (where the diffraction shift is the same). The Search shift is somewhat independent to allow compensation of diffraction shifts introduced by strong defocussing (as often applied in the Search state).

1.1.5 Working with Low Dose in diffraction For Low Dose diffraction the software provides special functionality in the form of the Peek function. This allows a quick 'peek' at the diffraction pattern under the Exposure conditions but with a strongly reduced beam intensity (achieved through a gun tilt, like the beam blanker). This function can be used (if necessary) to insert the beam stop accurately at the transmitted (central) beam. The 'normal' way of operating the peek function is:

a) While in the Focus state switch the Beam blanker on. b) Switch to the Exposure state. c) Switch Peek on (the beam blanker will switch off). d) Shift the diffraction pattern or beam stop as needed. e) Switch Peek off (the beam blanker will be re-activated. f) Take the exposure (Expose button). g) The Peek function must be calibrated before it can be used.

Note: The Peek function is only enabled when: • Low Dose is in the Exposure state. • The function itself is enabled (Options tab). Special note about focussing the diffraction pattern It may be noticed that the diffraction pattern moves sideways strongly when the diffraction focus is adjusted in the Focus state. This is a normal consequence of the off-axis shift employed - the ray path goes at an angle through the diffraction lens, which is the focussing lens in diffraction. To focus the diffraction pattern for Focus and Exposure states, always go to an area that may be damaged and focus the pattern in the Exposure state. The sideways movement of the pattern may not be gone totally but it will be considerably less than in the Focus state.


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1.1.6 TEM control buttons and knobs Low Dose uses a number of buttons and knobs on the Control Pads of the TEM microscope. The assignment of the user buttons is flexible and can be switched on/off at will: • Search state (default L1) • Focus state or toggle between Focus 1 and 2 substates (default L2) • Exposure state (default R1) • Beam blanker toggle on and off (default R2) • Peek (default R3) • Exposure starts the actual Low Dose exposure • Multifunction X and Y

- Align the image in the Search state - Set off-axis shift for the Focus state

These buttons and knobs are assigned in the Options tab of the Low Dose flap-out. Low Dose keeps track of any other assignment to these buttons and knobs (e.g. on alignment or stigmation). If a button or knob is given another function, its will no longer affect Low Dose (so pressing L1 when the L1 function has been changed doesn't switch to Search anymore). As soon as the button or knob has been released, it gets back its Low Dose functionality. When Low Dose is stopped (by pressing the Low Dose button or exiting the TEM User Interface), the buttons and knobs get back their original setting.

1.1.7 User levels in Low Dose The Low Dose software supports two user levels, 'user' and 'supervisor'. Supervisor settings (both Low Dose settings and calibrations) are stored separately from those of the user. When a new user starts Low Dose for the first time, the settings and calibrations used are those defined by the supervisor. Thereafter the user has set his/her own settings and these will be retrieved when the software is started. The only way thereafter to go back to the supervisor settings is to have the supervisor save settings and calibrations files and load these files as a user. Low dose settings and calibrations saved by Factory or Service are equivalent to Supervisor settings.

1.1.8 Spotscan Spotscan is an option for the Low Dose software. It relies on hardware installed in the microscope PC and microscope and will not function without the proper hardware. The Low Dose version with spotscan supports two modes of exposure: • The normal 'flood-beam' exposure wherein the whole exposure area is illuminated by a broad beam

and exposed together. • The spotscan exposure wherein a smaller beam is stepped across the area to be exposed and each

spot is exposed individually. Spotscan settings are defined in the Spotscan tab of the Low Dose Control Panel flap-out.


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1.2 Low dose Control Panel The Low Dose Control Panel contains the functions used for Low Dose electron microscopy. A more extensive description of Low Dose and its use is given on the Low Dose page. Important notes: Low Dose only supports operation in a restricted set of optical conditions. Note supported are the STEM mode on any microscope and the Porbe and Free Condenser Control modes on Titan. Low Dose does not support EFTEM handling. You can use the EFTEM lens series but the actual series (normal or EFTEM) is not stored with the Low Dose settings and Low Dose does not switch between the two lens series during operation. Low dose button The Low Dose button activates or deactivates Low Dose. The status can be seen from the color of the button (gray is off, yellow is on). When Low Dose is activated, it saves the currently active settings of the microscope and changes them to Low Dose settings (Search at start-up, any other state when Low Dose has been active previously). When Low Dose is switched off, it restores the microscope to the status it had before Low Dose was switched on.

Note: The magnifications (or cameral lengths) listed for the three Low Dose states are the so-called reference magnifications (the values on the plate camera, that is, the ones displayed by the TEM User Interface with the main screen up). These fixed values are used to avoid confusion (which could occur when Low Dose would continuously adjust values when the screen goes up and down). The values with the screen down are roughly 10% lower than with the screen up. Blank button The Blank button toggles the beam blanker on and off. The beam is blanked (that is, no electrons come down the column) when the button is yellow. The beam blanker uses an offset on the gun tilt which is calibrated (by supervisor or user). Note that the beam blanker may also used by other software (e.g. during normalizations). The status of the Blank button will reflect that. Peek button The Peek button toggles the diffraction peek function on and off. When Peek is active the beam blanker is used with a reduced blanking amplitude that should be calibrated so just enough electrons are coming down the column to allow centering or focusing of the diffraction pattern. The button is visible only when the function is enabled in the Options tab of the Low Dose flap-out. The Peek button is only enabled if the following two conditions are met: 1. The Exposure state is active. 2. The microscope is in diffraction.


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The 'normal' way of operating the peek function is: 1. While in the Focus state switch the Beam blanker on. 2. Switch to the Exposure state. 3. Switch Peek on (the beam blanker will switch off). 4. Shift the diffraction pattern or beam stop as needed. 5. Switch Peek off (the beam blanker will be re-activated). 6. Take the exposure (Expose button). The Peek function must be calibrated before it can be used. Status The status line displays the current Low Dose status as well as any error messages.. Search button The Search button switches Low Dose to the Search state. Search settings The settings used for the Search state (microscope mode, magnification or camera length, spot size, intensity and image shifts) are displayed underneath the Search button. Search start button The Start button starts image acquisition on the CCD camera in the Search state. It is enabled only when a CCD has been identified, selected for operation in Search and Low Dose is in the Search state. Focus button Pressing the Focus button switches the Low Dose function to the Focus state. Focus substates The Focus state has two substates, 1 and 2, that can have different settings for the off-axis beam and image shifts. Switching between the two substates is done by selecting the corresponding radio button on the Low Dose control panel or pressing the TEM User Button selected when the Focus state is already active. Focus settings The settings used for the Focus state (magnification or camera length, spot size, intensity and image shifts) are displayed underneath the Focus button. The microscope mode is not displayed because it is, by definition, the same as for the Exposure state. Focus start button The Start button starts image acquisition on the CCD camera in the Focus state. It is enabled only when a CCD has been identified, selected for operation in Focus and Low Dose is in the Focus state. Exposure button Pressing the Exposure button switches the Low Dose function to the Exposure state. This is not the same as making the plate-camera exposure! The Exposure state activated by the Exposure button is meant for setting up the conditions under which the Low Dose exposures will be made. Exposure settings The settings used for the Exposure state (microscope mode, magnification or camera length, spot size, and intensity; in addition the exposure time is shown at the bottom) are displayed underneath the Exposure button.


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Flap out button Pressing the flap-out button leads to the Low Dose flap-out, containing the Spotscan (if present), Settings, Calibrate, Options and TV /CCD tabs.

1.2.1 Expose panel

Expose button When the Expose button (or the TEM Exposure button on the left-hand Control Pad) is pressed, the actual Low Dose exposure is started. The progress of the exposure is displayed in the status as well as by the status of the state and blank buttons. The medium for exposure (plate camera or CCD) depends on the setting chosen. Series The Series check box enables the automatic exposure of a series. The settings for the series are defined in the Series panel. The Series check box is only enabled when at least one setting has been defined for series. Series and Double cannot be used together. Double The Double check box enables double exposure (multiple exposures on a single plate). The exposure times for the double exposures are defined in the Double panel. The Double check box is only enabled when at least one setting has been defined for double. Double and Series or Use spotscan cannot be used together. Use spotscan Only on systems where spotscan is installed : When the Use spotscan option is enabled the exposure(s) will be taken as spotscans, not flood-beam exposures. The Exposure time will change to spot dwell time (exposure time per spot). The spotscan as executed is defined in the Spotscan tab of the flap-out. Spotscan and pre-exposure or Double cannot be combined. Dim screen In order to prevent light from the monitor from striking the plate (which is lying open in the projection chamber during the exposure; it is therefore always important to switch room lights off and cover the windows of the projection chamber), the monitor screen can be blanked during the exposure. The blanking will initially display text in dark gray (instructions on how to remove the screen dim if it doesn't remove itself after exposure). This text can be removed by unchecking the Text visible check box in the top left corner. The setting will be remembered from one microscope session to the next.


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Notes: • Should the screen dimmer fail to be removed after the exposure, then it can always be removed by

pressing Shift+Esc on the keyboard. Do not use Alt+Tab to switch to another window because that will (except in exceptional cases) not work.

• This option does not work when a CCD is used as recording medium (there is no danger of light leaking from the monitors onto the CCD).

Exposure time Because the conditions during operation (search and focus) are not suitable for measuring the exposure time to be used during the actual Low Dose exposure (due to differing magnification/camera length, spot size and intensity settings), the exposure time must be set by the operator. Switch to the Exposure state initially and determine the exposure time as indicated by the plate camera of the microscope. If necessary, adjust the exposure settings (spot size, intensity) and then enter the desired exposure time on the Plate camera panel (type or enter by pressing the up or down spin buttons). If the CCD is used for exposure, the proper exposure time must be entered in the camera setup (NOT in DigitalMicrograph but in the TEM Control Panel for MSC cameras). Low dose itself does not define CCD settings. While Low Dose is active, the manual exposure of the plate camera is set to the same setting as the exposure time for Low Dose plate exposures. When Low Dose is switched off, the plate camera manual exposure time setting that was active before Low Dose was switched on is restored. If spotscan is used : The dwell time (in milliseconds) determines how long each individual spot will be exposed. The scan board that drives the beam itself allows dwell times down to 1 microsecond. The beam deflection coils of the microscope cannot support such high scan rates (the scan will not consist of individually stepped spots but a blurred line) and advised dwell times are 10 milliseconds or higher. To estimate the dwell time, select the required spotscan settings, go to the Exposure state and insert the small viewing screen. Read off the auto exposure time in the Plate camera control panel of the microscope. Estimate the ratio of the spot diameter and the small screen diameter. Divide the measured exposure time by the square of the ratio (it goes by area). This will be the dwell time. If the spotscan spot is much smaller than the small screen, increase the magnification so the spot makes up about 1/3 to 1/2 of the small screen. Estimate the ratio as described above but modify it further by dividing it by the square of the ratio of the measurement magnification and the true exposure magnification (once again it goes by area). For good results, you may want to bracket initial attempts (take one exposure at a 3x shorter dwell time, one at the dwell determined and another at a 3x longer dwell time) and thereby calibrate the required dwell times. If Double is active : The exposure time as indicated is not used. Instead the exposure times as defined for double exposure are used. Wait time In order to allow the microscope to stabilize after the mechanical 'shocks' from the loading of the plate a waiting time is set before the exposure is taken. For high resolution allow at least 5 seconds waiting time. At lower magnification or lower resolution a shorter waiting time can be set. Pre-exposure The Low Dose mode allows pre-exposing the specimen (the beam is put on the specimen but the exposure shutter of the microscope stays closed so this exposure has no effect on the plate) to allow stabilization (in case of charging). When this function is enabled, it is possible to choose a pre-exposure time.


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Note: When pre-exposure is active, the resolution of the exposure may not be as good as ultimately achievable in Low Dose due to drift of the shutter (below the specimen) during the actual exposure, especially if the waiting time after the pre-exposure is short (1 second or less). During Low Dose exposure without pre-exposure the shutter is opened several seconds before the beam is unblanked for the Low Dose exposure. Wait time after pre-exposure Unless a waiting time is set after the pre-exposure, the real Low Dose exposure takes place as quickly as possible after the pre-exposure. When the waiting time is enabled (through the checkbox), the waiting time can be set.

1.2.2 Focus panel

The off-axis shift for the Focus state is controlled by distance and (rotation) angle. Thereby it is possible to circle around an area of interest by simply changing the rotation angle, without any danger of accidentally moving towards the real area of interest. Circling may be necessary, e.g. in the case where the off-axis area lies on a grid bar. Once calibrated the 0 and 180° angles will lie along the tilt axis. Focussing with a tilted specimen can be done either at 0 or 180° (where the focus will be the same as on the area of interest provided the specimen is flat) or at 90 and 270° with an equal off-axis distance and taking the focus setting halfway in between. Distance The off-axis shift distance can be set with the distance trackbar and the Multifunction X knob. The distance should be set such that the off-axis shift is sufficient to ensure that the Focus-state beam does not overlap onto the Exposure area, while as the same time keeping it as small as possible. The trackbar is active only in the Focus state. Angle The off-axis shift rotation angle can be set with the angle trackbar and the Multifunction Y knob. The trackbar is active only in the Focus state.


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1.2.3 Series panel

The series panel contains settings used for automated through-focus series. The through-focus series is totally flexible. It simply will execute as many exposures as there are entries in the list, changing the focus by the amount specified for the particular setting. Multiple entries with the same value, or the value 0 are allowed. It is also allowed to enter a single non-zero value In that case each Low Dose exposure will be made with the focus between Focus state and Exposure state offset by the specified amount (making it e.g. possible to focus in the Focus state to minimum contrast - close to zero or Gaussian focus - and have each Low Dose image recorded at -1000 nm). Note: The focus settings are not absolute but are values relative to the focus set when the exposure series is started. Focus setting The Focus settings list is filled by entering values for the focus (underfocus is taken as negative) in the Focus setting edit control in nanometers and pressing the Add button. Focus settings list The Focus settings list contains the list of values used for the through-focus series. Add button The Add button allows insertion of a new setting into the focus settings list. The value for the new focus must be inserted into the Focus setting edit control. The maximum number of values is 20. Delete button The Delete button allows removal of settings from the list. Select a setting and press Delete. Move up The through-focus exposures are recorded in the sequence as they occur in the list. To change the sequence, select a setting and press Move up to move it up in the list. Move down The through-focus exposures are recorded in the sequence as they occur in the list. To change the sequence, select a setting and press Move down to move it down in the list.


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1.2.4 Double panel

The double panel contains settings used for recording double exposure (multiple exposures on a single plate). The double exposure setup is totally flexible. It simply will execute as many exposures as there are entries in the list, changing the exposure times as indicated in the list for the particular setting. The double exposure sequence is similar to that for a normal low-dose exposure except that the plate is not removed until the last double exposure has been done (or the exposure is cancelled). After one exposure has been recorded, further execution pauses until the operator presses the Expose button (on the Low Dose Control Panel) or the Exposure button (on the left-hand TEM Control Pad) again. Pressing either of these buttons while an exposure is taking place (the Expose button is yellow) cancels further exposure, otherwise it starts the next exposure in the sequence. When dim screen is active, the dimmed screen will display a message alerting the operator to press Exposure to start the next exposure. Between individual exposures of a double exposure sequence, it is possible to lower the screen and look at the image. Be aware, however, that the Low Dose state remains in Exposure. The Peek and Beam Blanker functions become accessible when the screen is lowered. When Double exposure is selected in the Expose tab, the Low Dose exposure time as listed becomes meaningless, and the double exposure times in the list are used instead. Exposure time The exposure times list is filled by entering values for the exposure time (in seconds) in the exposure time edit control and pressing the Add button. Exposure times list The exposure times list contains the list of values used for the double exposure times. The maximum number of values is 20. Add button The Add button allows insertion of a new setting into the exposure times list. The value for the new exposure time must be inserted into the exposure time edit control. Delete button The Delete button allows removal of settings from the list. Select a setting and press Delete. Move up The double exposures are recorded in the sequence as they occur in the list. To change the sequence, select a setting and press Move up to move it up in the list.


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Move down The double exposures are recorded in the sequence as they occur in the list. To change the sequence, select a setting and press Move down to move it down in the list.

1.3 Low Dose Spotscan tab Only if spotscan is installed: The Spotscan tab of the Low Dose Control Panel contains the controls for defining and inspecting the spotscan. Note: Spotscan has typically been used on plate negatives. Low Dose allows the use on CCD cameras. In that case, make sure the area covered by the spotscan is larger than the area covered by the CCD. Also set a CCD exposure time that is sufficient to allow effective exposure of the whole spotscan array (since Low Dose does not control the actual hardware shuttering during a CCD exposure, there are uncertainties in the timing and if you set the CCD exposure time too short, the CCD image may not have all spots exposed). A typical value of 5 seconds is usually sufficient. Since the beam will be positioned away from the center before the spotscan exposure starts and only a single scan is executed, a longer CCD exposure time has no negative side effects (e.g. beam damage).

Pattern The spotscan can be executed in either a hexagonal pattern or a square pattern. In the case of a hexagonal pattern, the closest packing of the spots is achieved, with each second row of spots displaced 1/2 spot (as a result each spot has six nearest neighbors, arranged in a hexagon around it). In the square pattern, the spots lie at the corners of squares (and each spot has four neighbors: above, left, right and below). Orientation The spotscan pattern can be rotated through 360 degrees. When calibrated, the 0 and 180° directions have the x direction of the spotscan coincide with the a tilt axis of the CompuStage. Focus correction An automatic focus correction can be applied to spotscan exposures for tilted specimens. The focus correction required is automatically derived from the CompuStage alpha tilt angle. If you use pre-tilted specimens or pre-tilted cartridges, specify an offset angle to compensate for the difference between the CompuStage alpha tilt value and the real tilt angle of the specimen.


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For focus correction to work properly, it is very important that the orientation angle is set to 0 and the following calibrations have been done: • Image/beam shift for the active mode (found in the Alignments Control Panel). • Spotscan Pivot points • Spotscan Beam shift • If the size of the spots is affected by the objective-lens current (especially smaller spots and in

nanoprobe), Spotscan Focus-Intensity. Setup The spotscan setup contains a number of functions to set the scan area and distance between spots. The total number of spots is indicated. Note: The TEM magnification to be used for the spotscan should be set before switching any of the spotscan setup functions on, because the optimum beam deflection range is not reset when the magnification is changed while spotscan setup functions are active (in this way the scan can be observed, for example, at lower magnification). None When None is checked, the spotscan setup is disabled and spotscan wobbles or beam shifts are switched off. This is necessary after a setup, otherwise the microscope is not usable. Outline To set the total area scanned by the beam, you can either use the Outline function or set X and Y separately. In the Outline function the beam is moved continuously to the four corners of the scan range (for a 'normal' setting, this usually implies that the spots are just off the viewing screen and thus invisible - you can lower the magnification to check). Adjust the X and Y scrollers to set the required scan frame (should be at least the size of a negative). The scan area size is indicated above the X and Y sliders (in arbitrary units when not calibrated, otherwise in micrometers). Static X To set the total area scanned by the beam, you can either use the Outline function or set X and Y separately. With the Static X function the beam is moved to the extreme X position of the scan range. Adjust the X scroller to set the required scan frame (should be at least the size of a negative). The scan area size is indicated above the X and Y sliders (in arbitrary units when not calibrated, otherwise in micrometers). Static Y To set the total area scanned by the beam, you can either use the Outline function or set X and Y separately. With the Static Y function the beam is moved to the extreme Y position of the scan range. Adjust the Y scroller to set the required scan frame (should be at least the size of a negative). The scan area size is indicated above the X and Y sliders (in arbitrary units when not calibrated, otherwise in micrometers). X, Y sliders The X and Y sliders become activated when Outline, Static X (X slider only) or Static Y (Y slider only) are on. They control the size of the scan range for the X and Y directions. Spot wobble To determine the distances between spots, click on Spot wobble. The spot will wobble back and forth and the scroller can be adjusted until the spots just touch each other. The spot distance is indicated above the spot slider (in arbitrary units when not calibrated, otherwise in micrometers). The number of spots in the total scan is also indicated.


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Note: The optimum beam deflection range (reset when spotscan setup functions are switched on) affects the spot distance. Therefore always check the spot distance after setting the scan range. Preview The result of the spotscan setup can be viewed on the viewing screen to check that everything is alright for the exposure. This function is available only in the Low Dose Exposure state (press the Exposure button). The progress of the scan is indicated by the progress bar in the top of the panel (blue increasingly fills the bar). View Pressing the View button starts and stops the spot scan. Slower When the Slower button is pressed, the spot dwell time is increased by a factor 2. Faster When the Faster button is pressed, the spot dwell time is reduced by a factor 2. To 0,0 When the button To 0,0 is pressed, the scan is stopped and the beam is moved to the 0,0 position (center of the scan frame). If the beam is not centered on the screen, it should be shifted to the center with the normal beam shift (or Align beam shift alignment). The control of the AC beam deflection coils (used by spotscan) remains on. Shift away When the Shift away button is pressed, the beam will be moved to an extreme X,Y position. It should no longer be visible on the screen, unless the magnification has been lowered from the exposure magnification used during the setup. The control of the AC beam deflection coils (used by spotscan) remains on. AC off To switch the AC coils (used by spotscan) control off after the preview, press the AC off button.


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1.4 Low dose Settings tab The Settings tab of the Low Dose Control Panel contains controls that are related to the Low Dose settings. In addition to settings related to the optics, Low Dose also allows a choice of media (screen, plate, TV or CCD) for each state independently. The accessibility of these media may change and is reflected in the lists (under "Use:"). The system used for the media is explained under the TV / CCD tab.

Search Reset Pressing the Reset button resets the currently active Search settings to zero. What gets reset depends on the Mode only / All choice. The button is only active in the Search state. Search Mode only / All When Mode only is selected, only those settings of the mode (HM, LM, or Nanoprobe; Imaging or Diffraction) currently active on the microscope are reset to zero. When All is selected, all settings are reset. Search Medium The choice between (Viewing) Screen, TV and CCD for the Search state is made by selecting one of the items in the drop-down list. (See picture below). Right: The list of possible media for the Search state (the items except for Screen are dependent on the microscope configuration).


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Focus Reset Pressing the Reset button resets the currently active Focus settings to zero. What gets reset depends on the Mode only / All choice. The button is only active in the Focus state. Focus Mode only / All When Mode only is selected, only those settings of the mode (HM, LM, or Nanoprobe; Imaging or Diffraction) currently active on the microscope are reset to zero. When All is selected, all settings are reset. Focus Medium The choice between (Viewing) Screen, TV and CCD for the Focus state is made by selecting one of the items in the drop-down list. Exposure Reset Pressing the Reset button resets the currently active Exposure settings to zero. What gets reset depends on the Mode only / All choice. The button is only active in the Exposure state. Exposure Mode only / All When Mode only is selected, only those settings of the mode (HM, LM, or Nanoprobe; Imaging or Diffraction) currently active on the microscope are reset to zero. When All is selected, all settings are reset. Exposure Medium The choice between Plate (Camera) and CCD exposure is made by selecting one of the items in the drop-down list. Display screen dim text With the option Display screen dim text the user decides whether the dimmed screen will carry text ("Press Shift + Esc to remove" or the progress of the series exposure) or not (dimmed screen is totally black). In case of double exposure the dimmed screen will always have text, otherwise the user would not know when to press the Exposure button for the next double exposure in the sequence. Filename Settings can be loaded from and saved to file. When a filename has been defined (through a Load or Save), the name will be listed. When a file is loaded, the entry also lists which file version was loaded. If the same settings are used all the time (for any particular user), there is no need to save and load settings since the currently active Low Dose settings are always saved upon exiting and restored upon opening of the program (for each user individually). Load button Opens a standard Open File dialog that allows selection of a file with Low Dose settings. This file will be read and settings in the file will be made active. The file name must have the extension ".lds". Save button If no file name for Low Dose settings has been defined, this opens a standard Save File dialog that allows entering a file name under which the currently active Low Dose settings will be stored. The file name must have the extension ".lds" (the extension will be added automatically by the software). Save As button Opens a standard Save File dialog that allows entering a file name under which the currently active Low Dose settings will be stored. The file name must have the extension lds (the extension will be added automatically by the software).


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1.5 Low dose Calibrate tab The Calibrate tab of the Low Dose Control Panel contains the functions necessary for the Low Dose calibrations.

Blanker button The Blanker button starts the beam blanker calibration procedure. The beam blanker relies on a user-calibrated offset of the gun tilt. Once the gun tilt has been aligned properly the beam blanker calibration can be done. The user then turns the Multifunction X knob until the electron beams is no longer visible. For safety with the beam blanker it is advisable to turn the gun tilt a bit further than the point where the light disappears (safety margin). Image / Tilt button The Image / Tilt button starts the Image shift / Alpha tilt calibration procedure. This calibration ensures that the Low Dose Focus substate off-axis shift angles 0° and 180° will be along the Alpha tilt axis of the CompuStage. The procedure (for the HM and LM modes) consists of: 1. Preparation, wherein the program sets the microscope to a specific mode, magnification and spot

size. The user must center a recognizable image feature. 2. The stage will move the feature along the X axis of the stage (which is parallel to the Alpha tilt axis)

by approximately 90% of the fluorescent screen. The user must now recenter the feature using the image shift.

Peek button The Peek button starts the peek calibration procedure. The peek function uses a user-calibrated offset of the gun tilt. Once the gun tilt has been aligned properly the peek calibration can be done. The user then


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turns the Multifunction X knob until the electron beam is about 10% of the initial setting (almost no visible light). Spotscan Pivot points button Only visible if Spotscan is installed: The Spotscan Pivot points button starts the pivot points alignment procedure (there is no such alignment procedure in the standard TEM software). For proper operation of the spotscan, the beam shift pivot points (in this case for the AC - or STEM - deflection coils) must be aligned for HM, LM and Nanoprobe (the latter not on a Tecnai 10). The pivot point aligned is similar to those in the normal TEM alignment procedures. This procedure is accessible only from the Low Dose Exposure state (press the Exposure button). Note: This is an alignment and not a Low Dose calibration. The results are stored in the operator's alignments. Spotscan Beam shift button Only visible if Spotscan is installed: The Spotscan Beam shift button starts the beam shift calibration procedure (there is no such alignment procedure in the standard TEM software). The spotscan beam shift calibration procedure determines the beam shifts for HM, LM and Nanoprobe (the latter not on a Tecnai 10). Because the spotscan uses different coils (AC) than the normal beam shifts (DC), these calibrations must be done separately. This procedure is accessible only from the Low Dose Exposure state (press the Exposure button). The procedure (for the three modes) simply consists of: 1. Preparation, wherein the program sets the microscope to a specific mode, magnification and spot

size. 2. The user must focus the beam and center it accurately. 3. Move the beam to the edge of the screen (the point where the phosphor stops) with the scroller at

the bottom. If the edge cannot be reached, move the scroller to its end point. The magnification will be changed for another attempt (until the edge can be reached). If too little of the range of the deflection is used (the scroller is at less than half of the range), a setting in the microscope will be changed and the calibration step is redone to get the proper definition.

4. The program recenters the beam. 5. Repeat with for Y. From the known screen size and magnification the program calculates a calibration factor that converts the beam shift values to micrometers. Note: Part of this adjustment is an alignment. Those results are stored in the operator's alignments. Spotscan Focus-Intensity button Only visible if Spotscan is installed: The size of the spots can be affected when the objective-lens current is changed (to some extent in normal Microprobe HM but especially in Nanoprobe) which can result in disturbing effects when Spotscan focus correction is applied for tilted specimens as the spots used for recording will get progressively smaller along the scan. When the Focus-Intensity calibration has been done, Low Dose will automatically compensate the spot size change with the Intensity. The procedure consists of focusing the spot for HM and Nanoprobe for two objective-lens settings (controlled by Low Dose). Calibration instructions Instructions during the calibration procedures will be displayed here. OK button Pressing the OK button (or the L1 user button on the TEM Control Pads) proceeds with the calibration procedure to the next step.


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Cancel button Pressing the Cancel button cancels the calibration procedure. All intermediate settings determined so far will be removed. The microscope returns to its starting position. Filename Calibrations can be loaded from and saved to file. When a filename has been defined (through Load or Save), the name will be listed. If the same calibrations are used all the time (for any particular user), there is no need to save and load calibrations since the currently active Low Dose calibrations are always saved upon exiting and restored upon opening of the program (for each user individually). Load button Opens a standard Open File dialog that allows selection of a file with Low Dose calibrations. This file will be read and calibrations in the file will be made active. The file name must have the extension cal. Save button If no file name for Low Dose calibrations has been defined, this opens a standard Save File dialog that allows entering a file name under which the currently active Low Dose calibrations will be stored. The file name must have the extension cal (the extension will be added automatically by the software). Save As button Opens a standard Save File dialog that allows entering a file name under which the currently active Low Dose calibrations will be stored. The file name must have the extension cal (the extension will be added automatically by the software).


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1.6 Low dose Options tab The Options tab of the Low Dose Control Panel contains a range of optional settings.

Enable peek If this option is checked, the Peek button becomes visible and, when appropriate, enabled. Multifunction knobs to Search shift When this option is selected the Multifunction knobs of the TEM Control Pads will be switched automatically to the Search image shift (in image mode). Note that there is no other way of setting the image shift in Low Dose (the Image shift alignment has the same result, though it uses a different parameter to achieve the effect). Multifunction knobs to Focus shift When this option is selected the Multifunction knobs of the TEM Control Pads will be switched automatically to the Focus image shift (in image mode). Another way to set the Focus image shift is though the track bar sliders in the Focus panel. Normalizations Magnetic lenses suffer from hysteresis - an effect that makes the magnetic field difficult to set reproducibly because the effective field depends on the direction of change (up or down). Hysteresis can affect the position of the beam (due to changes in C1 - spot size and C2 - intensity) as well as the image or diffraction pattern (mostly the projector system consisting of the diffraction, intermediate and projector 1 and 2 lenses). The magnetic field of a lens can be made reproducible by normalization, a procedure that erases the recent history of a lens by running it to maximum and minimum power. This forces any


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setting in between to be reached through a well-defined and reproducible path, starting always from the same point. In Low Dose the normalizations are applied to achieve a reproducible swithing between the states. A drawback of the normalization is that it is time-consuming. In order to allow full flexibility in this, the user can select which lenses should be normalized during the possible switches. The default settings are displayed in the panel above. It is NOT advised to normalize the objective lens when switching between Focus and Exposure (this can affect the focus) and only normalize the objective lens in the switch from the Search to the Focus/Exposure state when Search is in LM and Focus and Exposure are in HM TEM. Normalize Normalizes the lenses selected by the check boxes to the right of the button. Switch Search to Focus via Exposure Another means of making settings more reproducible (especially when the Objective lens is normalized between Search and Focus) is to always switch from Search to Focus via the Exposure state (the beam will remain blanked during all the transitions). The actual sequence followed is then Search > Focus > Exposure > Focus. Note that this procedure typically takes a long time because of the intermediate normalizations involved. User buttons Under User buttons, the user defines which TEM Control Pad User Buttons (L1 .. L3, R1 .. R3) are used by Low Dose and which button does what. If a user button name (L1..R3) is listed in the drop-down list of a function, that function is assigned to a TEM User Button (otherwise the list will display None). If a new selection is made by selecting another entry in a list, the software will re-assign any function previously assigned to the particular User Button (if present) by shifting it to the next empty User Button. Dose measurement In the Exposure state you can measure the dose on the specimen. The dose is derived from the screen current and the magnification. The dose is reported in electrons per square nanometer per second. In order to derive the dose for the actual exposure, multiply by the exposure time used. If the small (focusing) screen is out, the diameter of the beam must be entered (unless it is larger than the main screen). If the small (focusing) screen is in, the beam must be larger than the small screen for a proper measurement. Dose measurement is continuous if the Continuously check box is checked. However, the Dose measurement will only run in the Exposure state (if Low Dose is switched to another state, the dose measurement is stopped). You can stop a continuous dose measurement by pressing the (now yellow) button again. If the Continuously check box is unchecked, a single dose measurement will be done. Should you find that the dose measurement on your microscope deviates significantly from measurements by other methods, then it is possible to adjust factors in the registry (all string values in HKEY_LOCAL_MACHINE\Software\Fei\LowDose) : • "BS Factor" for microscopes with maximum high tension values less than 200 kV or high-tension

settings less than 200 kV. • "BS Factor 200" for high-tension settings less than 300 kV. • "BS Factor 300" for the high-tension setting of 300 kV.


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1.7 Low dose TV / CCD tab The TV / CCD tab of the Low Dose Control Panel contains the information that Low Dose uses with regard to cameras (TV and CCD). The system works in combination with the settings defined for the Search, Focus and Exposure states. In some cases Low Dose automatically detects what cameras are on the system, while in other cases the user has to add some.

The following are detected automatically: • If an off-axis TV is present in the microscope software configuration, the off-axis TV-rate camera box

is always checked (the user cannot remove the check mark). The same applies to the "TV-rate camera present check box". If a Gatan Imaging Filter is present, the GIF TV camera (retractable) is assumed to be present and the box (bottom left) is checked. Once again, the user cannot remove this check.

• All CCD cameras are automatically detected and their location is either retrieved from the stored microscope configuration (e.g. Tietz cameras) or from their name (e.g. Gatan). In the case when the naming of a camera is possible, the names must be as follows: if the CCD camera is in the wide-angle port (above the projection chamber), the name must have the character sequence "WA"; if the camera is that of a GIF (Imaging Filter), the character sequence "GIF" must be in the name; if it is an off-axis CCD, the character sequence "off-axis" must be present in the name. Upper- or lowercase does not matter. All cameras not having the above character sequences are assumed to be on-axis below the projection chamber. Cameras identified are indicated by check marks in the boxes on the right-hand side of the schematic microscope picture. Once again, these check marks cannot be changed.

Low Dose State media Because CCD cameras are not always accessible (depending on whether the controlling software is running and/or the camera is switched on), the system used for selecting the media for the different Low Dose states has been set up as flexibly as possible. The accessible media are always shown in the media lists on the Settings tab.


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There are potentially ten media: • Screen - for Search and Focus • Plate - for Exposure • Four different TV positions - for Search and Focus • Four different CCD positions - for all states A medium is identified by its position, not its name. Thus, if you can access a particular CCD through different controllers (e.g. Gatan DigitalMicrograph and TIA), the medium will be chosen on the basis of where it is located, even if you change controller. If a medium is not accessible (e.g. a CCD controller has not been started or shut down), it will disappear from the lists. The selection in the list will temporarily default to screen or plate. When the medium becomes accessible, it will re-appear in the lists and the selection will change back to the medium as before - unless the user changes the medium selection in the list. The same logic applies to the selection when loading settings files. If the medium is available, it will become selected. If not, it will only become selected once the medium is available and the default medium is selected in the meantime). Note that what is saved in the settings files are the media that would be selected when the proper CCD controller(s) is(are) present, and not necessarily what is currently displayed in the lists as chosen. An example In the example we have a system with an Imaging Filter plus an off-axis TV camera. The user sets up the system such that: • Search is on the off-axis TV camera. Because the off-axis shift in EFTEM is too limited at low

magnifications, the Search magnification is chosen sufficiently high that the off-axis image shift can reach the camera (note that Low Dose does not change lens series - normal to and EFTEM or vice versa).

• Focus is defined on the GIF TV-rate camera. • Exposure is on the GIF CCD camera. When changing states, Low Dose will automatically select the appropriate detector position, lift the viewing screen, and insert or retract the GIF TV camera. When DigitalMicrograph is not available, the media will default to off-axis TV (since this does not rely on DigitalMicrograph), screen (the GIF TV camera control is only through DigitalMicrograph) and plate (the CCD camera is not available). If DigitalMicrograph becomes available again, the media will automatically be switched back to the previous selection. TV-rate camera present If a TV-rate camera is present, it can be used in the Search and Focus states. Whenever possible, the Low Dose software will help in using the TV-rate camera, either by reminding the user to insert the camera or by automatically lifting the screen (when the TV-rate camera is located underneath the viewing screen) and/or inserting the camera. CCD camera controller If a CCD camera is present, it can be used in the Search and Focus state for viewing and in the Exposure state for recording. In all cases the Search, Focus and Acquire setups must be done in the regular way: • For TIA in the TEM CCD/TV Control Panel. • For DigitalMicrograph in DigitalMicrograph itself (use the floating windows: for the exposure use the

"Camera Acquire Record" setting, for Search and Focus the "Camera View" Search and Focus, respectively).

Low dose simply uses the predefined setups (Search = Search, Focus = Preview or Focus, Exposure = Acquire or Record) and does not change any settings (except the exposure time used for the Low Dose Exposure). If more than one controller is present, you can change the controller to be used by selecting


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one from the drop-down list (this function is identical to that in the CCD/TV Control Panel). The controller is not specific to Low Dose. Low Dose does not automatically select a controller. TV rate In the schematic diagram of part of the microscope there are four possible locations for TV-rate cameras, the Wide-Angle TV port (above the viewing screen), directly below the viewing screen (on- and off-axis) or further below (e.g. a GIF). If more than one TV-rate camera is present, select the particular option for the TV-rate camera that will be used during Low Dose imaging. CCD In the schematic diagram of part of the microscope there are four possible locations for CCD cameras, the Wide-Angle TV port (above the viewing screen), directly below the viewing screen (on- and off-axis) or further below (e.g. a GIF).

1.8 Low Dose Settings The following values are stored with the Low Dose Settings, either as user settings or in a file : Options • Reset Search, Focus, Exposure settings for current mode or all • Peek enabled • TV/CCD use per state • TV/CCD settings • Multifunction knobs enabled or disabled in Search and Focus states • Normalizations selected for Low Dose state transitions • Switch from Search to Focus via Exposure • TEM User Button assignment Exposure settings • Exposure time • Waiting time after plate in • Dim screen enabled/disabled • Pre-exposure used and time • Pre-exposure waiting time • Exposure series enabled/disabled • Exposure series defocus values • For spotscan: use spotscan for exposure, and spotscan settings like dwell time and amplitudes Search state • Mode • Magnification • Camera Length • Spotsize • Intensity • On Titan: Illuminated area • Focus of Objective lens (for HM image and LAD), of Diffraction lens for (HM D and LM Image) • Beam Shift • Image Shift • Diffraction Shift


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Focus state • Substate (1 or 2) • Magnification • Camera Length • Spotsize • Intensity • On Titan: Illuminated area • Beam Shift (both substates separately) • Image Shift (both substates separately) Exposure state • Mode • Magnification • Camera Length • Spotsize • Intensity • On Titan: Illuminated area • Focus of Objective lens (for HM image and LAD), of Diffraction lens for (HM D and LM Image)

1.9 Low Dose calibrations The following values are stored with the Low Dose Calibrations, either as user settings or in a file : Image shift / A Tilt : • LM • HM • Beam Blanker amplitude Peek amplitude Spotscan beam shift • HM • LM • Nanoprobe


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2 TEM Photomontage

2.1 Introduction A photomontage consists of a series of images that together make one large image. The advantage of a montage over a lower-magnification image is the fact that the individual images of the montage are recorded at higher magnification and thus allow resolving finer details. By photomontage it is possible, for example, to record detailed images and still allow an overview of the whole specimen area investigated. A photomontage can in principle be executed by hand. It is, however, difficult to ensure that sufficient overlap exists between the images to avoid gaps in the montage and at the same time minimize the number of images needed. The TEM Photomontage option makes it possible to define and automatically execute recording of a complete photomontage.

2.2 Getting started The TEM Photomontage option consists of two parts: • The Photomontage Control Panel. • The Photomontage Display window. To use TEM Photomontage: • Open the Workspace layout in the TEM UI (bottom-right selection list) to drag the Photomontage

Control Panel into a workset. • Go to the tab of the workset and press the Display button in the Photomontage Control Panel. The

Photomontage Display window will be loaded and its functionality become available. You can leave the Photomontage Control Panel in your workset for future use. The Display window will only be loaded when the Display button is pressed. The major part of the Photomontage functionality is present only in the Display window. The Control Panel also provides rapid access to the more often-used functions.

2.3 Photomontage Control Panel Photomontage control panels for Plate (left) and for CCD (right) as recording medium.

The Photomontage Control Panel contains two sets of functions for photomontage : • Functions that load the Photomontage Display window (a separate window that is typically positioned

in the data space of the TEM User Interface) or determine its size. • Functions that give rapid access to often-used photomontage functions. These functions may change

as a function of the medium selected (Plate or CCD).


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Display The Photomontage Display window is only loaded on operator request. To load it press the Display button. A message will appear that the Photomontage server is being loaded. Once the server is running, the window will be displayed, the Display button will turn yellow, and the message will disappear. You are now ready to start working with Photomontage. If the Display button is pressed again, the Display window will disappear. It is, however, not unloaded but simply hidden. If you display it again (by once more pressing the button), it will still have kept all the photomontage settings as set previously. The window is only unloaded when you close the TEM User Interface. Size adjustable When this option is checked, the Display window can be positioned anywhere you like (the software will remember your settings and restore them) and you can define its size in the standard Windows way (click and drag on a border). If the option is off, the Display window will be fixed in size and position to fill the data space of the TEM User Interface. The following controls will be enabled or disabled when their counterpart in the Display window is enabled or disabled. Add point / Acq image For Plate medium, adds a point to the series of boundary points that define the photomontage area. For CCD, it starts the sequence for obtaining the reference image. Set magn. Starts the procedure to define the magnification to be used for the montage. Calib rot. Starts the rotation calibration procedure (Plate only) for the currently active magnification. Modify Switches montage modification (Plate only) on or off. When modification is active, the Modify button will be yellow. CCD / Plate Changes selection of the recording medium (CCD or Plate). If a montage has been defined for the other medium, that montage will be removed. Auto Starts the Automatic Exposure procedure for Plate or CCD as medium . When Photomontage is in the Automatic Exposure procedure, the Auto button will be yellow. Manual Starts the Manual Exposure procedure (Plate only). When Photomontage is in the Manual Exposure procedure, the Manual button will be yellow. Mount Starts the Mount procedure for CCD Photomontage. When Photomontage is in the Mount procedure, the Mount button will be yellow.


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2.4 Photomontage display The Photomontage Display window is a window that is typically located in the data space of the TEM User Interface. It can either be freely positioned and sized or fixed. The choice between the two is made in the Photomontage Control Panel. The display contains two essential elements, the menu and the display itself. TEM Photomontage has two major modes of operation : • Recording of the images on plate negatives. This mode of operation is the same as on older software

versions, except that the possibility of manually recording CCD images is no longer available. • Recording of the images on CCD. This mode of operation is new to software version 2.1.8/3.0. It is

only available in combination with TIA CCD. Because the two modes of operation are substantially different, they are described separately below.

2.5 Recording on plate negatives Photomontage with recording on plate will always use the stage for moving from one exposure to another. The area covered by the montage is determined by driving the stage to a series of positions at the perimeter and adding these to the boundary points. The main functionality of Photomontage resides in the menu of the display window. A typical sequence for setting up and executing a plate-negative photomontage is as follows: • If necessary, check that all options are defined as needed. • Define the photomontage area by bringing a series of points on the perimeter of the area to the

center of the screen with the specimen stage and using Add boundary point to enter it. • Select the magnification to be used for the photomontage. • If necessary, calibrate the rotation. • If necessary, modify the montage suggested by the software. • Execute the exposure of the montage, either automatically or manually. • Store and/or print the data for later reference.


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2.5.1 The display

The window will display the photomontage, showing the area overlaid with the individual exposures. Initially the exposures are simply labelled by their sequence number. Once an exposure has been recorded, it number will be replaced by the exposure number (provided there is enough room for the longer labels). The colors used for the display are freely user-configurable. Menu The menu gives access to all photomontage functionality. Montage The montage is displayed in the window. The gray area is the area of the specimen covered. The green rectangles are the individual exposures, labelled here still with sequence numbers. Stage axes The x,y directions of the stage axes are indicated on the screen. Scale bar The scale bar gives an indication of the sizes of the photomontage area and individual exposures. Status bar The status bar lists on the left the current stage position. On the right it will display hints for getting started, messages in case of errors, or the current setting of the magnification used for the montage and the number of exposures in the montage. Note: The magnification listed is the so-called reference value (the value on the plate camera, that is, the one seen with the screen up). This fixed value is used to avoid confusion (which could occur when


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photomontage would continuously adjust values when the screen goes up or down). The values with the screen down are roughly 10% lower than with the screen up. Border icons The border icons behave as the standard Minimize, Maximize and Close buttons. The Close button, however, does not truly close the window but hides it. The window is closed only upon closing the TEM User Interface. The Maximize button is only visible if the window size can be adjusted freely.

2.5.2 The menu The menu consists of a main menu with submenus: • File • Medium • Calibrate • Setup • Montage • Exposure • Help

2.5.2.1 File menu The File Menu provides the operations that are concerned with file operations, the settings and printing. New montage Starts a new montage (resetting all previous montage values) Open list Not enabled in on-line version (off-line only). Save list, Save list as

Saves the list of exposures with their X,Y locations and exposure numbers in a text file, Save as allows definition of different file name

Save graphic, Save graphic as

Saves the current display as a bitmap, Save as allows definition of a different file name

Control options Leads to the Control options dialog Load options Loads options from file Save options Saves options to file Printer setup Leads to printer setup dialog Print list Prints the list of exposures with their X,Y locations and exposure numbers Print graphic Prints the current graphical display

2.5.2.2 Medium menu With the Medium Menu, the operator chooses which of the two media (Plate, CCD) is active. CCD will only be enabled if TIA CCD is present and running.

2.5.2.3 Calibrate menu The Calibrate Menu has functions for CCD Photomontage only.


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2.5.2.4 Setup menu With the Setup Menu, the operator defines the boundaries of the photomontage, sets the magnification and, if needed, determines the rotation angle between the specimen-stage axes and the camera. Add boundary point Insert the current specimen-stage X,Y location into the list of points that define

the photomontage boundary. Set magnification Defines the magnification to be used for the photomontage. If the rotation

calibration has already been done and the rotation calibration option is off, the photomontage will be calculated immediately after this procedure.

Calibrate rotation Starts calibration procedure for determining the rotation angle between the photomontage boundaries and the camera. The photomontage will be (re)calculated immediately after this procedure.

2.5.2.5 Montage menu The Montage Menu contains a number of functions that are related to the exposures: modifying the photomontage and displaying a list of the montage X,Y locations. Modify montage Starts or stops modification of the photomontage: mouse double-click or click

and drag, Cursor keys, Ctrl Cursor keys, Insert, Delete and Undo changes become active or inactive. When the menu item is checked, modification is active. Modification will automatically be switched off when certain other procedures like Exposure are started.

Insert Inserts a new exposure at the cross set by mouse double click. The new exposure will be fit to the existing montage, not centered at the cross.

Delete Deletes the exposure at the cross set by double clicking the left-hand mouse button.

Delete from list Allows deletion of one or more exposures by number from a list displayed. Undo changes Undo changes will revert to the photomontage defined before Modify montage

was switched on. Display list Shows the list of exposure, with sequence number, and X,Y locations, and if

available the exposure number. The list can be printed directly from here (as well as from the File menu).

Back to setup Removes the currently defined montage and switches back to the situation where boundary points can be defined or deleted.

2.5.2.6 Exposure menu The Exposure Menu contains the functions that are related to the performing the exposure of the photomontage. Automatic Starts automatic exposure procedure. Manual Starts manual exposure procedure.

2.5.2.7 Help menu The Help menu contains two items, Show and About. Selecting Show (or pressing F1) will display this Help file in the TEM User Interface. About will display the About TEM Photomontage dialog listing the current version number of the software. When reporting bugs, always include the version number in the report.


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2.5.3 Control options The software supports a range of options, defined in the Control options dialog. Options are stored as set for each user separately and recalled upon restart. Options can be saved to file and reloaded.

Plate type The software supports rapid selection of exposure sizes from any of the three pre-defined plate camera formats (the inch size, European cm format and Japanese cm format). The Camera dimensions consists of two values, stored separately for each camera, for the horizontal and vertical size of the exposure. In the case of the film cameras, horizontal is left to right for the microscope operator (longest dimension for plate film), vertical is towards and away from the operator. It is advisable to check the values for the system used. Measure on a used negative the longest (= horizontal) and shortest (= vertical) exposed area (discounting the plate number). Subtract 2 to 3 mm to account for reproducibility of the plate camera. CCD Not relevant for plate montage. Overlap The overlap between the exposures is the percentage of the horizontal or vertical axis that must be common to both exposures to ensure fit of the photomontage without gaps. The overlap can be set either automatically or at a fixed value. If done automatically the program will adjust the overlap for the magnification used (a higher value at higher magnification, typical values are 10% at 5000x or below, 20% at 30kx, 30% at 50kx). Note: It is allowed to define a negative fixed overlap value. In that case the individual exposures will not overlap but instead be spaced apart. In this way an area can be covered by a regular grid of exposures without requiring them to fill the whole area. This function can be used for example for statistical analysis.


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A regular grid of non-overlapping exposures on an area.

Exposure The Exposure options concern whether a sound signal should announce start and finish of individual exposures and how long the software should wait after stage movement before taking an exposure. Rotation If the Rotation option Always calibrate is switched on, the software will start the rotation calibration procedure automatically whenever a new montage is defined (directly after Set magnification). Otherwise the software will use a stored value for the current magnification. If no such value exists, the rotation must be calibrated before the montage is calculated. Note: Although the magnifications are in principle rotation-free on most instruments, deviations up to several degrees can exist and these can affect the accuracy of photomontage, especially with small overlaps. The rotation calibration must be still be done therefore. Colors The four types of colors (Background, Area, Exposures, Labels) for the display can be freely selected. The selection is made in the Control options dialog or by clicking on the window with the right-mouse button. The latter will popup a menu that allows definition of the colors. Note: There is one exception to free color selection: if hyperlabels are used the Label color cannot be blue (this is the flash color of the hyperlabel). Use hyperlabels The software provides a special feature in the form of hyperlabels (labels that act like hyperlinks: when the cursor moves over them they flash their color to blue and a mouse-click brings up a dialog with information about the point, with additional functionality such as Go to or Delete). The labels are used in the boundary point definition phase to display the boundary points themselves. Thereafter the labels are those of the exposures.


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2.5.4 Setting up for a montage The setup for a montage follows these steps: • Define the boundary points for the montage area. • Define the magnification to be used. • If required, calibrate the rotation between the stage axes and the image axes.

2.5.5 Define boundary points The boundary points define the perimeter of the photomontage area. The perimeter can be any shape defined by a polygon of up to 500 points. These points themselves are determined simply by moving the specimen stage so a point on the boundary lies in the center of the viewing screen. After a minimum of two points have been loaded, the program will display a gray area that represents the photomontage area (for two points it will be a line). One aspect is critical in determining the boundary points. They must follow the perimeter of the photomontage in their proper sequence (starting at one point and going around until the same starting point is encountered again). If a point is out of sequence the program may display the photomontage area with gaps as in the left-hand image below.

Improper sequence. Point 4 from left figure removed. The strange shape of the area in the left-hand picture is the result of intersecting lines and does not properly represent the photomontage because point 4 was loaded between points 3 and 5 instead of between points 1 and 2. If a point is found to be out of sequence, then use the hyperlabel option to delete that point.


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2.5.6 Set magnification Set magnification is the Setup step where the magnification for the photomontage is defined. The operator is asked to set the microscope magnification to the desired value, after which the program will read the value form the microscope. If no rotation calibration is necessary the software will proceed by calculating the photomontage. First it quickly estimates the number of exposures required to cover the montage area. If the estimate indicates more exposures than the default plate stock of 56 exposures, a warning is given. The operator can then proceed or reset the magnification to a lower value. After the estimate the montage is calculated. Dependent on a variety of factors the suggested montage may be good enough or it may require modification. In the former case you can proceed directly to automatic exposure (plate camera only) of manual exposure.

2.5.7 Calibrate rotation Rotation calibration is the setup step wherein the program is informed about the rotation angle between the specimen stage X,Y axes and the camera. This is done simply by moving a recognisable image detail from one screen marker on the left to one on the right .

The Rotation calibration dialog during the first step. In the second step the feature selected is moved to the marker on the right-hand side.

Once a rotation value has been determined for a particular magnification, it will be stored by the software.

2.5.8 Modifying a montage The montage suggested by the software can be modified in various ways. In order to modify the montage, you have to switch modification on. During a modification procedure, the changes can be undone. The Undo will revert the montage to its configuration before modification was switched on. You can go through repeated cycles by switching modification on and off again. During modification mouse double-click and mouse click-and-drag operations on the window have the following meaning. • Mouse double-click puts a cross marker at the point where the cursor was during the double click. If

the cross lies inside an exposure (don't go too far towards the edge; also don't click on the label if it is a hyperlabel, because then the hyperlabel dialog comes up) that exposure can be deleted. If the cross lies near a potential exposure location, that location can be inserted as a new exposure.


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• Mouse click-and-drag shifts the exposures relative to the montage area (the actual shift is only displayed once the mouse button is released.

Exposure modification can be one of the following: • Shift the whole montage • Insert a new exposure (after a cross has been positioned with a double-click) • Delete an existing exposure (after a cross has been positioned with a double-click; by clicking on a

hyperlabel and selecting Delete; or Deleting one ore more exposures from a list. In the list multiple exposures can be selected by Click - Shift-Click or Click - Ctrl+Click).

Shifting the whole montage can be done in various ways: • Click the left-hand mouse button somewhere on the montage and drag the corresponding montage

point to a new location. The new montage will be redrawn as soon as the mouse button is released. • Use the cursor (arrow) keys to move the montage up, down, left or right. Regular cursor keys move

one pixel, Pressing the Control (Ctrl) simultaneously with the cursor key will shift by ten pixels.

2.5.9 Automatic exposure After a photomontage has been defined, it is possible to have the program record the whole montage automatically. When Automatic exposure is started, the following dialog is brought up.

The Automatic exposure progress dialog.

At the top the software indicates the progress of the procedure (in this case no exposures have been recorded yet). An estimated total duration is given. This is subject to considerable uncertainty because various procedures that can be time-consuming (stage movement, screen up/down) are only accounted for in a general overhead on top of the exposure time. The Sound on checkbox allows changing the Sound option during the actual procedure. Underneath the progress bar various values are listed such as the currently available exposure stock, exposure number and exposure time. For the exposure time two options exist if automatic exposure time is selected on the microscope. By default the software simply uses the exposure time as currently measured (this implies that during the whole exposure sequence the viewing screen will remain up). If the Measure for each checkbox is checked, the screen is lowered for each exposure to allow the microscope to measure the exposure time for each individual exposure. Since the small focussing is not motorized, the exposure time will always be read off the main screen then.


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Finally at bottom right there are two selections (for plate camera only) that allow automatic modification of the film text on the plate. If the first is checked, a line is entered in the film text with 'Montage exposure #' where # is replaced by the sequence number of the exposure. If the second is checked a line is entered with 'Loc X xxx.xx Y yyy.yy' where xxx.xx and yyy.yy are replaced by the stage x,y location of the exposure. Both options can be used together. Existing film text will be shifted down (if more than 4 lines exist after insertion of the montage data, the excess lines will be lost and not restored afterwards). After the exposure procedure is stopped, the film text is restored to its original setting (minus any excess lines lost). Exposures that have been recorded will have their plate exposure numbered entered in the list and, if sufficient space is available, their label on the screen is also replaced by the exposure number. Note 1: The procedure will stop or pause when an error is encountered, e.g. plate stock zero. In that case the operator can rectify the condition leading to the error and continuing with the procedure. Note 2: While exposing (both automatic and manual), the program uses a backlash correction procedure to make the positioning the exposures more accurate. However, because the backlash correction (probably) wasn't done during definition of the boundaries, there may be a systematic shift of the whole pattern of exposures of up to about 500 nm. For montages at high magnifications it is important therefore to check the exposure locations (e.g. go to selected exposure locations through hyperlabels), especially if the edge of the area covered by the exposures is close (less than 1 um) to the intended photomontage area.

2.5.10 Manual exposure After a photomontage has been defined, the montage can be exposed manually. When Manual exposure is started, the following dialog is brought up.

The Manual exposure progress dialog.

At the top the software indicates the progress of the procedure (in this case no exposures have been recorded yet). Underneath the progress bar various values are listed such as the currently available exposure stock, exposure number and exposure time. At bottom right there are two selections (for plate camera only) that allow automatic modification of the film text on the plate. If the first is checked, a line is entered in the film text with 'Montage exposure #'


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where # is replaced by the sequence number of the exposure. If the second is checked a line is entered with 'Loc X xxx.xx Y yyy.yy' where xxx.xx and yyy.yy are replaced by the stage x,y location of the exposure. Both options can be used together. Existing film text will be shifted down (if more than 4 lines exist after insertion of the montage data, the excess lines will be lost and not restored afterwards). After the exposure procedure is stopped, the film text is restored to its original setting (minus any excess lines lost). Exposures that have been recorded will have their plate exposure numbered entered in the list and, if sufficient space is available, their label on the screen is also replaced by the exposure number. IF the exposures are recorded on a CCD camera the exposure numbers given are 'CCD1', 'CCD2', etc. Note: While exposing (both automatic and manual), the program uses a backlash correction procedure to make the positioning the exposures more accurate. However, because the backlash correction (probably) wasn't done during definition of the boundaries, there may be a systematic shift of the whole pattern of exposures of up to about 500 nm. For montages at high magnifications it is important therefore to check the exposure locations (e.g. go to selected exposure locations through their hyperlabels), especially if the edge of the area covered by the exposures is close (less than 1 um) to the intended photomontage area.

2.6 Recording on CCD and automatic montage The area covered by the montage is determined by recording a reference CCD image at a magnification where the whole area of the montage is within the field of view and marking the perimeter of the montage in that image. Note 1: Two magnifications are involved in the montage, the magnification at which the reference image is acquired and the magnification at which the individual montages images are acquired. In order to ensure the software is able to record the montage images at the correct locations, it is very important that these two magnifications are accurately aligned to each other. To check, it is best to find a recognizable image feature, center that in the CCD (search) image at one magnification (mark the position by putting in an image marker), then change to the other magnification (you may need to use normalization to obtain accurate image positioning) and check that the feature is also exactly in the center at this magnification. Note 2: Recording and mounting a CCD montage requires a good deal of memory. If there are too many images the system will slow down very much. Before starting a montage, check the amount of memory available in Task Manager (right-click on an empty part of the Taskbar and select Task manager, select the Mem usage in the Performance tab. After the montage exposure, check again. If the amount of memory remaining is not at least as much as was needed for the montage acquisition, do not mount the montage). Note 3: As an alternative to image acquisition into a data series in TIA and final assembly of the images into a montage image by the photomontage software, you can acquire the images for storage in files. In this way you can acquire any number of images, unlimited by memory, but you cannot have the software assemble the images into a montage image. Supported file formats for image acquisition to file are Extended-header and Small-header binary, TIFF, and MRC file format, the latter either into separate images or a single file containing the whole series.


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The main functionality of Photomontage resides in the menu of the display window. A typical sequence for setting up and executing a plate-negative photomontage is as follows: • If necessary, check that the options are defined as needed. • Define the photomontage area by acquiring a CCD image - the reference image - that contains the

whole area that the photomontage should cover and using Acquire image to retrieve the image from TIA.

• Select the magnification to be used for the photomontage. • Execute the exposure of the montage. • Execute the montage mount.

2.6.1 The display The window will display the reference image with the photomontage image boundaries overlaid.

Menu The menu gives access to all photomontage functionality. Montage A schematic view of the montage is displayed in the window. The reference image is in the background, with overlaid on it the boundary as defined in red outlined by white. The blue-white lines indicate are the individual exposures. The individual montage images will be in an image series in TIA. After mounting the mounted montage will also be in TIA. Both image series and montage are located in a display window titled "Montage". Scale bar The scale bar gives an indication of the sizes of the photomontage area and individual exposures.


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Status bar The status bar lists on the left the current stage position. On the right it will display hints for getting started, messages in case of errors, or the current setting of the magnification used for the montage and the number of exposures in the montage. Note: The magnification listed is the so-called reference value (the value on the plate camera, that is, the one seen with the screen up). This fixed value is used to avoid confusion (which could occur when photomontage would continuously adjust values when the screen goes up or down). The values with the screen down are roughly 10% lower than with the screen up. Border icons The border icons behave as the standard Minimize, Maximize and Close buttons. The Close button, however, does not truly close the window but hides it. The window is closed only upon closing the TEM User Interface. The Maximize button is only visible if the window size can be adjusted freely.

2.6.2 The menu The menu consists of a main menu with submenus: • File • Medium • Calibrate • Setup • Montage (montage modification is not available for CCD montage) • Exposure • Mount • Help

2.6.2.1 File menu The File Menu provides the operations that are concerned with file operations, the settings and printing. New montage Starts a new montage (resetting all previous montage values). Open list Not enabled in on-line version (off-line only) Save list, Save list as

Saves the list of exposures with their X,Y locations and exposure numbers in a text file, Save as allows definition of different file name

Save graphic, Save graphic as

Saves the current display as a bitmap, Save as allows definition of a different file name.

Control options Leads to the Control options dialog Load options Loads options from file Save options Saves options to file Printer setup Leads to printer setup dialog Print list Prints the list of exposures with their X,Y locations and exposure numbers Print graphic Prints the current graphical display Export series Exports the series of images in TIA to one of the supported formats (Extended

Header Binary, MRC, TIFF).


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2.6.2.2 Medium menu With the Medium Menu, the operator chooses which of the two media (Plate, CCD) is active. CCD will only be enabled if TIA CCD is present and running.

2.6.2.3 Calibrate menu The Calibrate Menu has functions for CCD Photomontage only. The execution of a CCD montage requires calibration of the TEM image, the image shift and stage shift. To avoid unnecessary duplication, Photomontage uses the calibration procedures and stored settings of TEM AutoAdjust. Which calibrations can done can be determined from the menu. Calibrations that are disabled require the calibration above it to be done. The Setup menu will only be enabled if all three calibrations have been done. Magnification Executes the magnification calibration Image shift Executes the image-shift calibration Stage shift Executes the stage-shift calibration Calibrations have a set hierarchy (some calibrations can only be done once others have been done). They are specific to a number of conditions : CCD camera used in combination with the microscope lens series (Normal or EFTEM) High tension Microscope mode (LM, Microprobe, Nanoprobe, Lorentz) For the microscope modes, only the Nanoprobe mode does not require full calibrations. For that mode only the Focus/Stigmator calibration must be done separately (the rest of the calibrations if the same as in the Microprobe mode). Calibrations can only be done by TEM Experts (private calibrations for these users) and Supervisor, Service or Factory (dependent on availability, these calibrations are what basic microscope Users get. Calibration is done on the basis of a standard specimen, a cross-grating. This specimen has squares on it with a spacing of 463 nm.


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2.6.2.4 Magnification calibration The software will acquire an image of the cross-grating and analyse that to find the basic spacing. The analysis is done using an auto-correlation (at high magnifications) or an FFT (at low magnifications) in which the two basic vectors will be outlined by circles.

An image of a cross-grating, showing the 463 nm squares. The result of the magnification calibration. The autocorrelation of the cross-grating image (zoomed in 2x) shows the central peak pointed at by the line (which has been added manually for clarity). The software adds the two circles around the two base vectors of the pattern. It is important that these circles are located at the correct positions and not e.g. at the diagonals of the squares. Additional circles are placed at the multiples of the base vectors used to show how many base vectors were used together for the analysis.

2.6.2.5 Image-shift calibration The image-shift calibration procedure performs a calibration of the image shift on the CCD. This calibration must be done for all magnifications in the required range (up to magnifications of ~150kx). Normally the whole range is calibrated in one procedure, but it also possible to (re)calibrate the image shift for an individual magnification (e.g. when it appears that the calibration in the complete procedure went wrong for one magnification, that magnification can be redone later). Note: If the image-shift calibration fails at low magnifications because of the presence of strong contrast from grid bars, go to the center of the cross-grating specimen and use the triangle area in the "A" to do the image-shift calibration.

2.6.2.6 Stage calibration The stage calibration calibrates the stage x and y shifts.


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2.6.2.7 Setup menu With the Setup Menu, the operator defines the boundaries of the photomontage, sets the magnification and, if needed, determines the rotation angle between the specimen-stage axes and the camera. Acquire image Starts the retrieval of the reference image from TIA. Set magnification Defines the magnification to be used for the photomontage. Calibrate rotation Not enabled for CCD. Calibrations are under the Calibrate menu.

2.6.2.8 Exposure menu The Exposure Menu contains the functions that are related to the performing the exposure of the photomontage. Automatic Starts automatic exposure procedure. Repeat at current stage position

Repeats the automatic exposure but allows you to do this at the current stage position (otherwise the stage will move back to the position where the montage was originally defined). This option only becomes enabled once the exposure (Automatic or Manual) has been done once. It may be useful in cases where you simply want to repeat recording the same pattern more than once.

Manual Starts manual CCD exposure procedure. Repeat (Manual) at current stage position

Repeats the manual exposure but allows you to do this at the current stage position (otherwise the stage will move back to the position where the montage was originally defined). This option only becomes enabled once the exposure (Automatic or Manual) has been done once. It may be useful in cases where you simply want to repeat recording the same pattern more than once.

2.6.2.9 Mount menu The Mount Menu contains the functions that are related to the mounting of the photomontage. Execute Pressing Execute will start the automatic mounting of the montage images. Show log Will display a log containing information about the mounting of the images.

2.6.2.10 Help menu The Help menu contains two items, Show and About. Selecting Show (or pressing F1) will display this Help file in the TEM User Interface. About will display the About TEM Photomontage dialog listing the current version number of the software. When reporting bugs, always include the version number in the report.


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2.6.3 Control options The software supports a range of options, defined in the Control options dialog. Options are stored as set for each user separately and recalled upon restart. Options can be saved to file and reloaded.

Plate type Not relevant for CCD montage. CCD With CCD acquisition, there are two ways to handle the images: • Collect the images into a data series in TIA. This series can then be used later as the basis for

generating the montage image. The number of images strongly depends on image size and memory available.

• Collect the images as a file series. Each image is saved to file but not kept in a data series in TIA. While there is no limit to the number of images (except hard-disk space), you have to use other software to assemble the images into a single montage image. Supported file formats are Extended Header Binary, MRC and TIFF. The former two contain information about microscope settings in their header, while for the TIFF images you only guide to the image position is the file name (which contains the x, y values separated by underscores).

• The CCD Store images only is disabled if you have already acquired images. To switch between data series in TIA and files, you have to redefine the montage (File, New montage).

Overlap The overlap between the exposures is the percentage of the horizontal or vertical axis that must be common to both exposures to ensure fit of the photomontage without gaps. The overlap can be set either automatically or at a fixed value. If done automatically the program will adjust the overlap for the magnification used. Note: It is allowed to define a negative fixed overlap value. In that case the individual exposures will not overlap but instead be spaced apart. In this way an area can be covered by a regular grid of exposures without requiring them to fill the whole area. This function can be used for example for statistical analysis.


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A regular grid of non-overlapping exposures on an area.

Exposure The Exposure options concern whether a sound signal should announce start and finish of individual exposures and how long the software should wait after stage movement before taking an exposure. Rotation Not relevant for CCD montage. Colors Not relevant for CCD montage. Use hyperlabels The software provides a special feature in the form of hyperlabels (labels that act like hyperlinks: when the cursor moves over them they flash their color to blue and a mouse-click brings up a dialog with information about the point, with additional functionality such as Go to). The labels are those of the exposures.

2.6.4 Setting up for a montage The setup for a montage follows these steps: • Acquire the reference image. • Define the area for the montage in the reference image. • Define the magnification to be used.


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2.6.5 Acquire image The reference image that is used to define the area covered by the CCD montage must be acquired in TIA under conditions defined in the CCD/TV control panel of the TEM user interface. Select a suitable magnification and press the Acquire button in in the CCD/TV control panel of the TEM user interface to acquire a CCD image. If the image does not cover the whole montage area, modify the conditions and repeat. When you select Acquire image in the Photomontage menu, instructions will appear in the Photomontage window. Photomontage will always used the most recently acquired CCD image as the reference. Once you click on OK, the image will be retrieved and displayed in the Photomontage window. You can then required to define the montage area.

2.6.6 Define area After the reference image has been retrieved from TIA, these instructions will be displayed. The operator is now required to define the montage area by double-clicking with the left-hand mouse button on the reference image. Each click will define one boundary point. Make sure to go round the perimeter in a regular sequence (starting at one point and going around until close to the starting point). It is possible to back up, deleting points by pressing the minus key on the keyboard. Pressing Enter finalizes the area definition and automatically closes the perimeter definition. To make changes to the area definition after pressing Enter, you have to re-acquire the image.


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The area is now defined. The next step is to set the magnification for the montage itself.

2.6.7 Set magnification Set magnification is the Setup step where the magnification for the photomontage is defined. The operator is asked to set the microscope magnification to the desired value, after which the program will read the value from the microscope. The montage is then calculated


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2.6.8 Automatic exposure After a photomontage has been defined, the recording step is done fully automatic. The exposure conditions (image size, binning and exposure time) are as defined for CCD Acquire in the CCD/TV control panel of the TEM user interface. When exposure is started, the following dialog is brought up.

The exposure progress dialog.

At the top the software indicates the progress of the procedure (in this case no exposures have been recorded yet). An estimated total duration is given once a few images have been recorded (prior to that the time cannot be estimated). The Sound on checkbox allows changing the Sound option during the actual procedure.

2.6.9 Manual exposure The manual CCD exposure procedure is very similar to the automatic procedure except that you have to press buttons to: 1. Have the stage or image shift move to each position. 2. Acquire the CCD image. Step 2 is optional, but if that is not done, the actual montage cannot be assembled by the photomontage software. Also, the software does not track that you acquire all the images in their proper sequence (so if you skip an image, the next image gets to be in the wrong location). The advantage of the manual exposure procedure over the automatic procedure is that you get the chance after each "image move" to check the settings (like focus) and to have the option to record the CCD images manually (through the Acquire function of the CCD/TV control panel of the TEM user interface). When manual exposure is started, the following dialog is brought up.

The exposure progress dialog.

Initially the button with the caption Next will display First. Press the button to get started. The stage or image shift will be set to the first image position. Once that is done the Expose to acquire the CCD image


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in the normal Photomontage display window in TIA and add it to the series of images. The Next button is re-enabled and you can go to the next position, and so on. If the Exposure function is not used, the Next button does not get disabled. If you do not acquire the CCD images through the Expose function, the progress bar will show only half of the progress (each progress step is a move or an image acquisition).

2.6.10 Mounting the exposures into the montage After the images have been recorded, they can be mounted into the montage. The mounting happens automatically, with the following restrictions: 1. If the images do not overlap, they are mounted in an uniformly gray image of suitable dimensions

(with some margins along the sides) in the nominal positions they have in the specimen. 2. If there is an image overlap but the amount of overlap is too small to check the registration between

the individual images, the images are mounted according to the nominal positions in the specimen. The overlap areas are faded together to reduce the visibility of sharp boundaries where the images may not match perfectly.

3. If there is sufficient overlap, the registration between the individual images is measured by cross-correlation and the images are mounted accordingly.

Note: If the mounted image with check on image positions by cross-correlation (case 3) shows large shifts or the faded image mounting (case 2) does not produce a good fit, there are two potential problems: • The calibrations are not good. Redo the calibrations. • The images are distorted (lens distortions). Because lens distortions are smaller closer to the central

axis of the microscope, use smaller CCD areas (if necessary in combination with more images) to record CCD montages.


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2.7 File formats The file formats supported by photomontage are : • Small-header binary : binary data are preceded by a 10-byte header containing the data type and

x,y dimensions (first three items of the extended-header format). • Extended-header binary : the binary data are preceded by an extended header that contains the

data type, image dimensions, and several types of information stored specifically for Focus series reconstruction (see extended-header definition below). Note that depending on the particular software that writes the file, not all values are necessarily defined.

• MRC file : see separate file format description

2.7.1 Binary data type 1 - unsigned 1-byte integer 2 - unsigned 2-byte integer 3 - unsigned 4-byte integer 4 - signed 1-byte integer 5 - signed 2-byte integer 6 - signed 4-byte integer 7 - 4-byte (single-precision) floating point 8 - 8-byte (double-precision) floating point

2.7.2 Extended-header format Data type Comment 2-byte integer data type of the image (see above) 4-byte integer image width in pixels 4-byte integer image height in pixels 4-byte integer total size of the header in bytes (including preceding values). In effect the offset to the

data. double Microscope type 0 = T10 and T12, 1 = T20, 2 = T30, for Titan add 10 to HT

equivalent double Gun LaB6 = 0, FEG = 1 double Lens type HC = -2, BioTWIN = -1, TWIN = 0, STWIN = 1, UTWIN = 2, XTWIN = 3,

CryoTWIN = 4 double D number of microscope double High tension (kV) double Focus spread double MTF double Starting defocus (nm) double Focus step (nm) double DAC setting double Focus value (nm) double Pixel size (nm) double Spherical aberration (mm) double Semi-convergence (mrad) double Info limit (nm-1) double Number of images double Image number in series double Coma x double Coma y double Astigmatism 2 X


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double Astigmatism 2 Y double Astigmatism 3 X double Astigmatism 3 Y double Camera type serial number double Camera position (WA, MSC, GIF) double Magnification double Camera length double STEM magnification double Lens series double Lorentz double Spot size double Microscope mode double Objective lens value three doubles Currently undefined double Image shift X (m) double Image shift Y (m) double Image shift X (pixels) double Image shift Y (pixels) double Stage X (m) double Stage Y (m) double Stage Z (m) double Stage A (rad) double Stage B (rad) double Image minimum level double Image maximum level double Image real (0) or reciprocal space further doubles undefined Many of the values stored in the extended header are used by the TrueImage reconstruction software as a first guess to optimize the reconstructed wave function.


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2.7.3 MRC file format The MRC file is a file with extension .mrc. It contains a primary header and an extended header as described below, followed by the data. The file format is derived from the MRC file format which was originated at the Medical Research Council in Cambridge, England. • The MRC format can contain a series of images in a single file. The format consists of: • A primary header. A 1024 byte section. • An extended header. Additional information about the individual images. • Image data.

2.7.3.1 The primary header. Data type Name Byte

positionComment

integer (4-byte) nx 0 number of colums integer ny 4 number of rows integer nz 8 number of sections (i.e. images) integer mode 12 data type, 1 = pixel value stored as short

integer integer nxstart 16 lower bound of colums integer nystart 20 lower bound of rows integer nzstart 24 lower bound of sections integer mx 28 cell size in Angstroms (pixel spacing=xlen/mx) integer my 32 integer mz 36 4-byte floating point xlen 40 4-byte floating point ylen 44 4-byte floating point zlen 48 4-byte floating point alpha 52 cell angles in degrees 4-byte floating point beta 56 4-byte floating point gamma 60 integer mapc 64 mapping colums, rows, sections on axis

(x=1,y=2,z=3) integer mapr 68 integer maps 72 4-byte floating point amin 76 4-byte floating point amax 80 4-byte floating point amean 84 2-byte integer ispg 88 space group number (0 for images) 2-byte integer nsymbt 90 number of bytes used for storing symmetry operators

4-byte integer next 92 number of bytes in extended header. This is an important number. It defines the offset to the first image

2-byte integer dvid 96 creator id char (byte) extra[30] 98 extra 30 bytes data (not used) 2-byte integer numintegers 128 number of bytes per section in extended

header 2-byte integer numfloats 130 number of floats per section in extended

header


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2-byte integer sub 132 2-byte integer zfac 134 4-byte floating point min2 136 4-byte floating point max2 140 extra 28 bytes data 4-byte floating point min3 144 4-byte floating point max3 148 4-byte floating point min4 152 4-byte floating point max4 156 2-byte integer idtype 160 2-byte integer lens 162 2-byte integer nd1 164 divide by 100 to get float value 2-byte integer nd2 166 2-byte integer vd1 168 2-byte integer vd2 170 4-byte floating point tiltangles[9] 172 used to rotate model to match rotated image 4-byte floating point zorg 208 origin of image; used to auto translate model 4-byte floating point xorg 212 to match a new image that has been translated 4-byte floating point yorg 216 4-byte integer nlabl 220 number of text labels with useful data (0-10) char data[10][80] 224 10 text labels with 80 characters

2.7.3.2 The extended header Total number of extended headers (one for each image) is 1024. Please note: Some values have rather odd numbering. That is because prior usage determined that certain values were fixed too low and the addition of more types dictated the necessity for numbers below, while 0 is generally avoided. The 0 is typically an undefined number. Name Byte position Comment a tilt 0 Alpha tilt in degrees b tilt 4 Beta tilt in degrees x stage 8 Stage position in um y stage 12 z stage 16 x shift 20 Image shift position in um y shift 24 defocus 28 Defocus in micrometers exposure time 32 mean intensity 36 tilt axis angle 40 pixel size 44 Image pixel size in nanometers magnification 48 Microscope type 52 Tecnai 10 = -2, Tecnai 12 = -1, Tecnai 20 = 1,

Tecnai 30 = 2, for Titan add 10 to HT equivalent Gun type 56 W = -2, Lab6 = -1, FEG = 1 Lens type 60 HC = -3, BioTWIN = -2, TWIN = -1, STWIN = 1,

UTWIN = 2, XTWIN = 3, CryoTWIN = 4 D number of microscope 64 High tension (kV) 68 Focus spread 72 MTF 76


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Starting Df (nm) 80 Focus step (nm) 84 DAC setting 88 Spherical aberration 92 Semi-convergence 96 Info limit (nm-1) 100 Number of images 104 Image number in series 108 Coma 1 112 Coma 2 116 Astigmatism 2 1 120 Astigmatism 2 2 124 Astigmatism 3 1 128 Astigmatism 3 2 132 Camera type number 136 Camera position 140 WA = 1, MSC = 2, GIF = 3 Unused 144 Unused 148


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3 TEM Grid Scanning

3.1 Introduction During an investigation of a specimen on a TEM grid, it is often difficult to keep track of grid holes already investigated and grid holes still to be done. Furthermore, moving from one grid to another usually requires lowering the magnification, moving the specimen stage to the new hole and increasing the magnification again for a more detailed investigation. Such systematic searches are made much easier by TEM Grid Scanning. Grid Scanning makes use of the fact that grids are regular arrays of square or hexagonal holes. Once a few essential points are known (through a calibration procedure in Grid Scanning), the software can calculate the position of other grid holes and the specimen stage of the TEM microscope (the CompuStage) can be moved from one grid hole to another by a simple keyboard or mouse command. Holes passed during the Grid Scanning are displayed on the screen, making it easy to keep track of areas already investigated. Interesting specimen locations can be stored with comments added. Stored locations can be retrieved easily. Grid Scanning data can be saved in a data file for later retrieval. If reference points are defined, the software recalculates old to new locations, making it easy to retrieve previously stored locations, even if the grid is later not exactly in the same position.

3.2 Getting started The TEM Grid Scanning option consists of two parts: • The Grid Scanning Control Panel. • The Grid Scanning Display window. To use TEM Grid Scanning first use the TEM Workspace layout to drag the Grid Scanning Control Panel into a workset. Go to the tab of the workset and press the Display button in the Grid Scanning Control Panel. The Grid Scanning Display window will be loaded and its functionality become available. You can leave the Grid Scanning Control Panel in your workset for future use. The Display window will only be loaded when the Display button is pressed. The major part of the Grid Scanning functionality is present only in the Display window. The Control Panel also provides rapid access to the more often-used functions.

3.3 Grid scanning Control panel The Grid Scanning Control Panel contains two sets of functions for grid scanning: • Functions that load the Grid Scanning Display window (a

separate window that is typically positioned in the data space of the TEM User Interface) or determine its size.

• Functions that give rapid access to often-used Grid Scanning functions.


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Display The Grid Scanning Display window is only loaded on operator request. To load it press the Display button. A message will appear that the Grid Scanning server is being loaded. Once the server is running, the window will be displayed, the Display button will turn yellow, and the message will disappear. You are now ready to start working with Grid Scanning. If the Display button is pressed again, the Display window will disappear. It is, however, not unloaded but simply hidden. If you display it again (by once more pressing the button), it will still have kept all the Grid Scanning settings as set previously. The window is only unloaded when you close the TEM User Interface. Freely sizeable When this option is checked, the Display window can be positioned anywhere you like (the software will remember your settings and restore them) and you can define its size in the standard Windows way (click and drag on a border). If the option is off, the Display window will be fixed in size and position to fill the data space of the TEM User Interface. The following controls will be enabled or disabled when their counterpart in the Display window is enabled or disabled. Next Moves the stage to the next hole. Previous Moves the stage back to the previous hole. Center Moves the stage back to the center of the current hole. Add Adds a stored location for the current stage position. If text has been inserted for a label, this text will automatically be added to the location. If labels are not used, the label text is disabled. If labels are used and the location label field in the Control Panel is empty, a dialog will be displayed in the Grid Scanning Display in which the label can be entered. (So the difference is that the label in the Control Panel is defined before storing the location while in the display it is done afterwards.) Location label Text entered here will be added as a label to a location stored.


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3.4 Grid scanning display The Grid Scanning Display window is a window that is typically located in the data space of the TEM User Interface. It can either be freely positioned and sized or fixed. The choice between the two is made in the Grid Scanning Control Panel. The display contains two essential elements, the menu and the display itself. The main functionality of Grid Scanning resides in the menu of the display window. A typical sequence for setting up and executing a Grid Scanning is as follows: • If necessary, check that all options are defined as needed (some options cannot be changed later

unless New grid is select first in the File menu). • Define the grid hole parameters by bringing three special points on a grid hole and its neighbor to the

center of the screen with the specimen stage during the calibration procedure. • Start moving from gird hole to grid hole, storing locations along the way. • If the data for the grid must be stored for later retrieval with the grid again in the microscope, define

the reference points. • Store and/or print the data for later reference.

3.5 The display The window will display the a schematic outline of the grid (circle) with the stage axes. The current location of the stage is indicated by a solid square (red in the image below). Grid holes passed and stored locations are also displayed, the grid holes in their proper orientation relative to the stage axes. Stored locations are indicted by their serial number. The colors used for the display are freely user-configurable.


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Menu The menu gives access to all Grid Scanning functionality. Status information Grid Scanning displays some status information in a legend at top left of the display. This includes the number of holes passed and of stored locations. Status bar The status bar lists on the left the current stage position. In the center it displays the major options chosen for the grid scan. Error messages can be displayed on the right. Border icons The border icons appear to behave as the standard Minimize, Maximize and Close buttons. The Close button, however, does not truly close the window but hides it. The window is closed only upon closing the TEM User Interface.

3.6 The menu The menu consists of five main menus with submenus: • File • Setup • Hole • Location • Help

3.6.1 File menu The File Menu provides the operations that are concerned with file operations, the settings and printing. New grid Starts a new grid scan (resetting all previous montage values). Open Opens an existing grid scanning file. Save, Save as

Saves the grid scanning data in a file, Save as allows definition of different file name.

Control options Leads to the Control options dialog. Load options Loads options from file. Save options Saves options to file. Printer setup Leads to printer setup dialog. Print list Prints the list of stored locations and labels. Print graphic Prints the current graphical display.


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3.6.2 Setup menu With the Setup Menu, the operator defines the parameters of the Grid Scanning, defines (or redefines in case of data reloaded from file) the reference points, and starts a new spiral scan. Calibrate grid Insert the current specimen-stage X,Y location into the list of points that define the

Grid Scanning boundary. Reference points Starts procedure for storing reference points. Show ref.pt. data After reference points from an old file have been found again, the software can

display the relation between old and new reference points, such as rotation angle and relative shifts.

New spiral Allows starting of a new spiral scan at a user-defined location.

3.6.3 Hole menu The Hole Menu contains the items that control movement of the specimen stage: to the next hole, back to the previous hole and back to the centre of the current grid hole. Where the next hole will be is dependent on the scan type chosen. Next hole Moves stage to next hole. How the movement is executed depends on the type of

scan chosen. Previous hole Moves stage back to previous hole. Hole center Moves the specimen stage back to the centre of the current grid hole. Start/resume auto In automatic movement mode the scan can be (re)started with start/resume auto. Stop auto In automatic movement mode the scan can be stopped with stop auto.

3.6.4 Location menu The Location Menu contains a number of functions that are related to the locations stored. Add Adds the current stage location to the stored locations list. Delete Deletes one or more locations from the stored locations list. Display list Displays a list with all stored locations (X, Y, label) and allows printing of the list. Show number Shows a list of stored locations (with number and label), allowing selection of one

location for which the data (X,Y, label) will be shown. A button Goto allows moving the stage to the stored location.

3.6.5 Help menu The Help menu contains two items, Show and About. Selecting Show (or pressing F1) will display this Help file in the TEM User Interface. About will display the About TEM Grid Scanning dialog listing the current version number of the software. When reporting bugs, always include the version number in the report.


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3.7 Control options The software supports a range of options, defined in the Control options dialog. Options are stored as set for each user separately and recalled upon restart. Options can be saved to file and reloaded.

Grid type The software supports two types of grid, with square and with hexagonal (honeycomb) holes. Identify the type of grid before the calibration is started Scan pattern The scan pattern is one of four:

A Spiral scan is a scan where the movement goes around in a spiral around the starting point. Spiraling around the first hole (in the centre of the grid at the intersection of the black lines that represent the X and Y axes), an increasing number of holes is passed (follow the red lines with arrows). Passed holes are shown in their proper orientation in green, the current stage position is shown by the red square.

A Linear scan is a scan where the holes are arranged in lines that stretch from one side of the grid to the other. These lines themselves are accessed once again in a spiral. On a linear scan an increasing number of holes is passed (follow the red lines with arrows). Passed holes are shown in their proper orientation in green, the current stage position is shown by the red square.


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A Cursor scan is a scan where the movement depends on the cursor keys pressed by the operator (up, down, left, right). Starting at the first hole an increasing number of holes is passed (follow the red lines with arrows) by pressing one of the keyboard cursor keys. The meaning of up, down, left, right is as seen on the display (not direction of motion on the microscope's fluorescent screen). Passed holes are shown in their proper orientation in green, the current stage position is shown by the red square.

A Random scan is a scan where the software calculates grid-hole positions on the basis of random moves in the X,Y direction. In this way it is possible to remove operator bias from the procedure and thus to reduce the number of grid holes that must be scanned for statistical confidence. Each hole passed is based on a random selection of X and Y position. Movement from one hole to the next is outlined by the red line and arrows.

Scan type The Scan type can be either manual or automatic. In manual scan, the operator must command the software to move the specimen stage from one hole to the next. In automatic scan, the software waits until the delay time has passed and then moves the stage to the next hole. Relocatable grid

A Relocatable grid is a specially shaped grid that has an asymmetric 'ear' that fits into the single-tilt specimen holders delivered since 1993. The 'ear' takes care that the grid can be loaded into the specimen in only one way, without rotation and very little sideways movement. The relocatable grid thus makes it easy to remove specimens from the microscope holder and putting them back in and still retrieve stored locations easily. If relocatable grids are used, switch on the option under Control Options. Grid Scanning will then make it possible to drive back directly to the old location of the first reference point which should be very close to the current location of that point.

Use labels The label refers to user-written comments (up to a maximum of 100 characters) than are stored together with stored locations. If Use labels is checked the software will bring up a dialog allowing insertion of the location label. Use hyperlabels The software provides a special feature in the form of hyperlabels (labels that act like hyperlinks: when the cursor moves over them they flash their color to blue and a mouse-click brings up a dialog with information about the point, with additional functionality such as Go to or Delete).


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Use Function keys Function keys can be used in addition to menu commands to control Grid Scanning. The following functions are used: • Up, down, left, right arrows in cursor scan (these have no menu equivalent). • F4: Add location • F6: Next hole • F8: Previous hole Use Other keys Additionally a few other keyboard keys can be used: • Enter key: Add location • Space key: Next hole Colors The four types of colors (Background, Foreground, Holes, Locations, Current location) for the display can be freely selected. The selection is made in the Control options dialog or by clicking on the window with the right-mouse button. The latter will popup a menu that allows definition of the colors. Note: There is one exception to free color selection: if hyperlabels are used the Location color cannot be blue (this is the flash color of the hyperlabel).

3.8 Calibrate grid Calibration is the setup step wherein the software is informed about the dimensions and orientation of the grid. This is done by moving three points to the center of the screen with the specimen stage. The software will show a dialog with one of the following schematic drawings of a part of a grid.

The three calibration points lie on opposite sides of a grid hole (1, 2) and in the next grid hole (3). The choice of the points doesn't matter (for instance, 1 and 3 can be closest to the operator, 2 furthest away); what counts is their relation. After the third point has been entered the software knows the size of a grid hole, the orientation and the shift necessary to go from one hole to the next. The software then will drive the stage to a grid hole 750 �m away from the current position, where a point similar to number 1 must be found. From the position of this point, the software fine-tunes the calibration. If no fourth point can be identified with certainty, simply press OK without moving the stage.

3.9 Reference points Reference points are locations on the specimen that are easily recognized and can be used as reference when the specimen is removed from the specimen holder and later put back in again. By finding the reference points again, the software can recalculate the old locations to new ones.


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Reference points should be located close to but not at the periphery of the specimen. Locations further from the center increase the accuracy of relocation but if a reference point later lies outside the reach of the stage, then it is not possible to recalculate the old locations to new ones. Because specimens are often shifted up to 0.2 mm when re-inserted into the specimen holder, keep the reference points less than 800 �m away from the center. Setting the reference points can be done at any stage of Grid Scanning (but before the data are saved into a file, otherwise the reference points are not included in the file). When an old file containing reference points is opened, the software will ask to redo the reference points immediately. If the reference points are not redone at this stage, the software will display the stored locations and tracks in their original configuration. The reference points can then still be done later. Setting reference points for new data

During the procedure wherein new reference points are defined, the operator is asked to drive the stage to 2 or 3 points that can be recognized. It is suggested to avoid points that lie on the opposite side of the center as this will make it much more difficult to decide later whether the specimen has been reloaded in the same way or upside down. If at least two points are defined lying at an angle of about 120°, then it will be easier to decide. Three points make recognition of an upside-down position of the specimen even easier. If no third reference point is to be set, then simply press OK

while keeping the specimen stage at the same location as for point 2. The third point will then be ignored. Redoing reference points for old data

If a file containing reference points is reopened, the software will suggest to go through the procedure for relocating the old reference points. The software will show the distribution of the old reference points and list the label of each point in turn. The reference point locations will be updated with the new ones, once a reference point has been found again.

During this procedure this software can assist in finding back the old reference points through the Assist function. The Assist function will list a number of possibilities depending on which reference points must be found again: • Move the specimen stage to the old location for point 1. • Move the specimen stage in a circle, around 0,0 for point 1, around 1 for point 2, etc. • Calculate alternative locations for points 2 and 3 on the assumption that the specimen is upside

down. After finding reference point 1, the software will calculate what the most likely new location is for reference point 2 and ask if the stage must be driven there. The same is done for point 3 (if a third reference point had been set originally). For each reference point found back as well as the whole set of the reference points the software will go through a number of consistency checks (distances between old points and new points comparable, angles from the center of gravity of the old three-point triangle and old points and the center of gravity of the new three-point triangle and new points comparable). The software can handle rotation, shift and mounting upside down of the specimen. The software also calculates a 'stretch' parameter that is related to the percentage difference in dimensions between old and new reference points. The software cannot however accommodate severe distortions due to bending of the specimen, so some care should be taken to ensure the integrity of the specimen. The various values can be displayed under Show ref. pt. data.


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3.10 Show ref.pt. data After reference points from an old file have been found again, the software can display the relation between old and new reference points, such as rotation angle and relative shifts. The data are displayed as follows: • Old reference points (1, 2, 3) X,Y locations. • New reference points (1, 2, 3) X,Y locations. • Specimen position normal or upside down. • Rotation angles for all three points and individual angles. • Relative shift. • Stretch factors. The two sets of reference points define two triangles. The first effect may be that one triangle is mirrored relative to the other (when the grid is upside down). In the calculation of the data the software will first 'undo' the mirror operation. After removing any mirror, we again have two triangles which are now (usually) rotated relative to each other. The software next 'undoes' the rotation. The final result will be two triangles that are somewhat shifted relative to each other. Rotation angles The software calculates the rotation angles for four points on each triangle: the center of gravity and the three reference points individually. The values for all angles should be consistent (larger deviations for individual points may occur when the points lie too close to 0,0 since the angles are defined as between the lines through New - 0,0 and old - 0,0). Relative shift The shift is can also be calculated for the centers of gravity of the triangles. Stretch factors The distances between reference points in the old and new triangles should be similar (e.g. Old 1 to 2 should be of the same length as New 1 to 2). Strong deviations in the stretch factors from 1.0 are an indication that either one or more reference points is wrong or the grid has been distorted considerably during unloading/loading. If there are serious errors in the reference points, note down the positions of the new reference points most likely to be correct and open the file again. Go through the reference points procedure again to find the proper reference points.

3.11 New spiral In spiral scanning it may be desirable to start a new spiral at a new location, for example to skip areas empty of specimen, as when several tissue sections lie dispersed on a grid with empty space in between or the support foil of part of the grid has disappeared. To start a new spiral scan, drive the stage to the center of the grid hole that will form the start of the new spiral.


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4 TEM Smart Tilt Diffraction experiments can be difficult, especially in combination with the non-eucentric �tilt. Inexperienced users often find it difficult to predict by how much the stage must be tilted to bring a certain feature of the diffraction pattern (zone axis or systematic row) into alignment with the optical axis. TEM Smart Tilt combines a range of tools that assist in diffraction experiments: • A representation of the TEM viewing screen with the orientation of the tilt axes and tilt angles

indicated. The display is coupled directly to the active camera length on the microscope. • The possibility to double-click with the left-hand button of the mouse anywhere on the 'viewing

screen' displayed and the feature corresponding to that location will be brought to the center of the diffraction pattern by tilting.

• Accurate measurement of tilts necessary to bring certain features in the diffraction pattern along the optical axis or tilt them half-way ('Bragg' tilting to go from a diffracted beam in Bragg condition to the systematic row). After Bragg tilting it is possible to tilt left or right around the systematic row afterwards in user-defined tilt steps.

• Rapidly toggle between focussed diffraction pattern and shadow image for both SAED and CBED.

4.1 Getting started The TEM Smart Tilt option consists of two parts: • The Smart Tilt Control Panel. • The Smart Tilt Display window. To use TEM Smart Tilt first use the TEM Workspace layout to drag the Smart Tilt Control Panel into a workset. Go to the tab of the workset and press the Display button in the Smart Tilt Control Panel. The Smart Tilt window will be loaded and its functionality become available. You can leave the Smart Tilt Control Panel in your workset for future use. The Display window will only be loaded when the Display button is pressed. The major part of the Smart Tilt functionality is present only in the Display window. The Control Panel also provides rapid access to the more often-used functions.

4.2 Smart Tilt Control Panel The Smart Tilt Control Panel contains two sets of functions for Smart Tilt: • Functions that load the Smart Tilt Display window (a


• Functions that give rapid access to often-used Smart Tilt functions.


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Display The Smart Tilt Display window is only loaded on operator request. To load it press the Display button. A message will appear that the Smart Tilt server is being loaded. Once the server is running, the window will be displayed, the Display button will turn yellow, and the message will disappear. You are now ready to start working with Smart Tilt. If the Display button is pressed again, the Display window will disappear. It is, however, not unloaded but simply hidden. If you display it again (by once more pressing the button), it will still have kept all the Smart Tilt settings as set previously. The window is only unloaded when you close the TEM User Interface. Freely sizeable When this option is checked, the Display window can be positioned anywhere you like (the software will remember your settings and restore them) and you can define its size in the standard Windows way (click and drag on a border). If the option is off, the Display window will be fixed in size and position to fill the data space of the TEM User Interface. The following controls will be enabled or disabled when their counterpart in the Display window is enabled or disabled. Full Starts the Full (zone-axis) tilt procedure. Bragg Starts the Bragg tilt procedure. Left Tilts one tilt step to the left. Right Tilts one tilt step to the right. Switch Starts the switch direction procedure. Tilt step Defines the step used for tilting right and left. Focus Sets beam or diffraction pattern focus to the focussed setting. Defocus Sets beam or diffraction pattern focus to the defocussed setting (shadow image). Flap-out button The flap-out leads to the Smart Tilt Options tab.


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4.3 Smart Tilt Options The Smart Tilt Options contains several settings for using Smart Tilt.

Use compucentric tilting Smart Tilt fully supports compucentric tilting (tilting where the non-eucentric behavior of the�tilt axis is compensated). The compucentric tilting checkbox allows selection or deselection of compucentric tilting. This choice is a system-wide setting. The same setting may be selected elsewhere, e.g in the Compucentricity Control Panel itself. Buttons visible The Smart Tilt display has the possibility to display a toolbar with buttons for often-used functions. The Buttons visible option decides if this toolbar is visible or not. Show reminder The various Smart Tilt procedure rely on a properly centered diffraction pattern at the start. The operator can either make sure that the pattern is always properly centered. Or the software can remind the operator in each. User buttons You can assign some of the Smart Tilt functions to User Buttons (L1..L3, R1..R3) on the TEM Control Pads. This may make it easier to control, especially when intensively observing the diffraction pattern on the viewing screen. Each function can be assigned to one of the User Buttons by selecting one from the drop-down list. If None is selected, the function is not connected to a User Button. When a function is assigned to a button already occupied, the function of the occupied button will be switched automatically to en empty user button. The buttons are disconnected as long as Smart Tilt is not loaded or when it is hidden (e.g. by pressing the Display button). Focus CBED and SAED For the focus/defocus function of Smart Tilt two choices exist, one being CBED (Convergent Beam Electron Diffraction), the other SAED (Selected Area Electron Diffraction). For CBED the Intensity (C2) is used to switch between focussed diffraction pattern and shadow image. For SAED it is the diffraction focus that is used.


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4.4 Smart Tilt display The Smart Tilt Display window is a window that is typically located in the data space of the TEM User Interface. It can either be freely positioned and sized or fixed. The choice between the two is made in the Smart Tilt Control Panel. The display contains two essential elements, the menu and the display itself. The main functionality of Smart Tilt resides in the menu of the display window.

4.5 The display The window will display a schematic representation of the TEM viewing screen (but in gray instead of yellow to reduce ambient lighting near the microscope).

Menu The menu gives access to all Smart Tilt functionality. Toolbar buttons Toolbar buttons giving rapid access to often-used functions can be found on the left-hand side of the Smart Tilt display (the display of the toolbar is optional). If there is sufficient room the buttons will be large, otherwise they are small (as shown here). From top to bottom the buttons are Full tilt, Bragg tilt, Left, Right, Switch direction, Undo, Redo, Tilt step, Focus and Defocus. Display The display is a representation of the viewing screen of the TEM microscope with its inscribed circles and plate markers. On top of this, Smart Tilt displays the tilt axes in their orientation and circles representing tilt angles. The tilt axes are shown in red, the blue circles on the screen mark tilt angles and the green line (if present) marks the direction of the diffraction spot used for Bragg angle tilting. Convention: The direction of the tilt axes (+/-) is displayed in such a way that the center of a Laue circle will move in the direction of the marker on the screen if that particular direction and sign of tilt is chosen.


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By double clicking with the left-hand mouse button on the area covered by the screen, the software is instructed to change the tilt of the CompuStage by the values corresponding to the point where the mouse button was clicked. Convention: The tilt under mouse-button control is such that the point corresponding to the cursor location is brought to the center of the diffraction pattern. Note that this tilt is in the opposite direction to the movement indicated by the tilt axes on the screen. Status bar The status bar lists on the left the current stage position. In the center it displays the major options chosen for the grid scan. Error messages can be displayed on the right. Border icons The border icons appear to behave as the standard Minimize, Maximize and Close buttons. The Close button, however, does not truly close the window but hides it. The window is closed only upon closing the TEM User Interface.

4.6 The menu The menu consists of five main menus with submenus: • File • Calibrate • Tilt • Focus • Help

4.6.1 File menu The File Menu provides the operations that are concerned with file operations and toolbar display. Load calibrations Opens an existing Smart Tilt calibration file. Save calibrations, Save calibrations as

Saves the Smart Tilt calibration data in a file, Save as allows definition of different file name.

Buttons visible Determines whether the toolbar buttons are visible (menu item checked) or not.

4.6.2 Calibrate menu In the Calibrate Menu the calibration procedures are found. The calibrations required by Smart Tilt are two-fold: • The apparent rotation between the diffraction shift and the viewing screen for the different camera

lengths. • The calibration of the diffraction shift (rotation and distance) against the alpha and beta tilts. • Both types of calibrations are necessary for proper operation of Smart Tilt. Calibrations can be saved

in a file and reloaded. Calibrations are also automatically stored for the user once a procedure has been finished successfully.

All camera lengths Calibration procedure going through all camera lengths. Current camera length Calibration procedure for the current camera length. Alpha tilt Calibration of the alpha tilt against the diffraction shift. Beta tilt Calibration of the beta tilt against the diffraction shift.


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4.6.3 Tilt menu The Tilt Menu contains the items that control the tilting operations. During tilting operations the software keeps track of (a limited amount of) past stage positions (approximately 50) so a tilting operation can always be undone. Once one or more operations is undone, it is also possible to step through them forward again. Show reminder The tilting operations rely on a measurement of the diffraction shift. Starting

position must therefore always be a well-centered diffraction pattern. If the reminder is on, the operator will be reminded before each diffraction-shift measurement to check the centering of the diffraction pattern.

Zone axis Starts the Full (zone-axis) tilt procedure. Bragg Starts the Bragg tilt procedure. Set step Sets the tilt step used for Left and Right tilting. The tilt step is the absolute

change in angle in space (so made up of the proper combination of alpha and beta tilt).

Left Tilts around the systematic row (defined by the Bragg tilt or Switch direction operation) to the left.

Right Tilts around the systematic row (defined by the Bragg tilt or Switch direction operation) to the right.

Switch Changes the direction of the systematic row. This procedure is similar in operation to the Bragg tilt except that only the measurement is executed and no change of tilt is made.

Undo Switches back to previous stage position. Redo Goes forward to next stored stage position. Display list Display the list of past tilting operations. Any of the items can be selected by

clicking. A right-hand mouse-button click will popup a menu with Hide list (equivalent to using Display list in main menu again), Go to selected position, Copy (as text).

Copy (as text) Copies contents of tilt list as text to the clipboard. It can then be pasted into any program that accepts text input.

Compucentric Enables (checked) or disables compucentric tilting. Function is disabled when TEM Compucentricity is absent.

4.6.4 Focus menu The Focus Menu contains a number of functions that are related to the use of the focus functionality for rapid toggling between focussed diffraction pattern and shadow image. The Focus and Defocus settings must be defined before these functions can be used. It is advised to define these on a regular basis (e.g. each microscope session). Focus Toggles to focus condition. Defocus Toggles to defocus (shadow image) condition. CBED Select CBED focus/defocus (Intensity is focussing lens). SAED Select SAED focus/defocus (Diffraction lens used for focussing). Define focus Defines focus condition (use Intensity for CBED, Diffraction focus for SAED). Define defocus Defines defocus condition (use Intensity for CBED, Diffraction focus for

SAED).


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4.6.5 Help menu The Help menu contains two items, Show and About. Selecting Show (or pressing F1) will display this Help file in the TEM User Interface. About will display the About TEM Smart Tilt dialog listing the current version number of the software. When reporting bugs, always include the version number in the report.

4.7 Camera length calibration The rotation calibration consists of a simple procedure to determine the orientation of the diffraction pattern relative to the viewing screen. The procedure for a single camera length consists of two steps. In the first step, the diffraction pattern must be centered accurately on the screen with the Multifunction X,Y knobs. Once that is done the following dialog appears.

The central beam in the diffraction pattern must now be shifted to the marker indicated (or as far as possible in the direction of the marker). Note that the distance shifted is not relevant, it is the direction that is important. In the procedure for all camera lengths, the software goes through the same procedure for all camera lengths.

4.8 Alpha and beta tilt calibration In order for the program to display the orientation of the tilt axes on the screen and determine the tilts required when the diffraction shift is used, it is necessary to calibrate the tilt axes. In these procedures (unlike the rotation calibration), it is not only the direction that is important but also the magnitude of the shift. The tilt calibration procedure consists of the following steps (all executed after instructions from the program): • Tilt a crystal to a zone axis that is well-defined and will be recognisable from the Kikuchi pattern even

when tilted off axis. • Center the diffraction pattern accurately. • Tilt the zone axis away as far as possible (the higher the tilt away from zone axis, the more accurate

the calibration will be) with one tilt axis only, while still being able to recognise it. • With the diffraction shift recenter the zone axis (the intersection of the Kikuchi bands) on the screen.


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This procedure provides the program with the necessary data to transform a diffraction shift (orientation and value) to a tilt (direction and value). A suitable specimen for the procedure is one that will show strong Kikuchi bands and one that isn't bent (bending of the specimen will change the apparent tilt of the specimen and thus lead to an incorrect calibration). The silicon substrate of semiconductor specimens (somewhat thicker areas tend to show good Kikuchi bands and are also usually straight, unlike the thin edge) has been found to be quite good.

4.9 Full (zone axis) tilt The displacement of the diffraction pattern is a measure of tilt. By centering a feature in the diffraction pattern on the viewing screen with the diffraction shift the software can convert the tilts required to bring the particular feature (e.g. the center of a zone axis) to the screen center. In the case of Full tilt, the tilts are set in such a way that the particular feature is centered fully. Sequence of diffraction patterns outlining the Full tilt method:

Starting point is a pattern where a zone axis is clearly recognisable (at the intersection of the Kikuchi bands) but off-center. The center of the viewing screen is marked by the black cross. Next the pattern has been shifted with the diffraction shift so now the zone axis has been centered on the screen. The center of the viewing screen is marked by the black cross. The displacement necessary to center the pattern is then translated into tilts and the displacement is reset to zero and as a result the zone axis is now centered. The center of the viewing screen is marked by the black cross.


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4.10 Bragg tilt The displacement of the diffraction pattern is a measure of tilt. By centering a feature in the diffraction pattern on the viewing screen with the diffraction shift the software can convert the tilts required to bring the particular feature (e.g. a diffracted beam in Bragg condition) to the screen center. In case of the Bragg angle tilt, the tilts are set in such a way that only half of the angle from diffracted beam to transmitted beam is done (that is, if a beam is in the Bragg condition, the pattern after tilting will be in the symmetrical condition). Besides bringing a crystal from the Bragg condition into the symmetrical condition, this option also defines the direction for right and left-perpendicular tilting (tilting around the systematic row that contains the diffracted beam just used for setting the Bragg angle). The direction of the original diffracted beam is indicated on the viewing-screen representation by a green line from the center. Sequence of diffraction patterns outlining the Bragg angle method:

Starting point is a pattern where a diffracted beam is in the Bragg condition (note placement of the Kikuchi lines). The center of the viewing screen is marked by the black cross. Next the diffracted beam is centered on the screen with the diffraction shift. The center of the viewing screen is marked by the black cross. The displacement necessary to center the diffracted beam is then translated into tilts, the displacement is reset to zero, and as a result the pattern on the right shows the pattern centered on the systematic row. The center of the viewing screen is marked by the black cross. Thereafter it is possible to select left or right perpendicular tilt which will tilt the crystal around the systematic row containing the diffracted beam.


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4.11 Focussed diffraction and shadow image The shadow image is often a handy tool during tilting. In normal operation it is unfortunately always necessary to refocus the diffraction pattern (for SAED: Selected Area Electron Diffraction) or the beam (for CBED: Convergent Beam Electron Diffraction) when the shadow image has been used. Not only is this cumbersome, it can also require changing to different conditions before changing back to the diffracting conditions required. Smart Tilt allows the operator to set a focus and a defocus condition (on the Intensity for CBED, on the diffraction lens for SAED) and then toggle rapidly and accurately between them. Shadow image: an explanation The shadow image essentially is a mixture of diffraction and image information. In a focussed diffraction pattern (either SAED or CBED), there is no image information. The diffraction information in the pattern is therefore at its optimum, but it is difficult to determine where exactly the information is coming from. In the shadow image all diffraction disks (even the SAED pattern no longer has real spots) show a small image containing the information contributing to the diffraction disk. This image information makes it thus possible to see where the diffraction pattern is coming from or to track and correct for sideways movement during tilting. In the shadow image Fresnel contrast can be visible. This Fresnel contrast (not the diffraction contrast - an area scattering strongly will remain dark in the transmitted beam) depends on whether the shadow image is underfocus or overfocus, both in SAED and CBED. Going from under- to overfocus results in a Fresnel contrast reversal as well as a flip of the image visible in the disks by 180°. In order to get consistent results, one should always choose one or the other.


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5 TEM Compucentricity

5.1 Introduction The β tilt of the double-tilt holder is not eucentric. Its motion therefore results (in the ideal case) in a change in the X and Z values. (In non-ideal cases also Y and α are changed a little.) This non-eucentric behaviour can make it hard to keep track of a feature of interest in the specimen during tilting. TEM Compucentricity makes it possible to reduce these non-eucentric motions and thereby makes it easier to use the β tilt.

Schematic diagram of the effect of the β tilt. The thick black lines show the locations of the X and Z stage axes, while the dotted black line indicates the eucentric height. The central blue circle is the β tilt axis, which is displaced from its 'ideal' X 0, Z 0 location. The dotted blue circles indicate the path traveled by a point when tilted around β. Tilting around β for the point lying on the smaller dotted blue circle requires a small correction to X and Z (red arrow lines) to bring the area back to the same position in space (but a different stage position). For the point lying on the larger dotted blue circle the correction (red arrow lines) is much larger because it is further away from the tilt axis itself. The corrections are applied automatically when compucentric tilting is used. Compucentricity consists of: • Establishing the current stage position and the required new tilts. • Calculating what corrections should be made to the stage axes to keep the feature of interest

centered on the screen. • Moving the CompuStage to the new position. Since specimen holders deviate from the 'ideal' model, a correction can (and for good results should) be made for this 'non-ideal' behaviour through a calibration procedure. Data are stored for each specimen holder separately. Prior to calibration holders must therefore be identified by name.


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5.2 Getting started The TEM Compucentricity option consists of two parts: • The Compucentricity Control Panel. • The Compucentricity server. This (hidden) server is accessible to other software that supports

compucentric tilting like TEM Smart Tilt. To use the TEM Compucentricity first use the TEM Workspace layout to drag the Compucentricity Control Panel into a workset. Go to the tab of the workset with the Compucentricity Control Panel. Select an existing holder or add a new one. If necessary calibrate the holder. From there on compucentric tilting is available. Note: Due to the construction of the CompuStage and the nature of the Goto procedure, the Y axis will change when the Z is changed. In order to ensure getting the correct stage position after compucentric tilting, the tilting is done in two stages, the first setting the X, Z, α and β positions, and the second the Y position.

5.3 Compucentricity Control Panel The Compucentricity Control Panel provides the controls necessary for using TEM Compucentricity. In essence this means identifying a holder data set and selecting it. Holder data sets do not necessarily have to be physically separate holders. The same physical holder can have several different holder data sets in the list (e.g. each user his/her own set).

Holder list The holder list gives an overview of all holders currently defined. The list gives the names and the Windows NT user name of the person who defined the holder. The holder list can be sorted alphabetically on holder name and user name by clicking on the buttons Holder and User at the top of the list. Repeated clicking on the same button reverses the sorting order. The Select button doesn't become enabled until one holder in the list is selected. One entry in the list is always Default. This entry defines the 'ideal holder' which has its β tilt axis defined as 0 for all parameters. Select The Select button can be clicked once a selection in the holder list has been made. When the Select button is pressed the calibration parameters of that holder are entered into TEM Compucentricity. To reset the parameters to 0, select the Default holder and press Select. Note: If no user-defined holder (any other selection than Default) is selected, the flap-out tabs Data, Calibrate and Cal. data are not accessible.


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Remove The Remove button allows removal of the holder selected from the list. Holders can only be removed by their owners (the user with the same name as in the list) or the supervisor. The software will ask for confirmation before a holder is removed. Copy The Copy button allows copying the parameters from one holder data set to a new holder data set. First enter a new name for the holder. Then select a holder from the list and press Copy. Holder name A new holder data set is defined by entering a name in the edit control at the bottom of the Control Panel and either pressing Add or Copy. Add When a name in entered for the holder in the edit control at the bottom the Add button becomes active. When the button is pressed a new data set is entered in the list. Add is equivalent to a copy of the Default holder data set. Flap-out button The flap-out leads to the Compucentricity Options, Data, Calibrate and Cal. data tabs.

5.4 Compucentricity Options The Compucentricity Options tab provides a checkbox to select or deselect compucentric tilting. This choice is a system-wide setting. The same setting may be selected elsewhere, e.g in the Smart Tilt Control Panel. When the choice is on, tilting operations by software that supports compucentric tilting will use compucentricity, otherwise tilting will be done without corrections.

5.5 Compucentricity Data The Compucentricity Data tab provides information on the calibration data for the holder selected. For the Default holder the tab is disabled (all settings are zero by definition).


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Note: If no user-defined holder (any other selection than Default) is selected, the flap-out tabs Data, Calibrate and Cal. data are not accessible. The holder data consist of the following parameters: • X shift: the displacement of the β tilt axis in the X direction of the stage. • Z shift: the displacement of the β tilt axis in the Z direction of the stage. • A skew: the rotation of the β tilt axis in the direction of the � tilt axis of the stage. • C skew: the rotation of the β tilt axis in the vertical direction (a non-existent γ tilt axis of the stage). • Residual: a measure of the deviation of all the individual position measurements of the calibrations

from the fitted data. • Points: the number of X, Y, Z, α points for which a calibration has been done (with a maximum of

10). • Tilts: the number of tilts for each calibration. For all microscopes this number will be 11 except for

U-TWIN instruments where it is 9 (because of the more limited tilt range of the U-TWIN lens). • X, Y, Z locations of the calibration points in a list. The data can be copied by either clicking with the right-hand mouse button in the tab (and selecting Copy to text) or clicking inside the tab and pressing Ctrl+C. The data are then copied to the clipboard as text and can be pasted into any software that accepts text input.

5.6 Compucentricity Calibrate The Calibrate tab contains the controls needed to perform a calibration of the β tilt axis of a holder. The calibration procedure consists of defining a series of locations of a point on the specimen for a range of β tilt values, starting at 0°, then on to -5°, -10°, etc. unto -25° (-20° for U-TWIN), then back to +5°, +10° and so on to +25° (once again +20° for U-TWIN). At each tilt the operator must recenter the feature in the specimen accurately at the center of the screen and at the eucentric height.

Note: If no user-defined holder (any other selection than Default) is selected, the flap-out tabs Data, Calibrate and Cal. data are not accessible. Note 1: It is important that all points are correct. Since it may be impossible to achieve the correct eucentric height (due to the limitations of the Z range) at high � tilts and high values of X (further from zero than -500 �m or +500 �m), calibration should in general not be attempted too far away from the center of the stage. The accuracy of the calibration depends, however, on the location where the calibration is executed, with improved accuracy if the calibration point is close to the area where compucentric tilting will be done. The worst case is to calibrate close to the � tilt axis (where the compensations are small so stage position measurement errors can dominate over the actual compensations) and then tilt much further away from the actual position of the tilt axis. A much better general procedure is to combine calibrations at about -400 �m and +400 �m from the �tilt axis position.


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A general procedure for establishing a suitable calibration would be as follows: • Find a recognizable specimen area at X ~ 0 and Y ~ 0. Execute a calibration. The data for the holder

will now list where the X position of the β tilt axis lies. • Find a recognizable specimen area at approximately X shift -400 μm. Select Replace to replace the

earlier calibration (whose X and Z positions are usually reasonable but the A and C skews tend to be larger than realistic) and perform a new calibration.

• Find another recognizable specimen area at approximately X shift +400 μm. This time select Add (or Compare) and Add another point to the calibration.

Note 2: All β tilts set by the calibration procedure will be executed compucentrically. That means that the image feature selected should stay reasonably close to the center of the screen, unless the current holder calibration is far off (which may be the case when starting from the default settings). Start button The Start button starts the calibration procedure. The software instructs the operator to set the�tilt close to 0 and center the recognizable image feature. Instructions panel The instructions panel gives instructions during execution of the calibration procedure. Continue button The Continue buttons leads to the next step of the calibration procedure. When the procedure is finished, the calibration parameters and residual are calculated, dependent on the option Replace, Add or Compare chosen. After the procedure the stage is reset to the position (and tilts) at which the calibration procedure was started. Cancel button The Cancel button cancels the calibration procedure. The stage is reset to the position (and tilts) at which the calibration procedure was started. Replace If a calibration already exists, the Replace option is enabled. When this is selected the new calibration will replace the earlier calibration (all points). Add If a calibration already exists, the Add option is enabled. When this is selected the new calibration will be added to the earlier calibration. If ten points were already present, the first point is deleted and all points are shifted down one to make room for the data of the new point. Compare If a calibration already exists, the Compare option is enabled. When this is selected the two new calibrations will be calculated, one in which the new calibration is used only (Replace) and one in which it is added to the earlier calibration (Add). The Cal. data tab will become active and the data are displaced side by side. Choose an option (Replace or Add) and press Continue.


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5.7 Compucentricity Cal. data The Compucentricity Cal. data tab is displayed automatically at the end of a calibration procedure when the Compare option was chosen (it can also be inspected afterwards). It compares the calibration parameters and residual for the case where the new calibration would replace all existing data (left) and where it would be added (right). The operator can now choose to Replace or Add and press Continue to finish the calibration.

Note: If no user-defined holder (any other selection than Default) is selected, the flap-out tabs Data, Calibrate and Cal. data are not accessible.


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6 TEM k-Space Control

6.1 Introduction TEM k-Space Control is software that uses an input set of crystal-lattice parameters and lattice type to Create a stereographic projection of the crystal’s zone axes Simulate the electron diffraction patterns and Kikuchi lines that would be seen in the TEM when the crystal is tilted to various orientations. The Kikuchi- and diffraction-pattern simulations can aid the identification of diffraction patterns from the crystal’s zone axes in the TEM. In conjunction with the automatic control of the CompuStage and the ability for on-line diffraction pattern measurement of the microscope, TEM k-Space Control becomes a powerful tool for crystallographic research with the TEM. The orientation of the crystal can be determined with respect to the tilt axes of the CompuStage and the crystal can then be tilted by the software to any zone axis within the range of the specimen tilts.

6.2 Getting started The TEM k-Space Control option consists of two parts: • The k-Space Control Control Panel. • The k-Space Control Display window. To use TEM k-Space Control: • Open the TEM Workspace layout (bottom-right selection list) and drag the k-Space Control Control

Panel into a workset. • Go to the tab of the workset and press the Display button in the k-Space Control Control Panel. The

k-Space Control window will be loaded and its functionality become available. You can leave the k-Space Control Control Panel in your workset for future use. The Display window will only be loaded when the Display button is pressed.

6.3 General introduction The flow of working with k-Space Control falls into three separate parts: 1. Definition of the crystal structure 2. Determination of the orientation of the crystal 3. Changing the orientation

6.3.1 Definition of the crystal structure In order for crystallographic calculations to be performed, the crystal structure (lattice parameters and type of structure - Primitive, Face-centered, etc.) must be known. These data are entered by the user, either by typing and selecting the relevant data or by loading from an existing crystal structure file. The crystal structure must therefore be known beforehand (k-Space Control is NOT a tool for matching crystal structure from a database with data derived from diffraction on the microscope).


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6.3.2 Determination of the orientation of the crystal When the data for the crystal structure have been entered, the software will assume a 'generic' orientation. Next the real orientation of the crystal must be determined. k-Space Control provides two methods for determining the orientation, one called two-zone indexing and the other manual indexing. With the two-zone method, we use two zone axes with their stage tilts for defining the orientation of the crystal. With the manual method, we use a single zone axis with an indication of the rotation of the diffraction pattern. You can use either of these methods or start with manual indexing and then use two-zone indexing for fine-tuning. The manual method is usually faster (only a single zone axis needed) but less accurate (because of the estimation of the rotation or the diffraction pattern). The manual method can therefore be used quite effectively to do a rough orientation and on the basis of that select two zone axes for the two-zone indexing method. k-Space Control provides assistance with the determination of the orientation by: • Automatic indexing of the zone axis on the basis of the on-line measurement or manual entry of two

diffraction vectors and their angle. • Display of the diffraction patterns and Kikuchi maps.

6.3.3 Changing the orientation There are three methods to change the orientation of the k-Space Control display: • Define a zone axis to go to, enter the zone axis in the Zone edit control and press Tilt to. • Enter values for a and b CompuStage tilts under Stage grid a and Stage grid b and press Set. • Click with the left-hand mouse button on a point inside the k-Space Control display. This point

(orientation) will be brought to the center. Please note: The intersection of Kikuchi lines on the display may not coincide with the displayed locations of the zone axes. In that case the location of the zone axis is correct. The intersections of the Kikuchi lines may come out in the wrong position because the software simplifies Kikuchi lines to straight bands. Clicking on a Kikuchi-line intersection will therefore not necessarily bring the intersection to center, because it is the crystallographic direction that is centered. To change the orientation of the CompuStage to bring it into agreement with the orientation as seen on the k-Space Control display, press the Stage to button.

6.4 Display elements One the k-Space Control window a number of elements can be selected for display: • Lab grid • Stage grid • Zone axes with or without indices • Kikuchi map • Diffraction pattern • Lattice • Markers

6.4.1 Lab grid The Lab grid displays a stereographic projection with lines at 10° intervals. For more information on stereographic projections, please refer to the standard crystallography literature textbooks (or, if you can get hold of a copy: J.W. Edington, Practical electron microscopy in materials science, Monograph Two. Electron diffraction in the electron microscope).


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6.4.2 Stage grid The stage grid is a grid of lines at 10° spacings that displays the tilt angles for the α and β tilt axes of the specimen stage. The maximum tilts are read directly from the microscope. In general this will be ±30° for the β tilt. For the α tilt this will generally be ±80°, which is the range accessible to the a tilt itself but for most microscopes it will be much more limited in practice due to space limitations in the gap between the objective-lens pole pieces. A practical limit is also set by the shadowing caused by the specimen-holder cup at high tilt angles (in general above ~50° the shadow cast by the specimen-holder cup completely blocks the electron beam). S-TWIN pole pieces generally do not allow more than ~40° of α tilt and U-TWIN pole pieces no more than 20°. Because it is difficult to predict what the effective limit will be for a certain combination of X, Y, Z, α and β, the stage limits in k-Space Control are set to the limits allowed by the stage. In practice you may find that you cannot reach all orientations.

6.4.3 Zone axes Overlaid on the stereographic projection can be shown the positions of the zone axes. Zone axes can be displayed with a label showing their indices or without (dependent on the status of the Display check box). The position of the zone axes on the stereographic projection is accurate (unlike that of intersections of Kikuchi lines, see below).

Zone axes with indices up to a maximum value of 4 shown with a small font size (8). Kikuchi map are also shown. The lab grid is shown in red, the stage grid at an orientation of 60° in blue.


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Zone axes with indices up to a maximum value of 1 shows with a large font size (20). Kikuchi map are also shown. The lab grid is shown in red, the stage grid at an orientation of 60° in blue. Zone axes indication without the index labels at a higher camera constant.

6.4.4 Kikuchi map Kikuchi lines can be displayed in various ways. The user can choose the maximum index of the Kikuchi lines (up to 6) and whether the intensity of the Kikuchi lines is scaled to their intensity on the viewing


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screen of the microscope. The maximum index for the Kikuchi lines and the scaling are changed on the Display tab of the Control Panel flap-out. Note that the Kikuchi lines themselves are not indexed. The Kikuchi line index determines up to what values of the hkl indices Kikuchi lines will be displayed. The acceptable maximum values will generally depend on the effective camera constant and whether the lines are shown with relative intensities or bright (see below). At low values, Kikuchi lines up to high indices will tend to fill the display completely and give a very messy result, while at high camera constants the use of low maximum indices may result in the conspicuous absence of lines expected. In general, a value of 3 is useful for lower camera constants.

Kikuchi map shown with indices up to a maximum value of 6, with relative (scaled) intensities. The lab grid is shown in red, the stage grid at an orientation of 60° in blue. The labels indicate the zone axes. Kikuchi map shown with indices up to a maximum value of 6, with all Kikuchi line intensities the same (making it impossible to distinguish individual lines). The lab grid is shown in red, the stage grid at an orientation of 60° in blue. The labels indicate the zone axes.


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Kikuchi map now shown with indices up to a maximum value of only 3, with all Kikuchi line intensities the same. The lab grid is shown in red, the stage grid at an orientation of 60° in blue. The labels indicate the zone axes. A display with only a Kikuchi map with relative intensities.

Please note: The intersection of Kikuchi lines on the display may not coincide with the displayed locations of the zone axes. In that case the location of the zone axis is correct. The intersections of the Kikuchi lines may come out in the wrong position because the software simplifies Kikuchi lines to straight bands.


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6.4.5 Diffraction pattern The spot diffraction pattern can be displayed with the zone axis shown at the center. Note that a camera constant larger than 10 is necessary to display a pattern (when the spots are too close together and overlap they disappear). The spot pattern can be quite sensitive to small deviations of the crystal from the zone axis (in order to set the crystal exactly to the zone axis, the Tilt to function can be used). The display of the labels of the spot indices can be suppressed by removing the check from the Diffraction Display check box. Please note: The diffraction pattern displayed is based on the crystallographic parameters as input. This may deviate from the true diffraction pattern as seen on the microscope because the k-Space Control software does not account for structure factors, multiple scattering and possible forbidden diffractions.

6.4.6 Lattice The display can be set so that a projection of the crystal unit cell is shown. In the center, the unit cell is outlined and circles display the generalized positions in the unit cell (so at the corners for Primitive, corners and faces for Face-centered, etc.). For unit cells beyond the central one (their number depends on the setting of the Lattice points on the Display tab of the Control Panel flap-out) only the circles are shown and no further lines.

A lattice display with a value of 1 selected for an all-face centered cubic structure in <100> orientation.


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The same structure now with a lattice display up to value 3.

6.4.7 Markers The markers show the positions of the reference axes used during two-zone indexing. The axes are displayed by squares with a number, easiest seen when zone axis and Kikuchi Line display is off. Displaying the reference axes makes it easy to tilt back to these axes.

The marker for zone memory 2 which represents the secondary zone axis from Two-Zone Indexing. The position of the marker differs from the zone axis (102) to which it corresponds for the reason detailed below.

The position of one of the markers may deviate from the exact position of the zone axis to which is corresponds because the primary zone axis is assumed to be absolute and any difference between the calculated and measured angles is taken up by giving the secondary zone axis some freedom to move (along the line connecting the two zone axes). Thus, in the example shown above, the 102 axis is displayed at its crystallographic correct position while the marker shows the measured stage position.


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6.5 Two-zone indexing With the two-zone axis indexing method, the orientation of a crystal is defined by identifying two zones. In each case, the crystal must be tilted by the operator to a first zone axis. The possible indices of the axis must then be identified. The software reads the tilt settings for the axis. Then the crystal must be tilted by the operator to a second zone axis (note: the larger the difference in tilt, the more accurate will be the determination of the orientation) which must then be identified. Usually two zone axes are sufficient for determining an orientation, but there may be cases where a third axis is needed. (E.g. when in a cubic crystal the <121> and <221> axes are identified, the <111> axis can be on either side of the plane through the two axes. In that case a third axis is required for confirmation.) If more than two axes are needed, the two-zone method can be extended simply by choosing a third zone axis. In Two-zone indexing, a number of dialogs is used: • Two-zone indexing dialog • Zone-axis calculator dialog • Match constraints dialog After these dialogs have been considered, a step-by-step procedure for Two-zone indexing is given.

6.5.1 Two-zone indexing dialog When Two-Zone Indexing is selected, the software will display the following dialog:

Calculator Pressing the Calculator button leads to the Calculator dialog for the zone memory selected. Zone memory A total of five memories is available for zone axes, which can be used in any combination (but the software always uses only a combination of the two axes currently selected). The memory is selected via the spin buttons next to the Calculator buttons for the primary and secondary zone axes. The spin


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buttons work such that the selection for the other zone axes (that is, secondary for the primary-axis spin button and vice versa) is always skipped. It is thus not possible to have the primary and secondary zone axes on the same zone memory. If you want to use zone memory 2 as the primary axis, while it currently is at the secondary axis, and another as the secondary, then first change the secondary zone from 2 to another value and then the primary axis can be changed to select 2. Zone axis The two zone axes used for the indexing are displayed in the edit controls just below the “Calculator” buttons. The zone index can be changed by clicking on the required index in the list underneath or by typing in the index.. The special keyboard keys are used to enter negative values. Zone axes list The zone axis lists contain the possible indices of the zone axes, as determined via the zone axis Calculator. In the current version of the software, there is no particular sequence in the lists and all entries will be found to occur more than once. To determine if a second axis is compatible with a solution after a first axis has been defined (click in the primary zone axis list), click somewhere in the secondary axis zone list, then use the up or down arrows to change the selection. The 'Degrees between zone' value will be updated after each change. when the value matches that measured with the stage, the match is correct. View pattern When the View pattern button is pressed, the diffraction display on the right will show the diffraction pattern for the primary of secondary zone axis. If the camera constant is on manual scaling, the value will be set to a camera constant of 10 to allow viewing of the pattern. Diffraction pattern The diffraction pattern is displayed to the right. This pattern either corresponds to the pattern determined by the current orientation of the Lab grid or to the pattern of the zone axis selected for which the 'View pattern' button was pressed. By viewing the diffraction pattern it is, for example, possible to compare the displayed pattern with that on the microscope. In this it is possible to distinguish between solutions that are mirror images of each other. Degrees between zones After 'Degrees between zones' the difference in angle between the two zones used for two-zone indexing are displayed. These tilts are calculated when the selection in the edit controls is changed (by make a change in the selection in one of the lists).. Degrees between stage After 'Degrees between stage' the difference in angle between the stage tilts for the two zones used for two-zone indexing are displayed. These tilts are read automatically from the microscope. Suggestion After two zone axes have been defined through the calculator, the software will determine a match between zone axes, based on the angle measured with the stage. The match is usually only one of a set of possible permutations. For example, the match suggested with 00-1 and 02-1 is equally valid to the match selected (of 100 and 102). Index Pressing Index exits from the Two-zone indexing dialog, while changing the orientation as defined by the selection for the two zone axes. Note: The position of the primary zone axis is assumed to be absolute (so it is made to coincide exactly with the stage position measured for it) and any difference between the calculated and measured angles


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is taken up by giving the secondary zone axis some freedom to move (along the line connecting the two zone axes). Such differences can occur because of the accuracy of the measurement of the stage position, including the effects of backlash on the tilts (which may result in deviations up to 0.2° or so) or because the crystal is bent and the second zone axis orientation was not determined from the exact same place in the crystal. Cancel Pressing Cancel exits from the Two-zone indexing dialog, while leaving the orientation as it was.

6.5.2 Zone axis calculator dialog When one of the Calculator buttons is pressed, the software brings up the Zone axis calculator dialog:

Measurement The Zone Axis Calculator can use the same method for measuring in diffraction as the standard Measuring control panel. Please see the description there as to the execution of such measurements. The results of the measurements are automatically inserted into the edit controls for d1, d2 and the interplanar angle. d1 d1 is the first d-spacing used for indexing of the zone axis. The value for the d-spacing (in nanometers) can be determined with the on-line measurement or entered by hand. d2 d2 is the second d-spacing used for indexing of the zone axis. The value for the d-spacing (in nanometers) can be determined with the on-line measurement or entered by hand. Ratio When Calculate is pressed, the ratio between the two d-spacings as measured is calculated. This value can be compared to the calculated value.


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Interplanar angle The interplanar angle is the angle between the two d-spacings. The value for the interplanar angle (in degrees) can be determined with the on-line measurement or entered by hand. Setup Pressing the Setup buttons brings up the Match constraints dialog in which the limits on deviations are set for the match between d-spacings and interplanar angle. Calculate When the Calculate button is pressed, the d1, d2 and interplanar angle are matched against calculations of d-spacings an their interplanar angles, and possible matching zone axes are determined. Calculated values Under calculated values are listed the matches of the calculations: • Indices for the best matches for d1 and d2 • Calculated d-spacings for d1 and d2 • Calculated interplanar angle between matches for d1 and d2 spacings • Ratio between matches for d1 and d2 spacings Resultant zone The resultant zone list contains all matching zone axes. The contents of this list is copied to the Two-zone indexing dialog when OK is pressed. OK Pressing OK exits from the Zone-axis calculator dialog, while keeping the newly defined match for the zone axis. The data like d-spacings, interplanar angle and stage tilts are stored in the zone memory. The stage axes are read when the OK button is pressed. It is important, therefore, to keep in mind that the stage tilts at that particular moment must be such that they coincide with the orientation of the zone axis. Cancel Pressing Cancel exits from the Zone axis calculator indexing dialog, without changing any of the values.

6.5.3 Match constraints dialog

Absolute Distance Error Limit The absolute distance error limit determines by how much a calculated d-spacing may differ from the measured one for it to be accepted as a possible match for one of the d-spacings. Ratio Distance Error The ratio distance error limit determines by how much the calculated ratio between d-spacing 1 and d-spacing 2 may deviate from the ratio of the measured spacings.


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Angular Error Limit The angular error limit determines by how much the interplanar angle between the two d-spacings calculated may differ from the measured angle. OK Pressing OK exits the match constraints dialog while keeping any changes made to the values of the match constraints. Cancel Pressing Cancel exits the match constraints dialog without changing the values of the match constraints.

6.6 Two-zone indexing procedure in steps The two-zone indexing procedure can be executed as follows: • Find a suitable first zone on the crystal with the stage tilts. Intermezzo: Making it easier to find a suitable second zone axis • Identify the zone axis and use the Manual indexing method to set the rough orientation of the crystal. • Store the stage position in the Stage control panel. • Change the orientation to a different zone axis on the k-Space display. • Press the Stage To button in the k-Space Control Panel to tilt the real crystal to the new orientation. • Adjust the stage tilts to bring the required zone axis to the center. • Store this stage position also in the Stage control panel. • Go back to the stored position for the first zone axis with the Move To or Go To function of the stage. • When the second zone axis is needed, use the same functions to recall that stage position. End of intermezzo • Press the 2-Zone button in the Crystal tab of the flap-out of the k-Space Control panel. • In the Two Zone Indexing dialog, select a zone-axis memory for the primary zone axis with the spin

buttons and press Calculator for the Primary zone axis.


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• In the Zone Axis Calculator, use the measuring procedure to define two d-spacings and their interplanar angle or enter the values by hand (the latter as in the example dialog below).

• If necessary, check the Match constraints by pressing the Setup button. • Press the Calculate button. Calculated values will now be inserted and the Resultant Zone axis list

will be filled. • Press OK to exit from the Zone Axis Calculator dialog and go back to the Two-Zone Indexing dialog.


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The Two-Zone Indexing dialog after the first zone axis has been established. Note that the diffraction pattern visible still corresponds to the default crystal orientation (100) and not yet to the 00-1 axis.

• If it is required to display the diffraction pattern for the select one axis, Press View Pattern for the

Primary zone axis. The Two-Zone Indexing dialog after pressing View Pattern. The camera constant has been changed to 10 and the diffraction pattern now visible corresponds to the 00-1 axis.


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• Change the stage orientation to a second zone axis. • In the Two Zone Indexing dialog, select a zone-axis memory for the secondary zone axis with the

spin buttons and press Calculator for the Secondary zone axis. • In the Zone Axis Calculator, use the measuring procedure to define two d-spacings and their

interplanar angle or enter the values by hand (the latter as in the example dialog below).

• Press the Calculate button. Calculated values will now be inserted and the Resultant Zone axis list will be filled.

• Press OK to exit from the Zone Axis Calculator dialog and go back to the Two-Zone Indexing dialog.

The Two-Zone Indexing dialog after the second zone axis has been established.


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• If it is required to display the diffraction pattern for the select one axis, Press View Pattern for the

Secondary zone axis. The Two-Zone Indexing dialog displaying the diffraction pattern for the 02-1 zone axis.

• If necessary, change the selected primary and secondary zone axes. • Press Index and the orientation of the crystal in the k-Space display will be changed to the orientation

established. If necessary, it is possible to refine the orientation by using the k-Space display to tilt to a more suitable zone axis, storing the relevant zone axis through the Stage Control Panel, then re-entering the Two-Zone Indexing dialog and either replacing one or both the existing zone axes in the existing zone memories or changing to different zone memories and storing the results of the new zone axes there.

6.7 Manual indexing The manual indexing requires setting the crystal to a known zone axis and identifying a g vector (indices of the diffraction spot). The direction of this g vector is defined relative to the zero-offset angle which in turn is defined by the top of the display (north). Negative crystallographic indices can be set by using the special keyboard keys.


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The Manual Indexing dialog.

6.8 Special keyboard keys In order to allow display of the special notations that crystallography uses for zone axes, with the ‘bar’ 1, etc. (with the - sign on top of the number), a special font (called k_font) has been developed for k-Space Control. This special font replaces a number of standard characters with ‘bar’ 1, ‘bar’ 2. The following characters on the keyboard give access to the special k-Space Control characters: Normal character How to get k_font ! Shift + 1 ‘bar’ 1 @ Shift + 2 ‘bar’ 2 “ Shift + ‘ ‘bar’ 2 # Shift + 3 ‘bar’ 3 $ Shift + 4 ‘bar’ 4 % Shift + 5 ‘bar’ 5 ^ Shift + 6 ‘bar’ 6

These characters are the first six characters in the normal fonts (it can be checked in the Windows program Character Map, available under Accessories). Except for the ‘bar’ 2 as “, most of these characters are above the corresponding number on many keyboards. (Note: there is no real consistency in this, due to the fact that many countries have their own keyboard lay-out). The same characters are also used when the keypad keys are used (when Num Lock is on). So the keypad key 1 gives the 'bar one' character while the key above the Q gives the normal one. The special font is used automatically where needed, as e.g. in the Zone edit control on the k-Space Control Panel and various dialogs of the k-Space Display.


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6.9 k-Space Control Control Panel The k-Space Control Control Panel contains two sets of functions for k-Space Control : • Functions that load the k-Space Control Display window (a


• Functions that control k-Space Control.

Display The k-Space Control Display window is only loaded on operator request. To load it press the Display button. Once the server is running, the window will be displayed, and the Display button will turn yellow. You are now ready to start working with k-Space Control. If the Display button is pressed again, the Display window will disappear. It is, however, not unloaded but simply hidden. If you display it again (by once more pressing the button), it will still have kept all the k-Space Control settings as set previously. The window is only unloaded when you close the TEM User Interface. Sizeable When this option is checked, the Display window can be positioned anywhere you like (the software will remember your settings and restore them) and you can define its size in the standard Windows way (click and drag on a border). If the option is off, the Display window will be fixed in size and position to fill the data space of the TEM User Interface. The following controls will remain disabled until a crystal structure has been defined through New on the Crystal tab of the flap-out. Stage to When Stage to is pressed, the CompuStage will be tilted to the currently active orientation on the k-Space Control display. If the orientation entered is out of reach for the stage tilts, the Stage to button will be disabled. The function is enabled once the orientation of a crystal has been determined. Zone In the Zone edit control the crystallographic notation of a zone axis can be entered. Individual indices cannot exceed a single character (so the 1001 for ten-zero-one is not allowed). Negative indices are entered using the special keyboard keys. Tilt to When Tilt to is pressed, the stereographic projection is tilted to the zone axis indicated in the Zone edit control. There are no limitations imposed on the tilting. The tilt applies only to the display, not to the stage. The function is enabled once the orientation of a crystal has been determined.


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Camera constant The magnification of the stereographic projection and the other elements displayed depends on the camera constant. This value can either be defined by hand, using the camera constant slider, or automatic (in which case it is linked to the active camera length). The range of camera constants used by k-Space Control is 1 to 20. The camera constant (the camera length*the electron wavelength) is in mm*nm, so a camera constant of 1 means a distance of 1 mm corresponds to 1 nm d spacing (on the printed page; due to monitor size difference these values may not be exact on the monitor display). The camera constant (L*λ) forms part of the formula for converting distances in a diffraction pattern to d spacings: D*d = L*λ or d = (L*λ) / D where D is the distance of the diffraction spot in the pattern to the origin of the pattern (the transmitted beam). Thus while 1 mm at a camera constant of 1 means 1 nm d spacing, 2 mm means 0.5 nm d spacing (as D occurs in the denominator). Table of (approximate) camera lengths against camera constant for different accelerating voltages. Cam. const. 120 kV 200 kV 300 kV 1 300 mm 400 mm 500 mm 5 1500 mm 2000 mm 2500 mm 10 3000 mm 4000 mm 5000 mm 20 6000 mm 8000 mm 10 000 mm

Note: For displaying the diffraction pattern, it is necessary to choose a larger camera constant, otherwise the spots will be too close together and invisible. If the diffraction pattern display is on and no pattern is visible, increase the camera constant.

The display at the smallest camera constant.


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The display at a camera constant of ~4 shows a detail of that in the image above.

Auto The magnification of the display of the stereographic projection and the other elements displayed depends on the camera constant. This value can either be defined by hand, using the camera constant slider, or automatic (in which case it is linked to the active camera length). If Auto is checked, the adjustment is automatic, otherwise it must be set with the Camera constant slider. Stage grid a The Stage grid a value defines the tilt angle given to the stage a axis of the k-Space display. The stage grid tilt is changed to the value defined when the Set button is pressed. Stage grid b The Stage grid b value defines the tilt angle given to the stage b axis of the k-Space display. The stage grid is changed to the value defined when the Set button is pressed. Set The stage grid tilts are changed to the values defined for stage grid a and b when the Set button is pressed. Flap-out button The flap-out leads to the k-Space Control Crystal, Info, Display and View tabs.


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6.10 k-Space Control Crystal The k-Space Control Crystal Control Panel contains several functions for using k-Space Control.

New In order to start working on a new crystal or phase, the software must know the crystal structure. To this purpose the lattice parameters and crystal-structure type must be entered in the Crystal data dialog. The program works on a triclinic basis so for crystals with higher symmetry, the symmetry derives simply from the lattice parameters (all angles 90° and dimensions the same for cubic, all angles 90° and a=b for tetragonal, etc.). For hexagonal crystals the rhombohedral notation must be used.

The Crystal data dialog.

For simplified entering of the data, a set of crystal structures has been predefined (cubic, tetragonal, etc.). These are used simply to avoid defining unnecessary entries (e.g. when cubic is selected all angles will be set to 90° and b and c will be equal to a). These crystal structures are not imposed on the underlying calculations (as stated above, all is done on a generalized, triclinic basis). Thus a selection of cubic with a value of 0.52 for a is equivalent to selecting triclinic and entering the 0.52 for a,b and c and setting all angles to 90° (but selecting the cubic crystal structure is less work than entering all the values by hand).


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Print Setup When the Print Setup button is pressed, the k-Space Control software displays the printer setup dialog (e.g. for selecting the paper size). Print Printing causes the current display to be printed on the printer selected (if necessary, changes to printer setup can be made under the Printer setup button). Note that printing is done on a white background so the color selection may need to be adjusted. Open Via the Open dialog an existing oriented crystal structure can be read from disk. Crystal structure files have the extension .xtl. In the crystal structure file, the program saves the lattice parameters and the crystal-structure type. Save Once defined, crystal data can be saved for later recall. If no filename has been defined yet, the program displays the Save As dialog in which the filename can be set. If a filename has already been defined, then using Save will overwrite the existing file while under Save As a new filename can be given. Before overwriting an existing file, the software will ask for confirmation. Save As Once defined, crystal data can be saved for later recall. For working with k-Space Control, the orientation of the crystal must be determined. For the determination, two indexing methods are available, the Two-zone and the Manual method. 2-Zone When the 2-zone button is pressed, the Two-zone indexing method of determining the orientation of a crystal, is started. Manual When the Manual button is pressed, the Manual indexing method of determining the orientation of a crystal is started. If the Compucentricity option is present on the microscope, the tilting can be done with the aid of Compucentricity whereby the non-eucentric behavior of the β tilt axis is corrected. Use compucentric tilting The Use Compucentric tilting checkbox is used to select or deselect compucentric tilting. This choice is a system-wide setting. The same setting may be selected elsewhere, e.g in the Compucentricity or Smart Tilt Control Panels. When the choice is on, tilting operations by software that supports compucentric tilting will use compucentricity, otherwise tilting will be done without corrections.


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6.11 k-Space Control Info The k-Space Control Info Control Panel contains information concerning the crystal currently defined in k-Space Control.

Parameters Under Parameters the crystal lattice parameters are listed. Type Under Type the type of crystal lattice is listed. Indexing Under Indexing the method used to determine the crystal orientation is listed. If the two-zone method was used, the primary and secondary zone axes used for the indexing are listed. Matrix The orientation matrix of the crystal is defined as the matrix that relates the crystal axes to the lab grid (see also V. Randle, 1992, Microtexture determination and its applications, The Institute of Materials, London). The orientation matrix can be copied to the clipboard by clicking with the right-hand mouse button on the Info Control Panel and selecting Copy matrix to clipboard in the popup menu. The format of the output is a nine numbers separated by tabs on a line and each line separately. You can copy these directly into programs like Microsoft Excel and you will get 3x3 cells filled with the matrix values.


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6.12 k-Space Control Display The k-Space Control Display Control Panel contains several settings for the k-Space Control display.

Crystal indices Crystal indices determines the maximum value used for the uvw indices of the zone axes displayed (a number between 1 and 6). The zone axes can be displayed with or without the index label (depends on the Display check box). Display (crystal indices) The status of the Display check box determines whether the zone axes displayed on the k-Space Control display are labeled with their uvw index (check box checked) or shown without label. Diffraction Diffraction determines the maximum value used for the hkl indices of the diffraction spots displayed (a number between 1 and 6). The diffraction-pattern spots can be displayed with or without the index label (depends on the Display check box). Display (diffraction) The status of the Display check box determines whether the spots of the diffraction pattern are shown with or without label. Lattice points The Lattice points value determines the number of lattice points used on the display (a number between 1 and 6). For lower numbers fewer unit cells are shown. Only the central unit cell is indicated by lines. Kikuchi line index The Kikuchi line index determines up to what values of the hkl indices Kikuchi lines will be displayed. The acceptable maximum values will generally depend on the camera constant and whether the lines are shown with relative intensities or bright (see below). At low values, Kikuchi lines up to high indices will tend to fill the display completely and give a very messy result, while at high camera constants the use of low maximum indices may result in the conspicuous absence of lines expected. In general, a value of 3 is useful for lower camera constants. A value of 6 can be used if the Bright option is switched off.


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Bright Kikuchi lines can be displayed with relative intensities (where Kikuchi lines that have lower intensities in the microscope are displayed with lower intensities on the k-Space Control display) or as bright. In the latter case all Kikuchi lines will have the same (green) intensity. Note that the relative intensities are not based on the structure factors that normally also determine the intensities of Kikuchi lines but on a general approximation. Stage orientation Generally, the orientation of the stage tilt axes does not correspond to the a tilt being E-W and the b tilt N-S as shown on the k-Space Control display. On most instruments the majority of camera lengths typically have a running about NNW-SSE and b ENE-WSW. You can adjust the stage grid display to reflect this deviation in orientation by entering a rotation angle for the stage orientation. How to determine the stage orientation The following procedure provides a method for determining which value to enter for the stage orientation: • Insert into the microscope a type of specimen that is relatively flat (not strongly bent) and with well-

visible Kikuchi lines. • Tilt the specimen to a clearly visible zone axis. • Press the New button and enter some kind of structure (it doesn't really matter what). • Press the Manual button and enter some values for the zone and g vector (once again, it isn't

important what you enter, as long as it is something possible). • The k-Space Control display will now show some kind of zone axis at the current stage position. • Change the a tilt angle of the stage back and forth to get a feeling of the direction in which the a tilt

affects the diffraction pattern (for the reference camera length ~500mm on many microscopes this will be close to NNW-SSE). Enter a value for the stage orientation (the angle rotates the tilts anti-clockwise from 0 where the a tilt is horizontal), e.g. 60°, and press the Enter button. The display of the stage grid will now be in the new direction.

• Change to a larger camera constant (10) or reasonably high camera length (~1000mm, with Auto checked).

• Click on the display in the direction of the plus or minus a tilt (just a little to the NNW or SSE in the example used here). The zone axis will be displaced form the center of the display.

• Now press the Stage to button. The a tilt should now be adjusted so that the zone axis becomes visible on the viewing screen displaced in the same direction as on the k-Space Control display.

• If the zone axis has moved into the wrong direction, add 180° to the stage orientation value (so it would become 240° in the example used here).

• If necessary, check again.


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6.13 k-Space Control View The k-Space Control View Control Panel contains several settings for the k-Space Control elements displayed. All colors (except for Kikuchi lines which is currently fixed to green) are defined by clicking on the colored rectangle to the right of the element. This brings up a standard color selection dialog wherein you can either select one of the predefined colors or add a custom color. Lab grid The Lab grid is the grid displaying the stereographic projection. The color can be selected by clicking on the colored rectangle to the right. Whether the Lab grid is displayed or not depends on the status of the check box (checked = display).

Stage grid The Stage grid is the grid displaying the orientation and tilts of the CompuStage a and b tilts. The color can be selected by clicking on the colored rectangle to the right. Whether the Stage grid is displayed or not depends on the status of the check box (checked = display). Kikuchi Kikuchi refers to the color of the Kikuchi lines. This is currently fixed to green. Whether the Kikuchi map is displayed or not depends on the status of the check box (checked = display). Zone axes Zone axes defines the color for the zone axis symbols. The color can be selected by clicking on the colored rectangle to the right. Whether the zone axes and their indices are displayed or not depends on the status of the check box (checked = display). Diffraction pattern Indices defines the color for the spots of the diffraction pattern. The color can be selected by clicking on the colored rectangle to the right. Whether the diffraction is displayed or not depends on the status of the check box (checked = display). Lattice Lattice defines the color for the display of the schematic lattice. The color can be selected by clicking on the colored rectangle to the right. Whether the lattice is displayed or not depends on the status of the check box (checked = display). Markers Markers defines the color for the markers that indicate the position of the zone axes used for defining the crystal orientation with the Two-zone indexing method. The color can be selected by clicking on the colored rectangle to the right. Whether the markers are displayed or not depends on the status of the check box (checked = display). Font size Under font size you select the size of the font used for displaying the zone axis and diffraction spots indices.


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7 TEM Free Lens Control

7.1 Introduction Free Lens Control allows control over individual lenses of the Tecnai microscope. The following limitations apply. Safety • The spot size (first condenser lens or C1) cannot be taken lower than the value for spot size number

1. • On 200 kV systems the spot size (first condenser lens or C1) cannot be taken lower than the value

for spot 5 when the high tension is higher than 120 kV and no holder is present in the CompuStage. • On 200 and 300 kV systems the spot size (first condenser lens or C1) and intensity (second

condenser lens or C2) are blocked when the gun is in conditioning. Operation • All lens settings range from 0 to 100%, except for the first condenser lens which has a minimum

value as decribed above and the minicondenser lens which ranges from -100 to 100%. • The changes in lens settings are not persistent. If microscope settings are changed that affect lens

settings (e.g. the switch between Microprobe mode and Nanoprobe mode), the Free Lens settings are overruled. In order to avoid interference as much as possible, the relevant knobs on the Control Pads of the microscope are disconnected. Thus the Intensity knob is disconnected when C2 is changed, the Focus knob when the objective lens is changed, and the Magnification knob when one of the projection system lenses(Diffraction, Intermediate, Projector 1, Projector 2) is changed. These knobs are reconnected when Free Lens Control is deactivated. There are, however, many controls that can interfere with Free Lens Control and not all can be blocked.

• As long as a particular lens has not been changed through Free Lens Control, its particular microscope control is not disconnected. Thus, if you only change settings of the projection system, the control of the condenser system (spot size, intensity) can still be done with the normal microscope controls.


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7.2 Free Lens Control Panel Activate When the Activate button is pressed the Free Lens Control is switched on (the button becomes yellow) or off. Get from 'scope When the Get from 'scope button is pressed, the current lens settings are copied from the microscope to the Free Lens settings. This function can be used before activating Free Lens Control to make the current lens settings on the microscope the starting settings for working with Free Lens Control. Safety : Various restrictions apply to the condenser system. The C2 lens setting may be automatically adjusted to a safe limit when C1 is changed.

For each lens the following controls are present Microscope value The microscope value lists the current lens value as it is on the microscope. The display will update each time a lens setting is changed, also when Free Lens Control is not active. Free Lens Control value The Free Lens Control value in the edit control displays the value used for a particular lens in Free Lens Control. You can change the value by typing another value. The value is transmitted to the Free Lens Control slider when the Enter button is pressed. If Free Lens Control is active, the new value is then set on the microscope (otherwise only the slider setting changes). Enter button When the Enter button is pressed, the Free Lens Control value is transmitted to the Free Lens Control slider. If Free Lens Control is active, the new value is then set on the microscope (otherwise only the slider setting changes). Free Lens Control slider The Free Lens Control slider controls the setting of the particular lens. When Free Lens Control is active any change of the slider is transmitted immediately to the microscope. The step sizes (small step by clicking on the left or right arrows, large step by clicking left or right of the thumb tab - the 'drag' rectangle) are controlled by the step size control to the right of the slider.


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Free Lens Control slider step size control The Free Lens Control slider step size control changes the size of the small and large step used by the slider. The meaning of the ranges (in percent) is as follows for all lenses except the minicondenser and objective lens: Step Small step Large step

1 0.00001 0.001 2 0.00003 0.003 3 0.0001 0.01 4 0.0003 0.03 5 0.001 0.1 6 0.003 0.3

For the objective lens all steps are ten times smaller, for the minicondenser they are two times smaller. Flap-out button The flap-out button leads to the Registers tab of the Free Lens Control Control Panel.

7.3 Free Lens Registers Introduction Free Lens settings can be stored in up to 10 registers. The settings of the registers are stored for each individual user when the user logs off and reloaded when the user logs on again. Sets of registers can also be stored in a file and loaded from file.

Register list The register list contains the labels of the registers defined by the user. The label of a register can be anything (up to 255 characters) except empty. If you do not specify a label yourself, the software will automatically call the register 'Register' with a serial number when a new register is added. Registers are selected by clicking on the entry in the list. The label of the selected entry is copied automatically to the register label edit control. Set to 'scope When a register has been selected, its settings can be transferred to Free Lens Control by pressing the Set to 'scope button. If Free Lens Control is active, these settings (all lenses together) are transferred immediately to the microscope. If Free Lens Control is not active, the settings are transferred to the sliders but not the microscope. To set only part of the register settings, first deactivate Free Lens Control, then use Set to 'scope. Re-activate Free Lens Control and press the Enter buttons of the lenses that must be set on the microscope to transfer those settings to the microscope. Register label The register label edit control contains the label of a register. It can be either a label typed in for a new register or the register


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currently selected. The label of a register can be anything (up to 255 characters) except empty. If you do not specify a label yourself, the software will automatically call the register 'Register' with a serial number when a new register is added. The software does not check for label contents (that is, whether two registers have the same label). Add When the Add button is pressed, a new register is added to the currently defined registers. The settings of the new register are those currently defined for Free Lens Control. Registers can be added irrespective of whether Free Lens Control is off or on. Overwrite When the Overwrite button is pressed, the currently selected register is overwritten with new settings. These settings are those currently defined for Free Lens Control. Registers can be added irrespective of whether Free Lens Control is off or on. Delete When the Delete button is pressed, the currently selected register is deleted. The software asks for confirmation. Open When the Open button is pressed, the Open file dialog is displayed in which a file can be selected (the extension for Free Lens Control files is .frl). The contents of this file are then loaded into the registers (overwriting anything that is there). Save, Save as Through the Save or Save as buttons the contents of the registers can be saved to a file (the extension for Free Lens Control files is .frl). When the Save button is used and a file name has been defined previously, the contents of the existing file are overwritten. When no file name has been defined or the Save as button is used, the Save file dialog is displayed, allowing definition of the file name under which the settings are stored. When the file name is the same as an existing file, the software will ask if that file must be overwritten.


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8 Magnification Calibration

8.1 Magnification Calibration Control Panel The Magnification Calibration Control Panel semi-automatically calibrates: • the HM magnifications on an embedded CCD camera • the LM magnifications on an embedded CCD cameraThe

Magnification Calibration Control Panel semi-automatically calibrates:

• the D camera lengths on an embedded CCD camera • the HM-STEM magnification • LM-STEM

The values measured are entered into the magnification tables of DigitalMicrograph™ and/or TIA CCD or in the TIA STEM magnification table. The control panel is accessible only to Supervisor, Service and Factory. Note: The magnifications as used by Magnification Calibration for CCDs always are the so-called reference magnifications. These are the magnifications as displayed by the microscope when the main viewing screen is up. This applies even to CCD cameras located in the wide-angle position (above the viewing screen at the top of the projection chamber), because it is the reference magnifications that are used by the CCD camera software to determine the effective magnifications from the magnification tables. There thus is an apparent discrepancy between the magnification value displayed by the microscope and by the Magnification Calibration software as well as the CCD magnification tables for wide-angle CCD cameras. Calibration selection list The calibration selection list allows selection of the calibrations to be executed. Currently the list only contains calibration of: • the HM (Mi, SA and Mh) magnifications • the Mi plus SA magnifications • the HR-TEM magnifications • the LM magnifications • the HM-STEM magnifications in TIA • if Microprobe STEM is installed, the calibration of the Microprobe STEM against the normal

(Nanoprobe) STEM • on FEG systems, the calibration of the LM-STEM against the normal (Nanoprobe) STEM The difference between the first and the second plus third is that it is not necessary to switch to another specimen during the procedure. Instructions The instructions field will display instructions on how to proceed or a status description of the function currently running or finished.


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Start button The Start button starts the calibration procedure. The software will link up to DigitalMicrograph or TIA and when successful, will display the name of the camera, the lens series (TEM or EFTEM) and high tension setting. If TIA CCD is present on a system with Gatan cameras, the software will also require that TIA is running (otherwise the TIA CCD magnification table will not be set).

The Start button will change to a yellow color and its caption to Stop. Pressing the Start button when it is yellow will stop the calibration procedure.

Starting the calibration procedure will open a log in a separate window. The log shows what is being done and what the results are. It can be saved in text format (under the File menu of the log window). The log can also be printed (if a printer is available to the system). Text files can also be opened in the log file window. Next button During the calibration procedure the operator will be expected to do certain things as setting up illumination, specimen area and focus, as well as change specimen (later in the procedure). Once these things have been done (as given by the instructions displayed), pressing the Next button will continue with the procedure. Flap-out button The flap-out button leads to the Report, File and Options tabs of the Automated Experiments Control Panel.

8.1.1 The HM magnification calibration procedure The calibrations procedure consists of a calibration for each magnification of the series. The following procedure is used: • Direct cross-grating calibration of the lowermost SA magnification. • Calibration of the image shift at the lowermost SA magnification (this does not calibrate a

magnification, but the image shift itself, used later). • Calibration of the Mi magnifications with the cross-grating method. • Calibration of the remaining SA magnifications, going up in magnification. The software detects the

magnification at which the cross-grating method does not work anymore and displays a warning in that sense. Then the software automatically switches to the image shift method. Once the magnification is high enough for high-resolution images (determined from the measured pixel size), the software will switch to the lattice-image method.

• Calibration of the Mh magnifications with the lattice-image method.


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8.1.2 The Mi plus SA magnification calibration procedure The calibrations procedure consists of a calibration for each magnification of the series. The following procedure is used: • Direct cross-grating calibration of the lowermost SA magnification. • Calibration of the image shift at the lowermost SA magnification (this does not calibrate a

magnification, but the image shift itself, used later). • Calibration of the Mi magnifications with the cross-grating method. • Calibration of the remaining SA magnifications, going up in magnification up to the point where the

magnification on the CCD camera is larger than 250kx. The software detects the magnification at which the cross-grating method does not work anymore and displays a warning in that sense. Then the software automatically switches to the image shift method which will be used for the remaining SA magnifications.

8.1.3 The HR-TEM magnification calibration procedure These calibrations cover the SA magnifications not done in the Mi plus SA magnification procedure plus the Mh magnifications. It thus complements the Mi plus SA magnification calibration procedure. All calibrations are done with with the lattice-image method. Since this requires a different specimen than the cross-grating, the separate HR-TEM procedure allows you to go through all the cross-grating calibrations and only then switch to silicon [110] or gold [001].

8.1.4 The LM magnification calibration procedure The calibrations procedure consists of a calibration for each magnification of the series down to a magnification of 200x on the CCD. The procedure uses an analysis of the FFT of the cross-grating to determine the magnifications.

8.1.5 The D camera length calibration procedure The calibrations procedure consists of a calibration for the camera lengths of the series (in the case of EFTEM the lowermost camera lengths are excluded from the calibration because they often are not usable - the projection system reduction is so strong that it becomes impossible to get the electron beam through the differential pumping aperture). The following procedure is used: • Direct calibration of the diffraction rings of a cross-grating at a camera length selected by software on

the basis of CCD camera size and high tension. • Calibration of the diffraction shift at that camera length (this does not calibrate a camera length, but

the diffraction shift itself, used later). • Calibration of the lowermost camera lengths with the diffraction shift (the rings are too close to the

center of the diffraction pattern). • Calibration of the rings for the camera lengths where the rings are well enough separated from the

center but not so large that they are no longer visible on the CCD. • Calibration of the higher camera lengths with the diffraction shift (the rings are too large to fit on the

CCD). If possible, the procedure will be run in the Nanoprobe mode, because the large illuminated area for parallel illumination in Microprobe is so large that it often leads to the appearance of severe distortions in the center of the pattern. It is important to focus the pattern so the rings becomes as sharp as possible. The focusing in Nanoprobe should be done with the Intensity - provided the camera lengths are focused prior, which should have been done in the Image HM-TEM Camera Length alignment procedure.


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Note: The software is not suitable for calibration of the LAD (LM) camera lengths nor for the Lorentz mode camera lengths.

8.1.6 The HM-STEM magnification calibration procedure The calibrations procedure consists of a calibration for selected magnifications (unlike the TEM magnifications, which are made from discrete lens settings with magnification-dependent random errors, the STEM magnifications are controlled by the amplitude of the scan which quite linear, so only a few magnifications suffice). The following procedure is used: • Direct cross-grating calibration of a lower HM-STEM magnification (not necessarily the lowermost

because this may be too low to resolve the grating squares effectively). If the image distortion is too high, the software will automatically adjust the distortions in an iterative procedure until the distortions are less than 0.1% (vector lengths of the grating square dimensions) and 0.1° (angle between vectors).

• Calibration of the image shift at the same HM-STEM magnification (this does not calibrate a magnification, but the image shift itself, used later).

• Calibration of several additional magnifications. The software detects the magnification at which the cross-grating method does not work anymore and automatically switches to the image shift method.

The STEM calibration procedure will be run for a total STEM rotation angle of 0°. This means that the free (user) STEM rotation is set to 0°, and the aligned value (which corrects for the mismatch between stage axes and STEM image axes) is also temporarily set to 0° (the latter value is reset at the end of the procedure to the starting value). Effectively this means that the smallest distortions and most accurate magnifications are achieved at the same STEM rotation angle. For this purpose it is necessary for users to set the free rotation to the compensating value of the alignment value (which is included in the calibration report). Note: The STEM magnifications are strongly dependent on the pivot points, perpendicular corrections and distortion adjustment. For STEM magnifications as accurate as possible, the same STEM settings should be used as during the calibration. It is therefore advised to adjust the pivot points and perpendicular corrections carefully before the calibration and store these settings in an alignment file (after the calibration, because that may have included adjustment of the STEM distortion) and having users always use those alignments. If the STEM distortions initially were a long way off, stop the procedure after the distortion adjustment and check the pivot points and perpendicular corrections, and then restart the procedure.

8.1.7 The LM STEM magnification calibration procedure LM-STEM is the STEM mode in which the objective lens is off. This mode is used for obtaining much lower magnifications than can attained in HM-STEM. LM-STEM can be reached from normal STEM by checking the Enable LMScan check box and turning the magnification down below the lowermost HM-STEM magnification. As for Microprobe STEM we make sure that the internal calibration in the Tecnai software is defined such that the same nominal magnification in normal and in LM-STEM gives the same actual magnification. This is done by the LM-STEM calibration procedure. The LM-STEM procedure is only available on FEG systems because on other instruments the cross-grating squares usually cannot be resolved reliably because of the large spot size in LM-STEM. The following procedure is used: • Direct cross-grating calibration of a lower HM-STEM magnification (not necessarily the lowermost

because this may be too low to resolve the grating squares effectively). If the image distortion is too high, the software will automatically adjust the distortions in an iterative procedure until the distortions


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are less than 0.1% (vector lengths of the grating square dimensions) and 0.1° (angle between vectors).

• Direct cross-grating calibration of a comparable LM-STEM magnification. If the image distortion is too high, once again the software will automatically adjust the distortions in an iterative procedure until the distortions are less than 0.1% (vector lengths of the grating square dimensions) and 0.1° (angle between vectors).

These STEM calibration procedures will be run for a total STEM rotation angle of 0°. This means that the free (user) STEM rotation is set to 0°, and the aligned value (which corrects for the mismatch between stage axes and STEM image axes) is also temporarily set to 0° (the latter value is reset at the end of the procedure to the starting value). Effectively this means that the smallest distortions and most accurate magnifications are achieved at the same STEM rotation angle. Note: Both types of STEM-mode magnifications are strongly dependent on the pivot points, perpendicular corrections and distortion adjustment. For STEM magnifications as accurate as possible, the same STEM settings should be used as during the calibration. It is therefore advised to adjust the pivot points and perpendicular corrections carefully before the calibration and store these settings in an alignment file (after the calibration, because that may have included adjustment of the STEM distortion) and having users always use those alignments. If the STEM distortions initially were a long way off, stop the procedure after the distortion adjustment and check the pivot points and perpendicular corrections, and then restart the procedure. The LM-STEM magnification calibration does not result in a calibration file. The calibration value for the LM-STEM magnification is directly set to the Tecnai software and also entered as a calibration value in the system branch of the microscope settings in the registry.

8.1.8 Measurement on cross-grating The magnification calibration is done on the basis of a standard specimen, a cross-grating. This specimen has squares on it with a spacing of 463 nm. The software will acquire an image of the cross-grating and analyse that to find the basic spacing. The analysis is done either using an auto-correlation or an FFT (at high and low magnifications, respectively) in which the two basic vectors will be outlined by circles. Additional circles may be present further away from the center to show the position of the multiple of the base vector that is actually used for further refinement of the measurement.

Image on left: An image of a cross-grating, showing the 463 nm squares.


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Image on left: The result of the magnification calibration. The autocorrelation of the cross-grating image (zoomed in 2x) shows the central peak pointed at by the line (which has been added manually for clarity). The software adds the two circles around the two base vectors of the pattern. It is important that these circles are located at the correct positions and not e.g. at the diagonals of the squares.

8.1.9 Measurement with image shift The image shift of the Tecnai is largely free of hysteresis and can be used to calibrate distances, from which magnifications can be determined. The cross-grating method is usable only at lower magnifications where a sufficient number of grid squares is present in the image. The lattice-image method can only be used at high magnifications where lattice fringes become visible. The indirect image-shift measurement must therefore be used in the gap left by these two methods, at intermediate magnifications. Before the image shift can be used to calibrate magnifications, it must first be calibrated itself. This is done at the lowermost SA magnification, immediately after the magnification there has been calibrated with the cross-grating. (The software thus does not rely on any existing - potentially inaccurate or even wrong - image shift calibrations in the system.) The procedure followed during image shift calibration is as follows: • Apply a negative shift from the current image position and acquire image 1, with a negative backlash

correction (first more negative, then to the desired position). • Apply a similar but now positive shift from the original position and acquire image 2. • Apply a positive shift from the original image position and acquire image 3 (position the same as

image 2), with a positive backlash correction (first more positive , then to the desired position). • Apply a similar but now negative shift from the original position and acquire image 4 (positions the

same as image 1). • Apply a negative shift from the original image position and acquire image 5 (position the same as

image 1), with a negative backlash correction (first more negative, then to the desired position). • The time at which each acquisition is finished is stored. • Determine the drift (if any) by measuring the shift between images 5 and 1 and normalising to the

time in between the acquisition of these images. • Determine the shift between images 1 and 2, and between 3 and 4, corrected for drift. These shifts

give the two vectors used for the calibration.

8.1.10 Measurement on lattice image A high-resolution image contains lattice lines whose apparent spacing is an accurate reflection of the magnification. Although the cross-grating is made of gold-palladium particles that will display a high-resolution image, the spacings found in the Fourier Transform of the image (the diffractogram) are poorly defined, mostly because of lattice relaxation and particle size effects. In addition, the spacing of gold-palladium is not known accurately (there is no guarantee that the alloy composition is constant from one


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cross-grating to another). Because of this, the magnification calibration uses one of two other types of easily available specimens: • Silicon in [110] orientation. • Single-crystal gold in [001] orientation. When appropriate in the procedure (dependent on magnification), the software will ask the operator to insert one of these specimens, orient it, and focus so a lattice image becomes visible. For silicon both <111> directions (0.31 nm) must be visible and for gold the <200> (0.204 nm). In the latter case, make sure that the <200> are well visible. If the <200> are weak or invisible and the <220> spacings (0.144 nm) well visible, the software may confuse these and give the wrong calibration. The software automatically will detect if silicon or gold is present (on the basis of the interplanar angles) and perform the calibration.

8.1.11 Diffraction-ring analysis Diffraction by the particles on a cross-grating results in rings in the diffraction pattern. The software analyses the diffraction pattern, finding the center and then determining the distance to four rings (0.23, 0.20, 0.14, 0.12 nm: note that these are not the spacings of gold, because the cross-grating is made with a gold-palladium alloy). If the pattern is not suitably centered, the software will center the diffraction pattern itself first. The ring used for the calibration is that of the 0.23 nm distance. The other rings are only used for crosscheck. In principle the 0.141 and 0.12 nm rings will give the same camera length as the 0.23 nm, while the 0.20 ring usually gives a value that is a bit off (because it forms a shoulder on the 0.23 nm ring, it is less well-defined). The distance of the rings is determined in six directions, perpendicular to the blooming (positive and negative direction) and at the four 45° angles. The error displayed is the standard deviation of the six distances. The log will list in how many profiles a particular ring was found. NOTE: ON 16-BITS CCD CAMERAS SEVERE OVEREXPOSURE IN THE CENTER IS NOT NECESSARY AND SHOULD BE AVOIDED TO PREVENT DAMAGE TO THE CAMERA. Important: The illumination conditions for diffraction-ring analysis must be set such that there is sufficient intensity in the rings, especially the most important one (0.235 nm). Especially on CCD cameras with a limited bit depth (12- or 14-bits), this means that the center of the diffraction pattern must be overexposed which will give blooming (the center shows a central overexposed blob, with two tails, usually up and down, a bit like the image of an aircraft propeller). The blooming is necessary and does not present a problem, neither for the CCD (which will NOT be damaged) nor for the ring analysis (which knows that blooming can be present and takes care of that). If you reduce the illumination intensity so that blooming is avoided, the rings analysis may fail.


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The images above show a diffraction pattern with a suitable amount of blooming (top left). The rings are invisible until the contrast levels in the image are adjusted (top right). After the analysis the software will convert the image to the log of itself, set the contrast levels and insert circles for the rings detected.


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8.1.12 Measurement with diffraction shift The diffraction shift of the Tecnai is largely free of hysteresis and can be used to calibrate diffraction spacings, from which camera lengths can be determined. The ring analysis method is usable only at camera lengths where the rings in the diffraction pattern are well-separated from the center and are not too large for the CCD. The indirect diffraction-shift measurement must therefore be used where the rings analysis cannot be done, that is for the lower and higher camera lengths. Before the diffraction shift can be used to calibrate camera lengths, it must first be calibrated itself. This is done at a suitable camera length, immediately after the camera length there has been calibrated with the rings. (The software thus does not rely on any existing - potentially inaccurate or even wrong - diffraction shift calibrations in the system.) The procedure followed during diffraction shift calibration is as follows: • Apply a negative shift from the current diffraction-pattern position and acquire image 1, with a

negative backlash correction (first more negative, then to the desired position). • Apply a similar but now positive shift from the original position and acquire image 2. • Apply a positive shift from the original diffraction-pattern position and acquire image 3 (position the

same as image 2), with a positive backlash correction (first more positive , then to the desired position).

• Apply a similar but now negative shift from the original position and acquire image 4 (positions the same as image 1).

• Determine the shift between images 1 and 2, and between 3 and 4. These shifts give the two vectors used for the calibration.

Since drift is not an issue in diffraction, no drift correction is done in the diffraction-shift based calibration.

8.2 Magnification Calibration Report Control Panel The Report tab of the Magnification Calibration Control Panel contains the controls used for report generation. All reports are in Acrobat Reader (pdf) file format. Two types of report can be created: • A tabular overview of the results of a calibration sessions,

displaying the TEM magnification, the measured value(s) according to the method used, which value is used for the magnification table, the associated consistency error, and the magnification factor from plate camera to CCD or the ratio between nominal and measured STEM magnifications..

• A graphical overview of the results of (at most) the last twelve calibrations done.

Note: the STEM magnification is a rather arbitrary value, based on the approximation that a full STEM frame should correspond to a frame covering 100x100mm at the same TEM magnification. Significant deviations form this arbitrary value can be found. However, such deviations are unimportant. The only really relevant value is the pixel size, which is what is calibrated. Create report When a calibration procedure has been executed successfully, the Create report button will become enabled and a report can be made of the calibration just done. Auto open report When the Auto open report checkbox is checked, the report will be opened automatically in Acrobat Reader (provided this is installed on the system). Otherwise the software will display a message when the report has been created successfully.


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Report from file Reports can be created from the files stored automatically by the software when a calibration procedure ahs been executed successfully. Select a file from the list underneath and press the Report from file button. History report A historical overview can be made of the (at most) twelve last calibrations done - for the combination of camera and microscope conditions. Click on one of the files in the list (this will define which conditions will be used for the selection). press the History report button. The software will find the result of the last twelve calibrations and display these in a graph. Calibrations file list The result of each successful calibration is always stored to file. These files are located in the folder Tecnai\Data\Magnification Calibration and have the extension tcl. It is advised to remove old files after a suitable period. When there are too many files present (more than 100), a warning will be displayed to remove some. These files can be read in by the software to create reports. The files are sorted in chronological order (the most recent at the top). The four columns list the creation date, the name of the CCD camera, microscope lens series, and high-tension setting.


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8.2.1 Tabular format report

The tabular format list the camera and microscope settings at the top. Below that follows a table with the following columns: • TEM - the magnification reported by the Tecnai microscope (the reference magnification on the plate

camera, displayed by the microscope when the viewing screen is up). • X-grating - the results of the magnification calibration from the cross-grating directly. • Im. shift - the result of the magnification calibration performed using the image shift of the

microscope. • Lattice - the result of the magnification calibration performed with high-resolution images of gold [001]

or silicon [110].


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• User - the value used for the magnification table. In most cases there will be only one calibration method for each magnification, but in some cases there may be more than one. In that case the used magnification indicates which of the method results is used (the other method then gives a cross-check).

• Error (%) - each result is based on the measurement of two vectors (see the description of the methods). The error indicates the consistency in the measurement of the two vectors and is NOT an indication of the absolute accuracy achieved.

• CCD/TEM - the ratio between the measured magnification on the CCD camera and the TEM reference magnification. On a perfect system, these values would all be the same.


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8.2.2 History format report

The history format list the camera and microscope settings at the top. Below that follows a graph displaying the CCD to TEM ratio or nominal to measured ratio for STEM images for each magnification. The last calibration is shown with filled red circles, the other calibrations have other symbols (unfilled, change of color and symbol type). A 2% error bar is shown in the top right-hand corner of the graph (not in the picture above). Note that the data of the above example are not real data. The graph allows rapid assessment of the quality of each calibration (measurement consistent with earlier or later calibrations) as well as (any) change in time of calibrations performed.


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8.3 Magnification Calibration File Control Panel The File tab of the Magnification Calibration Control Panel contains the controls used for setting magnification tables from stored calibrations.

Set Mag Table from File When a calibration file is selected in the Calibrations file list and DigitalMicrograph is running, it is possible to set the magnification table from the data in the file. The software will if TIA CCD (when present) should be updated as well and give a warning when TIA is not running. Next the software checks that the camera currently selected in DigitalMicrograph matches that in the calibrations file. If not, the software will switch cameras. The calibration data are then loaded into the magnification tables. Calibrations file list The result of each successful calibration is always stored to file. These files are located in the folder Tecnai\Data\Magnification Calibration and have the extension tcl. It is advised to remove old files after a suitable period. When there are too many files present (more than 100), a warning will be displayed to remove some. These files can be read in by the software to create reports. The files are sorted in chronological order (the most recent at the top). The four columns list the creation date, the name of the CCD camera, microscope lens series, and high-tension setting.

8.4 Magnification Calibration Options Control Panel The Options tab of the Magnification Calibration Control Panel defines the automatic behavior during focusing.

Focus on : For focusing there is a choice between the main screen (the main screen is lowered automatically to allow focusing) and the CCD camera (Preview mode; CCD image acquisition in the Preview mode will start automatically). To use the latter, set the CCD Preview mode to a suitable setting (continuous acquisition, half or quarter size, exposure time ~0.25 sec).


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With a GIF With a GIF there is a choice for focusing between the TV camera (the camera is inserted automatically to allow focusing) and the CCD camera (Preview mode; CCD image acquisition in the Preview mode will start automatically). To use the latter, set the CCD Preview mode to a suitable setting (continuous acquisition, half or quarter size, exposure time ~0.25 sec).


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9 TEM AutoGun (FP5460/20)

9.1 AutoGun procedures The AutoGun Control Panel provides a number of automated procedures. A short description of these procedures is given below.

9.1.1 Auto Conditoning Conditioning can be used to stabilize the high tension. It is mostly used after an exchange of a filament or on higher-voltage (200kV or higher) instruments. Conditioning takes the high tension up in controlled steps while monitoring the vacuum pressure in the gun. If the pressure remains more than 2 log steps above the previous attained level, the procedure will wait for it to drop again. If the pressure exceeds the previously attained value by a large amount, the procedure will step the high tension back a bit and wait. Conditioning goes up to the specified level with the delay step, both as defined by the user. On 100 and 120kV conditioning takes place by slowly increasing the high tension up to the maximum of the instrument (these microscopes do not have the ability to increase the high tension beyond the highest value). Once the user-defined goal has been reached, the conditioning procedure is finished. If the currently active high-tension setting is not one of the fixed high-tension steps, the high tension will be set to the closest fixed high-tension step below the current value. On 200 and 300kV instruments, the conditioning will take place in the normal regime at low high tension settings, but switch to true conditioning when an overvoltage (a high tension value beyond the normal maximum of 200 or 300kV) is required. After the user-define goal has been reached, the procedure will keep the high tension at that value for 10 more minutes for stabilization. Afterwards the high tension is set to the closest fixed high-tension step equal to or below the current value. The starting point for the conditioning depends on the current high-tension status : • If the high tension is off, it will be changed to lowermost fixed high-tension step. • If the high tension is on, the current high tension value defines the starting point. During conditioning the control panel displays how much time conditioning is estimated to take. The estimate is based on the assumption that there will be no delays due to vacuum pressures increases. Conditioning is accessible only to Expert user level or higher.

9.1.2 Auto Align The gun alignment can be performed automatically. The alignment is split into three possible procedures: • Gun tilt - the alignment that is the most frequently needed. • Gun tilt plus gun shift - since the gun shift is much more stable, this alignment is really only needed

after a change of filament (thermionic gun). • Gun tilt, gun shift plus spot size-dependent gun shift - the full procedure which is really only needed

after a filament change. Notes:


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• The alignments make use of the measurement of the current on the large fluorescent screen. This screen must there be down and the small screen out. If the beam current is less than the minimum detectable screen current, the procedure will either terminate or (in the spot size-dependent gun shift) skip the smaller spot sizes.

• Some of the measurements take place at high magnifications and require a reasonably focused beam. The latter is set through the focused beam function that is derived from the spot-size intensity calibration (in the HM Beam and LM Beam alignment procedures). Because on a FEG the size of the beam additionally depends on the extraction voltage and gun-lens setting, the software will ask the user to focus the beam. The aforementioned calibration should nevertheless be done correctly, because the software only asks for one spot, the others should then automatically be focused as well.

• For a FEG, the is one more important alignment - the gun tilt pivot points. These cannot be aligned on the basis of the screen-current measurement and are therefore not included.

• For a FEG the alignment depends on the gun settings, so all alignment values may change as a function of extraction voltage and gun-lens setting - though in general the same sensitivity is present as with the thermionic filament, with gun tilt most frequently needed and spot size-dependent gun shift the least.

• The accessibility of alignments depends on user level. For 'Users' only the gun-tilt procedure is accessible (the inherent assumption is that the remainder of the alignment is derived from Supervisor or higher user levels). All other levels have access to all three procedures.


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9.2 AutoGun (User) – (Thermionic) The AutoGun Control Panel provides functionality to control the electron gun and related functions that are required to switch 'light' on or off on the microscope. It combines functionality from the high-tension, filament, alignment and vacuum controls.

Note: The AutoGun control panel covers all available functionality of the normal Filament (User) control panel (and has more). It is suggested that you remove the normal Filament (User) control from your user interface setup and insert the AutoGun control panel instead. Light The Light button starts the procedure to make light : • Check high tension status and switch high tension on if needed. • Check the status of the filament and switch it on if needed. • Check the status of the Column valves and open these if needed. • Switch to a low magnification in the HM range with suitable illumination settings. If the first three conditions are fulfilled the button is yellow. If it is impossible to attain 'Light', the status will display why. When the yellow Light button is pressed, the light is 'switched off'. This means that the column valves are closed and the filament is switched off. When the make light (or switch off) is active, the button will be orange (a transitional status). Align The Align button starts or stops the gun-tilt alignment procedure. The color of the button indicates whether the procedure is active (yellow). Note: The gun-tilt pivot points can be difficult to align (especially on FEG microscopes) because the beam moves when the pivot points are changed. AutoGun has a special feature that will detect the change in pivot point and automatically compensates the gun-deflection settings to keep the beam centered. This makes the alignment of the gun tilt pivot much easier. You do not have to activate anything in AutoGun, just make sure it is in the user interface. Status The status label provides feedback on status and operation of the AutoGun functionality.


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High tension Pressing the High tension button switches the high tension on and off. The color of the button indicates the high-tension status (yellow = on). The high-tension setting is the one shown in the drop-down list box on the right. The High tension button has three possible settings: • The high tension is enabled but off : the button is 'normal' gray. • The high tension is on : the button is yellow. • The high tension is disabled : the text in the button is gray. The high tension is enabled through the High tension enable button on the System On/Off Panel. High-tension selection The high tension setting is by clicking in the drop-down list box and selecting the required setting (a range of fixed settings, comprising : • 40, 60, 80 and 100 kV for Tecnai 10 • 20, 40, 60, 80, 100 and 120 kV for Tecnai 12 • 20, 40, 80, 120, 160 and 200 kV for Tecnai 20 • 50,100,150,200, 250 and 300 kV for Tecnai 30 Filament The Filament button switches the filament on or off. The color of the button indicates the filament status (yellow = on). The button is only enabled when the high tension is on. Current filament setting The value gives the currently active filament setting (corresponding to the value displayed underneath the progress bar). User filament setting The User filament index is set with the spin control. Spin the value to the required setting. The value cannot exceed the filament limit set by the Supervisor. When the Enter button is enabled (which occurs after the value has been changed by the user), the value currently displayed can be sent to the server by pressing the button (as long as the button is enabled, the server has not been updated). Filament setting display The Filament setting display shows the currently active setting of the filament. The value is displayed in numerical format underneath as well as in a progress bar. The progress bar always has a range from 0 to a value that is a multiple of ten equal to or above the filament limit,. The filament limit is shown by a red bar coming from the right. Emission current The emission current display shows the value of the emission current. Emission setting The emission setting can be changed with the spin control within the range 1 to 6.


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9.3 AutoGun (Expert) – (Thermionic) The AutoGun Control Panel provides functionality to control the electron gun and related functions that are required to switch 'light' on or off on the microscope. It combines functionality from the high-tension, filament, alignment and vacuum controls.

Note: The AutoGun control panel covers a major part of the functionality of the normal Filament (Expert) and High tension (Expert) control panels (and has more). It is suggested that you remove these control from your user interface setup and insert the AutoGun control panel instead. The functionality not covered is that related to the ability to set the high tension to other values then the preselection (Free High Tension control). Light The Light button starts the procedure to make light: • Check high tension status and switch high tension on if needed. • Check the status of the filament and switch it on if needed. • Check the status of the Column valves and open these if needed. • Switch to a low magnification in the HM range with suitable illumination settings. If the first three conditions are fulfilled the button is yellow. If it is impossible to attain 'Light', the status will display why. When the yellow Light button is pressed, the light is 'switched off'. In the case of the FEG this means that the column valves are closed. When the make light (or switch off) is active, the button will be orange (a transitional status). Align The Align button starts or stops the alignment procedure selected from the drop-down list on the right. The color of the button indicates whether the procedure is active (yellow). Note: The gun-tilt pivot points can be difficult to align (especially on FEG microscopes) because the beam moves when the pivot points are changed. AutoGun has a special feature that will detect the change in pivot point and automatically compensates the gun-deflection settings to keep the beam centered. This makes the alignment of the gun tilt pivot much easier. You do not have to activate anything in AutoGun, just make sure it is in the user interface. Status The status label provides feedback on status and operation of the AutoGun functionality.


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High tension Pressing the High tension button switches the high tension on and off. The color of the button indicates the high-tension status (yellow = on). The high-tension setting is the one shown in the drop-down list box on the right. The High tension button has three possible settings: • The high tension is enabled but off : the button is 'normal' gray. • The high tension is on : the button is yellow. • The high tension is disabled : the text in the button is gray. The high tension is enabled through the High tension enable button on the System On/Off Panel. High-tension selection The high tension setting is by clicking in the drop-down list box and selecting the required setting (a range of fixed settings, comprising : • 40, 60, 80 and 100 kV for Tecnai 10 • 20, 40, 60, 80, 100 and 120 kV for Tecnai 12 • 20, 40, 80, 120, 160 and 200 kV for Tecnai 20 • 50,100,150,200, 250 and 300 kV for Tecnai 30 The actual high tension value is displayed on the right. Normally this is the same as the value from the drop-down list, but during conditioning it may differ. Filament The Filament button switches the filament on or off. The color of the button indicates the filament status (yellow = on). The button is only enabled when the high tension is on. Current filament setting The value gives the currently active filament setting (corresponding to the value displayed underneath the progress bar). User filament setting The User filament index is set with the spin control. Spin the value to the required setting. The value cannot exceed the filament limit set in the flap-out. When the Enter button is enabled (which occurs after the value has been changed by the user), the value currently displayed can be sent to the server by pressing the button (as long as the button is enabled, the server has not been updated). Filament setting display The Filament setting display shows the currently active setting of the filament. The value is displayed in numerical format underneath as well as in a progress bar. The progress bar always has a range from 0 to a value that is a multiple of ten equal to or above the filament limit,. The filament limit is shown by a red bar coming from the right. Emission current The emission current display shows the value of the emission current. Emission setting The emission setting can be changed with the spin control within the range 1 to 6. Flap-out button The flap-out button leads to the Control and Saturate tabs of the AutoGun Control Panel.


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9.4 AutoGun Control (Expert) – (Thermionic) The AutoGun Control Panel provides a number of system settings concerning the filament as well as access to the Conditioning procedure. Filament The filament type can either be Tungsten or LaB6. The type of filament can only be chosen by the Supervisor.

Filament limit The filament limit is the value up to which the filament setting can be increased before it stops automatically. The value can be adjusted by Expert user and Supervisor. Compensate HT change with emission When the high tension is changed, the emission from the filament changes (lower when the high tension goes down). The normal way to compensate this (in order to achieve usable "light" levels) is to change the emission setting of the the gun. When the Compensate HT change with emission, the software will automatically adjust the emission setting to achieve a comparable level of emission. Due to the fact that the emission settings and HT steps are not perfectly matched, the change will generally not be one to one and some variation in emission may be the result. Condition status It is not always possible to start the conditioning procedure. The Condition status indicates why conditioning is not possible (apart from another procedure currently running). If conditioning is possible, the condition status is not visible and the Condition button is enabled. Condition Pressing the Condition button starts or stops the Conditioning procedure. The color of the button indicates the status (yellow = active). Condition to The value for condition to is the goal used by the conditioning procedure. For Experts the suggested value is the maximum high tension plus 5% if an overvoltage is possible (200 and 300kV), otherwise it is the maximum high tension. Condition delay step The condition delay step defines the number of seconds the conditioning procedure will before it takes another step (subject to the vacuum pressure being low enough). Filament hour counter The filament hour counter displays the date the filament was installed and the number of hours the filament has been in operation.


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9.5 AutoGun (Supervisor) – (Thermionic) The AutoGun Control Panel provides functionality to control the electron gun and related functions that are required to switch 'light' on or off on the microscope. It combines functionality from the high-tension, filament, alignment and vacuum controls.

Note: The AutoGun control panel covers a major part of the functionality of the normal Filament (Supervisor) and High tension (Expert) control panels (and has more). It is suggested that you remove these control from your user interface setup and insert the AutoGun control panel instead. The functionality not covered is that related to the ability to set the high tension to other values then the preselection (Free High Tension control). Light The Light button starts the procedure to make light: • Check high tension status and switch high tension on if needed. • Check the status of the filament and switch it on if needed. • Check the status of the Column valves and open these if needed. • Switch to a low magnification in the HM range with suitable illumination settings. If the first three conditions are fulfilled the button is yellow. If it is impossible to attain 'Light', the status will display why. When the yellow Light button is pressed, the light is 'switched off'. In the case of the FEG this means that the column valves are closed. When the make light (or switch off) is active, the button will be orange (a transitional status). Align The Align button starts or stops the alignment procedure selected from the drop-down list on the right. The color of the button indicates whether the procedure is active (yellow). Note: The gun-tilt pivot points can be difficult to align (especially on FEG microscopes) because the beam moves when the pivot points are changed. AutoGun has a special feature that will detect the change in pivot point and automatically compensates the gun-deflection settings to keep the beam centered. This makes the alignment of the gun tilt pivot much easier. You do not have to activate anything in AutoGun, just make sure it is in the user interface. Status The status label provides feedback on status and operation of the AutoGun functionality.


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High tension Pressing the High tension button switches the high tension on and off. The color of the button indicates the high-tension status (yellow = on). The high-tension setting is the one shown in the drop-down list box on the right. The High tension button has three possible settings: • The high tension is enabled but off : the button is 'normal' gray. • The high tension is on : the button is yellow. • The high tension is disabled : the text in the button is gray. The high tension is enabled through the High tension enable button on the System On/Off Panel. High-tension selection The high tension setting is by clicking in the drop-down list box and selecting the required setting (a range of fixed settings, comprising : • 40, 60, 80 and 100 kV for Tecnai 10 • 20, 40, 60, 80, 100 and 120 kV for Tecnai 12 • 20, 40, 80, 120, 160 and 200 kV for Tecnai 20 • 50,100,150,200, 250 and 300 kV for Tecnai 30 The actual high tension value is displayed on the right. Normally this is the same as the value from the drop-down list, but during conditioning it may differ. Filament The Filament button switches the filament on or off. The color of the button indicates the filament status (yellow = on). The button is only enabled when the high tension is on. Cold run up The Cold run up button starts or stops the cold run up procedure, which is a combination of conditioning, filament autosaturation and full gun alignment. After the procedure has finished it will close the column valves and switch the filament off again. The procedure is meant to be run after exchange of a filament and the button is only enabled when the filament hour counter has been reset to 0 or a gun air has been done. The color of the button indicates whether the procedure is active or not (yellow = active). Current filament setting The value gives the currently active filament setting (corresponding to the value displayed underneath the progress bar). User filament setting The User filament index is set with the spin control. Spin the value to the required setting. The value cannot exceed the filament limit set in the flap-out. When the Enter button is enabled (which occurs after the value has been changed by the user), the value currently displayed can be sent to the server by pressing the button (as long as the button is enabled, the server has not been updated). Filament setting display The Filament setting display shows the currently active setting of the filament. The value is displayed in numerical format underneath as well as in a progress bar. The progress bar always has a range from 0 to a value that is a multiple of ten equal to or above the filament limit,. The filament limit is shown by a red bar coming from the right. Emission current The emission current display shows the value of the emission current. Emission setting The emission setting can be changed with the spin control within the range 1 to 6.


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Flap-out button The flap-out button leads to the Control tab of the AutoGun Control Panel.

9.6 AutoGun Control (Supervisor) – (Thermionic) The AutoGun Control Panel provides a number of system settings concerning the filament as well as access to the Conditioning procedure.

Filament The filament type can either be Tungsten or LaB6 (CeB6 can be treated as equal to LaB6). The instrument has two sets of values for controlling the filament. These are exchanged when the other filament type is chosen. The type of filament can only be chosen by the Supervisor from the drop-down list. Filament limit The filament limit is the value up to which the filament setting can be increased before it stops automatically. The value can be adjusted by Expert user and Supervisor. Note that the Supervisor setting is automatically used for the 'User' user level. Compensate HT change with emission When the high tension is changed, the emission from the filament changes (lower when the high tension goes down). The normal way to compensate this (in order to achieve usable "light" levels) is to change the emission setting of the the gun. When the Compensate HT change with emission, the software will automatically adjust the emission setting to achieve a comparable level of emission. Due to the fact that the emission settings and HT steps are not perfectly matched, the change will generally not be one to one and some variation in emission may be the result. Condition status It is not always possible to start the conditioning procedure. The Condition status indicates why conditioning is not possible (apart from another procedure currently running). If conditioning is possible, the condition status is not visible and the Condition button is enabled. Condition Pressing the Condition button starts or stops the Conditioning procedure. The color of the button indicates the status (yellow = active). Condition to The value for condition to is the goal used by the conditioning procedure. For Experts the suggested value is the maximum high tension plus 5% if an overvoltage is possible (200 and 300kV), otherwise it is the maximum high tension.


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Condition delay step The condition delay step defines the number of seconds the conditioning procedure will before it takes another step (subject to the vacuum pressure being low enough). Filament hour counter The filament hour counter displays the date the filament was installed and the number of hours the filament has been in operation. Reset count Pressing the Reset count button resets the filament counter to zero (that is, the date becomes today and the hour counter becomes zero). This function is only accessible to the Supervisor. Pressing the Reset count button should be done after the filament has been exchanged.


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AutoGun (User) – (FEG) The AutoGun Control Panel provides functionality to control the electron gun and related functions that are required to switch 'light' on or off on the microscope. It combines functionality from the high-tension, FEG, alignment and vacuum controls.

Note: The AutoGun control panel covers all available functionality of the normal FEG (User) control panel (and has more). It is suggested that you remove the normal FEG (User) control from your user interface setup and insert the AutoGun control panel instead. Light The Light button starts the procedure to make light: • Check high tension status and switch high tension on if needed. • Check the status of the FEG and switch to Operate if needed. • Check the status of the Column valves and open these if needed. • Switch to a low magnification in the HM with suitable illumination settings. If the first three conditions are fulfilled the button is yellow. If it is impossible to attain 'Light', the status will display why. When the yellow Light button is pressed, the light is 'switched off'. In the case of the FEG this means that the column valves are closed. When the make light (or switch off) is active, the button will be orange (a transitional status). Align The Align button starts or stops the gun-tilt alignment procedure. The color of the button indicates whether the procedure is active (yellow). Note: The gun-tilt pivot points can be difficult to align (especially on FEG microscopes) because the beam moves when the pivot points are changed. AutoGun has a special feature that will detect the change in pivot point and automatically compensates the gun-deflection settings to keep the beam centered. This makes the alignment of the gun tilt pivot much easier. You do not have to activate anything in AutoGun, just make sure it is in the user interface. Status The status label provides feedback on status and operation of the AutoGun functionality.


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High tension Pressing the High tension button switches the high tension on and off. The color of the button indicates the high-tension status (yellow = on). The high-tension setting is the one shown in the drop-down list box on the right. The High tension button has three possible settings: • The high tension is enabled but off : the button is 'normal' gray. • The high tension is on : the button is yellow. • The high tension is disabled : the text in the button is gray. The high tension is enabled through the High tension enable button on the System On/Off Panel. High-tension selection The high tension setting is by clicking in the drop-down list box and selecting the required setting (a range of fixed settings, comprising 20, 40, 80, 120, 160 and 200 kV for Tecnai F20 and 50,100,150,200, 250 and 300 kV for Tecnai F30). Operate The Operate button switches between the Operate and Standby FEG states. The button is only enabled when the FEG is on. Current extraction voltage The value gives the currently active extraction voltage (corresponding to the value displayed on the progress bar). User extraction voltage The Extraction voltage is set with the spin control. Spin the value to the required setting. The value of the Extraction voltage cannot exceed the Extractor limit set by the Supervisor. When the Enter button is enabled (which occurs after the value has been changed by the user), the value currently displayed can be sent to the server by pressing the button (as long as the button is enabled, the server has not been updated). Extraction voltage display The Extraction voltage display shows the value of the extraction voltage. The value is displayed in numerical format on the left as well as in a progress bar. The progress bar always has a range of 1000 Volts, changing dynamically from 2000-3000 to 3000-4000, etc. The extraction limit is shown by a red bar coming from the right. Emission current The FEG Emission current display shows the value of the emission current. Gun lens The Gun lens setting can be changed with the spin control within the range 1 to 8.


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9.7 AutoGun (Expert) – (FEG) The AutoGun Control Panel provides functionality to control the electron gun and related functions that are required to switch 'light' on or off on the microscope. It combines functionality from the high-tension, FEG, alignment and vacuum controls.

Note: The AutoGun control panel covers a major part of the functionality of the normal FEG (Expert) and High tension (Expert) control panels (and has more). It is suggested that you remove these control from your user interface setup and insert the AutoGun control panel instead. The functionality not covered is that related to powering the FEG up or switching it off and the ability to set the high tension to other values then the preselection (Free High Tension control). Light The Light button starts the procedure to make light: • Check high tension status and switch high tension on if needed. • Check the status of the FEG and switch to Operate if needed. • Check the status of the Column valves and open these if needed. • Switch to a low magnification in the HM with suitable illumination settings. If the first three conditions are fulfilled the button is yellow. If it is impossible to attain 'Light', the status will display why. When the yellow Light button is pressed, the light is 'switched off'. In the case of the FEG this means that the column valves are closed. When the make light (or switch off) is active, the button will be orange (a transitional status). Align The Align button starts or stops the alignment procedure selected from the drop-down list on the right. The color of the button indicates whether the procedure is active (yellow). Note: The gun-tilt pivot points can be difficult to align (especially on FEG microscopes) because the beam moves when the pivot points are changed. AutoGun has a special feature that will detect the change in pivot point and automatically compensates the gun-deflection settings to keep the beam centered. This makes the alignment of the gun tilt pivot much easier. You do not have to activate anything in AutoGun, just make sure it is in the user interface. Status The status label provides feedback on status and operation of the AutoGun functionality.


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High tension Pressing the High tension button switches the high tension on and off. The color of the button indicates the high-tension status (yellow = on). The high-tension setting is the one shown in the drop-down list box on the right. The High tension button has three possible settings: • The high tension is enabled but off : the button is 'normal' gray. • The high tension is on : the button is yellow. • The high tension is disabled : the text in the button is gray. The high tension is enabled through the High tension enable button on the System On/Off Panel. High-tension selection The high tension setting is by clicking in the drop-down list box and selecting the required setting (a range of fixed settings, comprising 20, 40, 80, 120, 160 and 200 kV for Tecnai F20 and 50,100,150,200, 250 and 300 kV for Tecnai F30). Operate The Operate button switches between the Operate and Standby FEG states. The button is only enabled when the FEG is on. Current extraction voltage The value gives the currently active extraction voltage (corresponding to the value displayed on the progress bar). User extraction voltage The Extraction voltage is set with the spin control. Spin the value to the required setting. The value of the Extraction voltage cannot exceed the Extractor limit set in the flap-out. When the Enter button is enabled (which occurs after the value has been changed by the user), the value currently displayed can be sent to the server by pressing the button (as long as the button is enabled, the server has not been updated). Extraction voltage display The Extraction voltage display shows the value of the extraction voltage. The value is displayed in numerical format on the left as well as in a progress bar. The progress bar always has a range of 1000 Volts, changing dynamically from 2000-3000 to 3000-4000, etc. The extraction limit is shown by a red bar coming from the right. Emission current The FEG Emission current display shows the value of the emission current. Gun lens The Gun lens setting can be changed with the spin control within the range 1 to 8. Flap-out button The flap-out button leads to the Control tab of the AutoGun Control Panel.


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9.8 AutoGun Control (Expert) – (FEG) The AutoGun Control Panel provides access to the extraction limit as well as the Conditioning procedure.

Extraction limit The filament limit is the value up to which the filament setting can be increased before it stops automatically. The value can be adjusted by Expert user and Supervisor. Condition status It is not always possible to start the conditioning procedure. The Condition status indicates why conditioning is not possible (apart from another procedure currently running). If conditioning is possible, the condition status is not visible and the Condition button is enabled. Condition Pressing the Condition button starts or stops the Conditioning procedure. The color of the button indicates the status (yellow = active). Condition to The value for condition to is the goal used by the conditioning procedure. For Experts the suggested value is the maximum high tension plus 5% if an overvoltage is possible (200 and 300kV), otherwise it is the maximum high tension. Condition delay step The condition delay step defines the number of seconds the conditioning procedure will before it takes another step (subject to the vacuum pressure being low enough). Operate and standby hours The FEG Timers control panel displays the date the timers were started (FEG tip installation) and the hours the FEG tip has run in Standby and Operate modes. The timers can be reset only by service.


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9.9 AutoGun (Supervisor) – (FEG) The AutoGun Control Panel provides functionality to control the electron gun and related functions that are required to switch 'light' on or off on the microscope. It combines functionality from the high-tension, FEG, alignment and vacuum controls.

Note: The AutoGun control panel covers a major part of the functionality of the normal FEG (Expert) and High tension (Expert) control panels (and has more). It is suggested that you remove these control from your user interface setup and insert the AutoGun control panel instead. The functionality not covered is that related to powering the FEG up or switching it off and the ability to set the high tension to other values then the preselection (Free High Tension control). Light The Light button starts the procedure to make light: • Check high tension status and switch high tension on if needed. • Check the status of the FEG and switch to Operate if needed. • Check the status of the Column valves and open these if needed. • Switch to a low magnification in the HM with suitable illumination settings. If the first three conditions are fulfilled the button is yellow. If it is impossible to attain 'Light', the status will display why. When the yellow Light button is pressed, the light is 'switched off'. In the case of the FEG this means that the column valves are closed. When the make light (or switch off) is active, the button will be orange (a transitional status). Align The Align button starts or stops the alignment procedure selected from the drop-down list on the right. The color of the button indicates whether the procedure is active (yellow). Note: The gun-tilt pivot points can be difficult to align (especially on FEG microscopes) because the beam moves when the pivot points are changed. AutoGun has a special feature that will detect the change in pivot point and automatically compensates the gun-deflection settings to keep the beam centered. This makes the alignment of the gun tilt pivot much easier. You do not have to activate anything in AutoGun, just make sure it is in the user interface. Status The status label provides feedback on status and operation of the AutoGun functionality.


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High tension Pressing the High tension button switches the high tension on and off. The color of the button indicates the high-tension status (yellow = on). The high-tension setting is the one shown in the drop-down list box on the right. The High tension button has three possible settings: • The high tension is enabled but off : the button is 'normal' gray. • The high tension is on : the button is yellow. • The high tension is disabled : the text in the button is gray. The high tension is enabled through the High tension enable button on the System On/Off Panel. High-tension selection The high tension setting is by clicking in the drop-down list box and selecting the required setting (a range of fixed settings, comprising 20, 40, 80, 120, 160 and 200 kV for Tecnai F20 and 50,100,150,200, 250 and 300 kV for Tecnai F30). Operate The Operate button switches between the Operate and Standby FEG states. The button is only enabled when the FEG is on. Current extraction voltage The value gives the currently active extraction voltage (corresponding to the value displayed on the progress bar). User extraction voltage The Extraction voltage is set with the spin control. Spin the value to the required setting. The value of the Extraction voltage cannot exceed the Extractor limit set in the flap-out. When the Enter button is enabled (which occurs after the value has been changed by the user), the value currently displayed can be sent to the server by pressing the button (as long as the button is enabled, the server has not been updated). Extraction voltage display The Extraction voltage display shows the value of the extraction voltage. The value is displayed in numerical format on the left as well as in a progress bar. The progress bar always has a range of 1000 Volts, changing dynamically from 2000-3000 to 3000-4000, etc. The extraction limit is shown by a red bar coming from the right. Emission current The FEG Emission current display shows the value of the emission current. Gun lens The Gun lens setting can be changed with the spin control within the range 1 to 8. Flap-out button The flap-out button leads to the Control tab of the AutoGun Control Panel.


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9.10 AutoGun Control (Supervisor) – (FEG) The AutoGun Control Panel provides access to the extraction limit as well as the Conditioning procedure.

Extraction limit The filament limit is the value up to which the filament setting can be increased before it stops automatically. The value can be adjusted by Expert user and Supervisor. For 'User' user level the extraction limit as set by the Supervisor is used. Condition status It is not always possible to start the conditioning procedure. The Condition status indicates why conditioning is not possible (apart from another procedure currently running). If conditioning is possible, the condition status is not visible and the Condition button is enabled. Condition Pressing the Condition button starts or stops the Conditioning procedure. The color of the button indicates the status (yellow = active). Condition to The value for condition to is the goal used by the conditioning procedure. For Experts the suggested value is the maximum high tension plus 5% if an overvoltage is possible (200 and 300kV), otherwise it is the maximum high tension. Condition delay step The condition delay step defines the number of seconds the conditioning procedure will before it takes another step (subject to the vacuum pressure being low enough). Operate and standby hours The FEG Timers control panel displays the date the timers were started (FEG tip installation) and the hours the FEG tip has run in Standby and Operate modes. The timers can be reset only by service.


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10 AutoAdjust

10.1 AutoAdjust (User) Control Panel The AutoAdjust Control Panel provides a number of automated functions: • AutoFocus with or without autostigmation • Auto beam center • Auto rotation center • Auto eucentric height

Note: AutoAdjust only works with the default CCD camera controller (TIA). TIA must therefore be running before AutoAdjust functionality is enabled. Focus When the Focus button is pressed, the AutoFocus procedure is started when the button is gray. When the procedure is already running (the button is yellow), the procedure is canceled. The Focus button is enabled when : • No other procedure is running. • The currently active microscope mode is compatible with the AutoFocus procedure (TEM imaging or

Nanoprobe imaging at magnifications <150kx). Δf The AutoFocus procedures measures the current defocus (with or without astigmatism). Once the procedure is finished, it will set the defocus to the user-defined value entered in the edit control. Note: There are three separate user-defined defocus values, one for LM, one for HM imaging (Microprobe) and one for Nanoprobe. The software will keep these separate. The value you enter is only for the currently active mode. The value will be changed automatically to the relevant one if the mode is switched. Include stigmator The AutoFocus procedure can be run with or without measurement and correction of astigmatism. The procedure without astigmatism is faster than the procedure with, because fewer measurements must be made. Note that the defocus measurement may be in error if astigmatism is present. It is advised to include stigmation at the start of a microscope session or, thereafter, when the microscope is switched to a different mode (in this case only LM or HM are relevant; Nanoprobe has the same stigmation as HM image). Tune When the Tune button is pressed, the tuning procedure selected in the drop-down list is started when the button is gray. When the procedure is already running (the button is yellow), the procedure is canceled.


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The available tuning procedures are : • All - in sequence - eucentric height, beam center, rotation center, focus and stigmation • Eucentric height • Beam center • Rotation center • Tune selection The drop-down list allows selection of one of the tuning procedures. Status The Status field shows information about the currently active procedure, feedback on possible failures, and instructions to be carried out. Next When the user must carry out some instruction (as given in the Status field), the Next button becomes enabled. Press the Next button to continue (after having carried out the instructions) or the Focus or Tune button to cancel further execution. Undo When the last action performed by AutoAdjust (the Undo button will be enabled after finishing a procedure) does not produce satisfactory results, you can go back to the starting position by pressing the Undo button. Generally only one action will be undone (so if you perform focus after eucentric height, it will only undo focus), except when the combined procedure (All) has been done, in which case all actions performed successfully in the combined procedure will be undone. Del Images In TIA a number of images are created by AutoAdjust. These images are called Image 1 to5 plus another image that can have various names, such as Cross-correlation. These images are necessary during execution of an AutoAdjust procedure. After that, you may want these images removed, which is what happens when you press the Del Images button. Flap-out The flap-out button leads to the Settings tab of the AutoAdjust Control Panel.


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10.2 AutoAdjust (Expert) Control Panel The AutoAdjust Control Panel provides a number of automated functions: • AutoFocus with or without autostigmation • Auto beam center • Auto rotation center • Auto eucentric height

Note: AutoAdjust only works with the default CCD camera controller (TIA). TIA must therefore be running before AutoAdjust functionality is enabled. Focus When the Focus button is pressed, the AutoFocus procedure is started when the button is gray. When the procedure is already running (the button is yellow), the procedure is canceled. The Focus button is enabled when : • No other procedure is running. • The currently active microscope mode is compatible with the AutoFocus procedure (TEM imaging or

Nanoprobe imaging at magnifications <150kx). Δf The AutoFocus procedures measures the current defocus (with or without astigmatism). Once the procedure is finished, it will set the defocus to the user-defined value entered in the edit control. Note: There are three separate user-defined defocus values, one for LM, one for HM imaging (Microprobe) and one for Nanoprobe. The software will keep these separate. The value you enter is only for the currently active mode. The value will be changed automatically to the relevant one if the mode is switched. Include stigmator The AutoFocus procedure can be run with or without measurement and correction of astigmatism. The procedure without astigmatism is faster than the procedure with, because fewer measurements must be made. Note that the defocus measurement may be in error if astigmatism is present. It is advised to include stigmation at the start of a microscope session or, thereafter, when the microscope is switched to a different mode (in this case only LM or HM are relevant; Nanoprobe has the same stigmation as HM image). Tune When the Tune button is pressed, the tuning procedure selected in the drop-down list is started when the button is gray. When the procedure is already running (the button is yellow), the procedure is canceled.


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The available tuning procedures are : • All - in sequence - eucentric height, beam center, rotation center, focus and stigmation • Eucentric height • Beam center • Rotation center • Tune selection The drop-down list allows selection of one of the tuning procedures. Status The Status field shows information about the currently active procedure, feedback on possible failures, and instructions to be carried out. Next When the user must carry out some instruction (as given in the Status field), the Next button becomes enabled. Press the Next button to continue (after having carried out the instructions) or the Focus or Tune button to cancel further execution. Undo When the last action performed by AutoAdjust (the Undo button will be enabled after finishing a procedure) does not produce satisfactory results, you can go back to the starting position by pressing the Undo button. Generally only one action will be undone (so if you perform focus after eucentric height, it will only undo focus), except when the combined procedure (All) has been done, in which case all actions performed successfully in the combined procedure will be undone. Del Images In TIA a number of images are created by AutoAdjust. These images are called Image 1 to5 plus another image that can have various names, such as Cross-correlation. These images are necessary during execution of an AutoAdjust procedure. After that, you may want these images removed, which is what happens when you press the Del Images button. Flap-out The flap-out button leads to the Settings and Calibrate tabs of the AutoAdjust Control Panel.


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10.3 AutoAdjust Calibrate (Expert) Control Panel The AutoAdjust Calibrate Control Panel provides the calibration procedures necessary for proper operation of the AutoAdjust functions.

Calibrate Pressing the calibrate button starts the calibration procedure selected in the drop-down list, if the button is gray. If a procedure is already running (the button is yellow), the procedure is canceled. Calibrations must be done in the proper sequence and if a procedure is selected that is not yet possible (because calibrations higher up in the hierarchy have not been done), the list will jump back to the lowermost procedure possible. Calibration selection The drop-down list allows selection of one of the calibration procedures. Calibration status The Status field shows information about the currently active procedure, feedback on possible failures, and instructions to be carried out. Next When the user must carry out some instruction (as given in the Status field), the Next button becomes enabled. Press the Next button to continue (after having carried out the instructions) or the Calibrate button to cancel further execution.

10.3.1 Calibrations Calibrations have a set hierarchy (some calibrations can only be done once others have been done). They are specific to a number of conditions : • CCD camera used in combination with the microscope lens series (Normal or EFTEM) • High tension • Microscope mode (LM, Microprobe, Nanoprobe, Lorentz) For the microscope modes, only the Nanoprobe mode does not require full calibrations. For that mode only the Focus/Stigmator calibration must be done separately (the rest of the calibrations if the same as in the Microprobe mode). Calibrations can only be done by TEM Experts (private calibrations for these users) and Supervisor, Service or Factory (dependent on availability, the se calibrations are what basic microscope Users get. Calibration is done on the basis of a standard specimen, a cross-grating. This specimen has squares on it with a spacing of 463 nm.


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The following calibration procedures are used : • Magnification calibration • Image-shift calibration • Stage calibration • Stigmator alignment (only supervisor level and higher) • Focus/stigmator calibration Magnification calibration The software will acquire an image of the cross-grating and analyse that to find the basic spacing. The analysis is done using an auto-correlation (at high magnifications) or an FFT (at low magnifications) in which the two basic vectors will be outlined by circles.

An image of a cross-grating, showing the 463 nm squares. The result of the magnification calibration. The autocorrelation of the cross-grating image (zoomed in 2x) shows the central peak pointed at by the line (which has been added manually for clarity). The software adds the two circles around the two base vectors of the pattern. It is important that these circles are located at the correct positions and not e.g. at the diagonals of the squares. Additional circles are placed at the multiples of the base vectors used to show how many base vectors were used together for the analysis.

Image-shift calibration The image-shift calibration procedure performs a calibration of the image shift on the CCD. This calibration must be done for all magnifications in the required range (up to magnifications of ~150kx). Normally the whole range is calibrated in one procedure, but it also possible to (re)calibrate the image shift for an individual magnification (e.g. when it appears that the calibration in the complete procedure went wrong for one magnification, that magnification can be redone later).


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Note: At low magnifications the grid bars may come into the field of view. The software will remove the grid bars from the images and replace them with random noise. This greatly improves the success rate of the shift measurement (especially if features like the central "A" in the cross-grating or large holes in the foil are in the field of view). Stage calibration The stage calibration calibrates the stage x and y shifts. This calibration is needed for consistency checks of the eucentric height procedure. See the note above on the removal of grid bars. Stigmator alignment The stigmator alignment ensures that image shifts caused by changes of the stigmator (objective in HM image and Nanoprobe; diffraction in LM image) are minimised. This procedure performs the same as the stigmator alignment procedures themselves (in the Alignment Control Panel) except in AutoAdjust the alignment is done automatically. It should be noted that the focus/stigmator calibration is dependent on this alignment and a change in the alignment will interfere with proper operation of the AutoAdjust functions. Focus/Stigmator calibration The focus/stigmator calibration procedure performs the calibration of beam tilt, focus/stigmator and image shift. Once this calibration has been done, the focus/procedure can be run for any magnification that is smaller than ~150kx (above the beam-tilt induced image-shift method doesn't work anymore because random fluctuations of image position start to play an important role and interfere too much with the focus/astigmatism measurement).


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11 TrueImage™ Focus Series Acquisition

11.1 Introduction The TrueImage™ suite of software provides the tools for the PAL-MAL focus series reconstruction of HRTEM images. The software consists of two parts, one for the acquisition and the other for the reconstruction. The acquisition on the TEM microscope is covered by the Focus series acquisition automated experiment. This software is the only reliable way for acquiring HRTEM through-focus series wherein the focus steps are kept the same throughout the series.

11.2 Automated Experiments Control Panel The Automated Experiments Control Panel with TrueImage Focus Series Acquisition active.

Active When the Active button is pressed the Automated Experiment currently selected is switched on (the button becomes yellow) or off. DigitalMicrograph must be running before the Focus Series Acquisition experiment can be activated. Euc Height Not available in Focus series acquisition. Auto Focus Not available in Focus series acquisition Start When the Start button is pressed, the function currently selected is started. The button remains yellow during execution of the function selected. When the button is pressed again while it is yellow, further execution of the current function is canceled. Next During some procedures, the user must set some conditions (specimen area, illumination, etc.) on the microscope, then press the Next button for the procedure to continue.


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Define File When the Define File button is pressed, the software brings up the standard Save File dialog in which you select a folder and file name for the focus series data. The images are saved in separate files, with the file name followed by an underscore ( _ ) and then a three-digit number (001, 002, etc.; for example test_001.ebn). If you collect another series without changing the file name, the second series is saved under the file name followed by an underscore, then a serial number (e.g. 02) followed again by the underscore and then the three-digit number (for example test_02_001.ebn). Experiment The Experiment drop-down list contains a list of all experiments available on the system. Select the experiment required and press Active to get started. Function For Focus series acquisition the only function currently available is Acquire series. Instructions The instructions field will display instructions on how to proceed or a status description of the function currently running or finished. Flap-out button The flap-out button leads to the Settings tabs of the Automated Experiments Control Panel.

11.3 TrueImage™ Focus Series Acquisition Settings The Automated Experiments Settings Control Panel.

Each Automated Experiment will typically have a number of associated functions, each of which will have its own settings. The settings are controlled on the Settings tab of the Automated Experiments Control Panel. Settings list From the drop-down list, a function can be selected, whose settings will then be listed in the property editor underneath. The settings list for tomography contains the following: Acquisition parameters Reconstruction parameters Settings The settings are displayed in a so-called property editor. This is a two-column grid, listing the setting on the left-hand side and its value on the right-hand side. The settings in the property editor are sorted alphabetically. You can change the settings by clicking on the value. Sometimes you have to type in a


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number or other setting. In the case where a limited selection is possible (for example with the boolean operators true or false) a small down arrow on the right-side allows selection of the one of the possibilities. Setting changes are updated as soon as you move to another setting value (e.g. by pressing the down or up arrow). If the new setting is not allowed, it will be switched back to a proper value. Tip: To change the settings for the function selected back to their default values, click on the property editor with the right-hand mouse button and select Reset to defaults in the popup menu. All settings of the current set will be restored to their software default values. Note: The property editor does not allow having some properties as read-only, but the Focus series acquisition has several such values that are either used for information display or which definition is restricted to some users. In practice this means that the value can be changed, but it will jump back to its original value.

11.4 Settings Below is given a description of the groups of settings used for Focus series acquisition and their meaning. When you change values, the software will do check on suitable values and if necessary, adjust the values. Note that you can change the values even when no experiment is currently active, but you will not see any update if the values you entered were incorrect. The update in the user interface is only done when the experiment is active.

11.4.1 Acquisition parameters These settings are stored and recalled for each individual user. Check Drift Before Drift can spoil the reconstruction of a focus series because there is not enough

overlap area between the first and last images. When the Check Drift Before option is switched on (true), the software will check the drift before acquiring the series, by recording two images with about 30 seconds between them and measuring the shift. When the drift exceeds a certain value, dependent on an estimate of the time needed to acquire the focus series, a warning is given and you can cancel further acquisition. Otherwise the value of the drift measured is given.

Data Path The path where the data will be stored. Define this path through the Define File function on the main control panel.

Exposure Time The exposure time (in seconds) used for the acquisition of the focus series images.

Filename The filename under which the data will be stored. Define this name through the Define File function on the main control panel. If the name has already been used in the same session, the software will automatically use the same name but with an additional serial number (02, 03, ..) to avoid overwriting the previously acquired focus series.


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File Type Images from the tomography tilt series can be saved as native server format (either DigitalMicrograph or TIA), as one of a series of binary formats, or both. In the latter case each image is saved in binary and native server format. Note that for TrueImage reconstruction the file format must be one of the binary formats, with the extended binary advised.

Focus Increment The true minimum focus step on the microscope, corresponding to a single step of the finest DAC available. This value is as currently set as calibration on the microscope. This value is read-only (you can change it, but it will jump back to its previous value).

Focus Step The number of focus increments you want to use as step between images.

Image Binning The binning value used for the acquisition of the focus series.

Image Height The height of the image in pixels for the acquisition of the tilt series. This number is adjusted for the binning used, so that at binning 1 a value of 1024 pixels becomes 512 at binning 2, etc. The only allowed values are 1024 or 2048. For a 2048x2048 camera both 2048 and 1024 are allowed. The latter can be binning 1 (center area of CCD) or binning 2.

Image Server A choice of DigitalMicrograph (DM) or TEM Imaging and Analysis (TIA).

Image Width The width of the image in pixels for the acquisition of the tilt series. This number is adjusted for the binning used, so that at binning 1 a value of 1024 pixels becomes 512 at binning 2, etc. The only allowed values are 1024 or 2048. For a 2048x2048 camera both 2048 and 1024 are allowed. The latter can be binning 1 (center area of CCD) or binning 2.

Last Image At A parameter used together with Start At Current Focus. If the latter is false, the series acquisition will not start at the focus you have currently set, but instead it will aim to have the last image of the series recorded at the value specified (usually this would be the Lichte or Alpha Null focus). In order to estimate where at which focus to start acquisition, the software assumes that the focus you have currently set is at the Minimum Contrast setting (~0.4 * Scherzer focus). The reason for using Minimum Contrast rather than any other value (such as 0 defocus) is that Minimum Contrast focus can be recognized reasonably well, whereas any other defocus cannot.

No Of Images The number of images recorded automatically in the series.

Start At Current Focus

This parameter defines whether you want to start the series at the focus as you have it currently set or whether the software should aim at recording the last image at the value specified for the last image (see Last Image At, above).


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11.4.2 Reconstruction parameters The reconstruction parameters are values that are stored in the extended-header binary file. These values are used as input for the TrueImage reconstruction software. Some of the values are system settings and as such can only be set by the special microscope users Supervisor, Service or Factory. Astigmatism 2 and 3 X and Y

The values for two-fold and three-fold astigmatism. For future use.

Coma X and Y The values for coma. For future use.

Focus Spread The values for the focus spread of the microscope. For future use. Value can only be set by Supervisor, Service or Factory.

Reconstruction Limit

The effective reconstruction limit to be used for focus-series reconstruction. This value is one of the factors determining the Lichte focus. It is in nanometer units.

Lichte focus A value calculated by the software from the value for the reconstruction limit and high tension on the microscope. Generally the focus series is aimed at ending at the Lichte focus value. Reference: H. Lichte (1993) Ultramicroscopy 51, 15.

Alpha Null focus A value calculated by the software from the value for the reconstruction limit and high tension on the microscope. The focus series can be aimed at ending at the Alpha Null focus value. Reference: M.A. O'Keefe (2001) 59th Ann. Proc. MSA 916.

MTF A value giving a single-number representation of the Modulation Transfer Function of the CCD camera. Value can only be set by Supervisor, Service or Factory.

Semi Convergence The measure of the incidence angle, relevant for the spatial-coherence envelope. For future use.

Spherical Aberration

The value of the spherical aberration of the microscope's objective lens.


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11.5 File formats You can save the image of the focus series in different binary file formats. However, only the extended-header binary stores all the information that helps to simplify TrueImage reconstruction. The binary file formats are : Binary : only data are stored in the file. Further information about data type (2- or 4-byte integer, single- or double-precision floats) and image dimensions is put into a separate .log file with same file name. Extended-header :the binary data are preceded by an extended header that contains the data type, image dimensions, and several types of information stored specifically for Focus series reconstruction (see extended-header definition below). Note that depending on the particular software that writes the file, not all values are necessarily defined.

11.5.1 Binary data type 1 - unsigned 1-byte integer 2 - unsigned 2-byte integer 3 - unsigned 4-byte integer 4 - signed 1-byte integer 5 - signed 2-byte integer 6 - signed 4-byte integer 7 - 4-byte (single-precision) floating point 8 - 8-byte (double-precision) floating point

11.5.2 Extended-header format Data type Comment 2-byte integer data type of the image (see above) 4-byte integer image width in pixels 4-byte integer image height in pixels 4-byte integer total size of the header in bytes (including preceding values). In effect the offset to the

data. double Microscope type 0 = T10 and T12, 1 = T20, 2 = T30, for Titan add 10 to HT

equivalent double Gun LaB6 = 0, FEG = 1 double Lens type HC = -2, BioTWIN = -1, TWIN = 0, STWIN = 1, UTWIN = 2, XTWIN = 3 double D number of microscope double High tension (kV) double Focus spread double MTF double Starting defocus (nm) double Focus step (nm) double DAC setting double Focus value (nm) double Pixel size (nm) double Spherical aberration (mm) double Semi-convergence (mrad) double Info limit (nm-1) double Number of images double Image number in series double Coma x double Coma y double Astigmatism 2 X


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double Astigmatism 2 Y double Astigmatism 3 X double Astigmatism 3 Y double Camera type serial number double Camera position (WA, MSC, GIF) double Magnification double Camera length double STEM magnification double Lens series double Lorentz double Spot size double Microscope mode double Objective lens value three doubles Currently undefined double Image shift X (m) double Image shift Y (m) double Image shift X (pixels) double Image shift Y (pixels) double Stage X (m) double Stage Y (m) double Stage Z (m) double Stage A (rad) double Stage B (rad) double Image minimum level double Image maximum level double Image real (0) or reciprocal space further doubles undefined Many of the values stored in the extended header are used by the TrueImage reconstruction software as a first guess to optimize the reconstructed wave function.

11.5.3 MRC file format The MRC file is a file with extension .mrc. It contains a primary header and an extended header as described below, followed by the data. The file format is derived from the MRC file format which was originated at the Medical Research Council in Cambridge, England. • The MRC format can contain a series of images in a single file. The format consists of: • A primary header. A 1024 byte section. • An extended header. Additional information about the individual images. • Image data.

11.5.3.1 The primary header. Data type Name Byte

positionComment

integer (4-byte) nx 0 number of colums integer ny 4 number of rows integer nz 8 number of sections (i.e. images) integer mode 12 data type, 1 = pixel value stored as short

integer integer nxstart 16 lower bound of colums integer nystart 20 lower bound of rows


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integer nzstart 24 lower bound of sections integer mx 28 cell size in Angstroms (pixel spacing=xlen/mx) integer my 32 integer mz 36 4-byte floating point xlen 40 4-byte floating point ylen 44 4-byte floating point zlen 48 4-byte floating point alpha 52 cell angles in degrees 4-byte floating point beta 56 4-byte floating point gamma 60 integer mapc 64 mapping colums, rows, sections on axis

(x=1,y=2,z=3) integer mapr 68 integer maps 72 4-byte floating point amin 76 4-byte floating point amax 80 4-byte floating point amean 84 2-byte integer ispg 88 space group number (0 for images) 2-byte integer nsymbt 90 number of bytes used for storing symmetry operators

4-byte integer next 92 number of bytes in extended header. This is an important number. It defines the offset to the first image

2-byte integer dvid 96 creator id char (byte) extra[30] 98 extra 30 bytes data (not used) 2-byte integer numintegers 128 number of bytes per section in extended

header 2-byte integer numfloats 130 number of floats per section in extended

header 2-byte integer sub 132 2-byte integer zfac 134 4-byte floating point min2 136 4-byte floating point max2 140 extra 28 bytes data 4-byte floating point min3 144 4-byte floating point max3 148 4-byte floating point min4 152 4-byte floating point max4 156 2-byte integer idtype 160 2-byte integer lens 162 2-byte integer nd1 164 divide by 100 to get float value 2-byte integer nd2 166 2-byte integer vd1 168 2-byte integer vd2 170 4-byte floating point tiltangles[9] 172 used to rotate model to match rotated image 4-byte floating point zorg 208 origin of image; used to auto translate model 4-byte floating point xorg 212 to match a new image that has been translated 4-byte floating point yorg 216 4-byte integer nlabl 220 number of text labels with useful data (0-10) char data[10][80] 224 10 text labels with 80 characters


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11.5.3.2 The extended header Total number of extended headers (one for each image) is 1024. Please note: Some values have rather odd numbering. That is because prior usage determined that certain values were fixed too low and the addition of more types dictated the necessity for numbers below, while 0 is generally avoided. The 0 is typically an undefined number. Name Byte position Comment a tilt 0 Alpha tilt in degrees b tilt 4 Beta tilt in degrees x stage 8 Stage position in um y stage 12 z stage 16 x shift 20 Image shift position in um y shift 24 defocus 28 Defocus in micrometers exposure time 32 mean intensity 36 tilt axis angle 40 pixel size 44 Image pixel size in nanometers magnification 48 Microscope type 52 Tecnai 10 = -2, Tecnai 12 = -1, Tecnai 20 = 1,

Tecnai 30 = 2, for Titan add 10 to HT equivalent Gun type 56 W = -2, Lab6 = -1, FEG = 1 Lens type 60 HC = -3, BioTWIN = -2, TWIN = -1, STWIN = 1,

UTWIN = 2, XTWIN = 3 D number of microscope 64 High tension (kV) 68 Focus spread 72 MTF 76 Starting Df (nm) 80 Focus step (nm) 84 DAC setting 88 Spherical aberration 92 Semi-convergence 96 Info limit (nm-1) 100 Number of images 104 Image number in series 108 Coma 1 112 Coma 2 116 Astigmatism 2 1 120 Astigmatism 2 2 124 Astigmatism 3 1 128 Astigmatism 3 2 132 Camera type number 136 Camera position 140 WA = 1, MSC = 2, GIF = 3 Unused 144 Unused 148 Many of the values stored in the extended header are used by the TrueImage reconstruction software as a first guess to optimize the reconstructed wave function.

TEM help Scripting Version Tecnai 4.0 / Titan1.1

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TEM help manual -- Scripting Table of Contents 1 TEM Scripting...................................................................................................................................... 4

1.1 Introduction .................................................................................................................................. 4 1.1.1 Tem objects .............................................................................................................................. 4

1.1.1.1 The ‘Instrument’ object .................................................................................................. 4 1.1.1.2 The secondary microscope objects ............................................................................... 4 1.1.1.3 The utility objects ........................................................................................................... 4

1.1.2 TEM Scripting Features: ........................................................................................................... 4 1.1.2.1 Synchronous functions .................................................................................................. 4 1.1.2.2 Events............................................................................................................................ 5 1.1.2.3 TEM constants............................................................................................................... 5 1.1.2.4 TEM error codes ............................................................................................................ 5

1.2 A first example ............................................................................................................................. 5 1.3 A second example (using utility objects) ...................................................................................... 5 1.4 TEM modes.................................................................................................................................. 6 1.5 Remote Scripting.......................................................................................................................... 7

1.5.1 Specifying the microscope server............................................................................................. 7 1.5.2 Receiving events from a remote microscope server................................................................. 7

2 The TEM Object Model ....................................................................................................................... 9 2.1 The microscope objects ............................................................................................................... 9 2.2 Utility objects .............................................................................................................................. 10

3 The Instrument object........................................................................................................................ 11 4 The microscope objects .................................................................................................................... 13 5 The Gun object.................................................................................................................................. 14

5.1 Gun object constants ................................................................................................................. 14 5.1.1 Enum HTState ........................................................................................................................ 14

6 The InstrumentModeControl object ................................................................................................... 15 6.1 InstrumentModeControl object constants:.................................................................................. 15

6.1.1 Enum InstrumentMode: .......................................................................................................... 15 7 The Illumination object ...................................................................................................................... 16

7.1 Illumination object constants ...................................................................................................... 17 7.1.1 Enum IlluminationMode ..........................................................................................................17 7.1.2 Enum CondenserMode (Titan only)........................................................................................ 17 7.1.3 Enum DarkFieldMode.............................................................................................................18 7.1.4 Enum IlluminationNormalization ............................................................................................. 18

8 The Projection object......................................................................................................................... 19 8.1 Projection object constants ........................................................................................................ 22

8.1.1 Enum ProjectionMode ............................................................................................................22 8.1.2 Enum ProjectionSubMode...................................................................................................... 22 8.1.3 Enum LensProg...................................................................................................................... 22 8.1.4 Enum ProjectionDetectorShift ................................................................................................ 22 8.1.5 Enum ProjDetectorShiftMode ................................................................................................. 22 8.1.6 Enum ProjectionNormalization ............................................................................................... 23

9 The BlankerShutter object................................................................................................................. 24 10 The Vacuum object ........................................................................................................................... 25

10.1 Constants: .................................................................................................................................. 25 10.1.1 Enum VacuumStatus: .............................................................................................................25


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11 The Stage object ............................................................................................................................... 26 11.1 StageAxisData object................................................................................................................. 26 11.2 Stage object constants............................................................................................................... 27

11.2.1 Enum StageStatus.................................................................................................................. 27 11.2.2 Enum StageAxes.................................................................................................................... 27 11.2.3 Enum StageHolderType ......................................................................................................... 28 11.2.4 Enum MeasurementUnitType................................................................................................. 28

12 The AutoLoader object ...................................................................................................................... 29 12.1 Cassette slot status constants ................................................................................................... 29

12.1.1 Enum CassetteSlotStatus....................................................................................................... 29 13 The TemperatureControl object ........................................................................................................ 30

13.1 Refigerant level constants.......................................................................................................... 30 13.1.1 Enum RefrigerantLevel ........................................................................................................... 30

14 The (Plate) Camera object ................................................................................................................ 31 14.1 Camera object constants ........................................................................................................... 32

14.1.1 Enum ScreenPosition ............................................................................................................. 32 14.1.2 Enum PlateLabelDateFormat ................................................................................................. 32

15 The Acquisition object ....................................................................................................................... 33 15.1 Usage example in a pseudo-programming language................................................................. 34 15.2 CCDCameras............................................................................................................................. 35

15.2.1 CCDCamera ........................................................................................................................... 35 15.2.1.1 CCDCameraInfo .......................................................................................................... 36 15.2.1.2 CCDAcqParams .......................................................................................................... 37

15.3 STEMDetectors.......................................................................................................................... 38 15.3.1 STEMDetectors ...................................................................................................................... 38 15.3.2 STEMDetector ........................................................................................................................ 38

15.3.2.1 STEMDetectorInfo ....................................................................................................... 38 15.3.2.2 STEMAcqParams ........................................................................................................ 39

15.4 AcqImages ................................................................................................................................. 39 15.4.1 AcqImage ............................................................................................................................... 39

15.5 Acquisition Constants................................................................................................................. 39 15.5.1 Enum AcqImageSize ..............................................................................................................39 15.5.2 Enum AcqImageCorrection .................................................................................................... 40 15.5.3 Enum AcqShutterMode .......................................................................................................... 40 15.5.4 Enum AcqExposureMode....................................................................................................... 40

15.6 SafeArray handling..................................................................................................................... 40 16 The UserButton object....................................................................................................................... 42 17 The utility objects............................................................................................................................... 43

17.1 The Gauge (utility object) ........................................................................................................... 43 17.1.1 Gauge object constants.......................................................................................................... 44

17.1.1.1 Enum GaugeStatus: .................................................................................................... 44 17.1.1.2 Enum GaugePressureLevel......................................................................................... 44

17.2 The StagePosition (utility object)................................................................................................ 45 17.3 The Vector (utility object) ........................................................................................................... 45

18 TEM constants .................................................................................................................................. 46 19 TEM error codes................................................................................................................................ 47

19.1 Enum TEMScriptingError: .......................................................................................................... 47 20 TEM-specific issues .......................................................................................................................... 48

20.1 Synchronous functions............................................................................................................... 48 20.1.1 Automatic normalization ......................................................................................................... 48 20.1.2 Pole touch............................................................................................................................... 48 20.1.3 Setting the high tension .......................................................................................................... 48

20.2 Normalizations ........................................................................................................................... 48


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20.3 TEM sessions............................................................................................................................. 49 20.4 Setting parameters out of range................................................................................................. 50

21 Property- and function- types ............................................................................................................ 51 22 TEM scripting in C++......................................................................................................................... 52

22.1 Import the dynamic link library.................................................................................................... 52 22.2 Create an ‘Instrument’ and get the secondary microscope object that you need....................... 52 22.3 Manipulate and read microscope parameters, invoke microscope actions................................ 53 22.4 Use the collections ..................................................................................................................... 54 22.5 Receive events from the user buttons........................................................................................ 55 22.6 Receive Events from a remote microscope server..................................................................... 56 22.7 Errors and error handling ........................................................................................................... 56

23 TEM scripting in Delphi ..................................................................................................................... 58 23.1 Introduction and package installation......................................................................................... 58 23.2 Events ........................................................................................................................................ 58 23.3 Using the dynamic link library .................................................................................................... 58 23.4 Example program....................................................................................................................... 58

23.4.1 Large fonts.............................................................................................................................. 59 23.4.2 About box and Version number .............................................................................................. 59

23.5 Create an ‘Instrument’ and get secondary microscope objects.................................................. 59 23.6 Manipulate and read microscope parameters, invoke microscope actions................................ 60 23.7 Use the collections ..................................................................................................................... 60 23.8 Receive events from the user buttons........................................................................................ 62 23.9 Receive events from a remote microscope server ..................................................................... 62 23.10 Errors and error handling ........................................................................................................... 63

24 TEM scripting in JScript..................................................................................................................... 64 24.1 Create an ‘Instrument’ and get the secondary microscope object that you need....................... 64 24.2 Manipulate and read microscope parameters, invoke microscope actions................................ 64 24.3 Use the collections ..................................................................................................................... 65 24.4 Receive events from the user buttons........................................................................................ 66 24.5 Errors and error handling ........................................................................................................... 66

25 TEM scripting in VBScript.................................................................................................................. 68 25.1 Create an ‘Instrument’ and get the secondary microscope object that you need....................... 68 25.2 Manipulate and read microscope parameters, invoke microscope actions................................ 68 25.3 Use the collections ..................................................................................................................... 69 25.4 Receive events from the user buttons........................................................................................ 69 25.5 Errors and error handling ........................................................................................................... 70

26 TEM scripting in Visual Basic ............................................................................................................ 72 26.1 Import the dynamic link library.................................................................................................... 72 26.2 Create an ‘Instrument’ and get the secondary microscope object that you need....................... 73 26.3 Manipulate and read microscope parameters, invoke microscope actions................................ 73 26.4 Use the collections ..................................................................................................................... 74 26.5 Receive events from the user buttons........................................................................................ 75 26.6 Receive events from a remote microscope server ..................................................................... 75 26.7 Errors and error handling ........................................................................................................... 76 26.8 Visual Basic-specific Errors........................................................................................................ 77


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1 TEM Scripting The TEM scripting adapter enables communication between a script and the TEM microscope (Tecnai or Titan). To make use of TEM scripting, it is not necessary to have a deeper understanding of the internal structure of the TEM software. The adapter's functionality should be understandable to somebody who is able to use a TEM microscope. Most scripting or programming languages (VBScript, JScript, Visual Basic, C++, Delphi, ...) are supported. The examples you find in the various chapters of this help file use JScript syntax, but the help also includes chapters that adress the peculiarities of other languages. You can also inspect the example programs that are delivered with TEM scripting (the default installation directory is tecnai\scripting or titan\scripting).

1.1 Introduction The TEM Scripting adapter supports its clients (in other words: your scripts) with a number of software objects that correspond to the various parts of the microscope. These objects are the main components of the adapter. The adapter further provides some other features such as User Button events and TEM constants for convenience.

1.1.1 Tem objects

1.1.1.1 The ‘Instrument’ object This is the main object. It manages access to all parts of the microscope, for example the gun, the camera and so forth, that are modelled as ‘secondary microscope objects’. Please note: Because of the introduction of the Titan microscope, the TEM scripting object (previously called "Tecnai") has become more generic : it is now called TEMScripting. Because of this change you may have to recompile any existing code. Functions previously present have not changed.

1.1.1.2 The secondary microscope objects These objects represent the parts of the microscope. They have properties and methods as is common for objects in most scripting languages. The properties directly correspond to microscope parameters (such as magnification, beam shift etc.). Together they define the state of the microscope that can be controlled through scripting. The methods generally invoke some actions (taking an exposure, normalizing the lenses etc).

1.1.1.3 The utility objects There are some TEM specific utility objects that are used to handle multidimensional properties (such as the stage position, which consists of 5 coordinates).

1.1.2 TEM Scripting Features:

1.1.2.1 Synchronous functions In order to support a simple, sequential way of scripting, all the adapters functions work synchronous, i.e. they will only return after they have finished executing. (The setting of properties is equivalent to a function call.)


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1.1.2.2 Events The adapter supports the usage of the six User Buttons L1..L3, R1..R3 (on the TEM control pads) and can, if desired, generate events when they are pressed. These events can be handled by the script. Thus a user can interact with the script via the control pads of the microscope.

1.1.2.3 TEM constants For ease of programming, a number of TEM-specific constants have been defined. These constants are accessed as elements of enumerations. For example, there exists an enumeration named ScreenPosition that enumerates the possible positions of the fluorescent screen. Using the values spUP, spDown, and spUnknown, that are elements of this enumeration, instead of direct values like 1 ,2 , or 3 makes programming easier and reduces the chance of programming errors. Some programming environments (such as the one for VB, for example) possess an IntelliSense mechanism that supports the use of enumerated constants via dropdown menus.

1.1.2.4 TEM error codes For the same reason the adapter includes some TEM-specific error codes, that represent physical error conditions.

1.2 A first example It is easy to start with TEM scripting. Suppose you want to know the actual magnification. You need to do three things: 1. create an ‘Instrument’ object 2. get access to the projection system 3. check the property ‘Magnification’ The following code does this (in JScript syntax): var MyTem = new ActiveXObject("TEMScripting.Instrument") var Proj = MyTem.Projection var m = Proj.Magnification Please note: Because of the introduction of the Titan microscope, the TEM scripting object (previously called "Tecnai") has become more generic : it is now called TEMScripting. The variable m now contains the magnification. That's it! In the same way you can set properties to new values. For example Proj.MagnificationIndex = Proj.MagnificationIndex + 1 is essentially the same as increasing the magnification by turning the button on the TEM control pad by one click clockwise.

1.3 A second example (using utility objects) Some properties do not consist of a ‘simple type’. For example, the image shift is a two-dimensional property, having an x- and an y-component. The ‘utility’ objects are used to handle these, because generally you would like to set and read all components of those properties simultaneously.


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The following example shows how to use them (JScript syntax): Start again by creating access to the projection system var MyTem = new ActiveXObject("TEMScripting.Instrument") var Proj = MyTem.Projection Suppose we want to shift the image in x-direction to +500nm, leaving the y-position at 0: var MyImageShift = new MyTem.Vector(0.5E-6,0) Proj.ImageShift = MyImageShift The first statement creates a ‘Vector’ object that is already initialized with the desired the image shift. (Since the ‘Vector’ object is TEM-specific, its creation is also handled by the main ‘Instrument’ object.) The second line uses the ‘Vector’ to set the image shift. The described way of creating a utility object currently only seems to work in JScript. Alternatively, if you read the image shift you will of course also get a ‘Vector’ returned: var MyImageShift = Proj.ImageShift MyImageShift.X = MyImageShift.X + 0.5E-6.0 Proj.ImageShift = MyImageShift This will result in a relative image shift in x-direction of 500nm. Note that setting the image shift x (changing the value of MyImageShift.X alone) to a different value does not do anything on the microscope itself. You have to set the modified vector as a whole back (in other words, in the previous example it is the third line of code that does it).

1.4 TEM modes There is one important feature of the TEM microscopes inner working that needs to be explained before the TEM objects and properties are described in detail. Certain microscope parameters such as the intensity setting, the focus value, the spotsize, etc., are dependent on the optical "mode" of the microscope. A "mode" is characterized by a specific setting of the lenses that interact to form the image and the beam. As such, the optical mode is determined by the following parameters that you probably know from working with the microscope: • the probe setting (microprobe/nanoprobe) • the projector mode (diffraction/imaging) • the range of magnification or diffraction (LM, Mi, SA, Mh, LAD, D) • the lens program (Regular or EFTEM) • the high tension range (there are three ranges named high, medium and low for microscopes with

maximum high tension greater or equal than 200kV) Some parameters of the optical system change on a transition between modes (i.e., internally, they exist ‘per mode’). For example, when you switch from low LM to Mi magnification, the focus setting will change. It will return to its old ‘LM-value’, if you lower the magnification again. Such parameters are called ‘mode dependent’. The same will happen with the corresponding properties of the TEM scripting adapter's microscope objects. If you change a mode dependent property, this will only affect the current mode and thus will not affect its values in the other modes. Only the values of the active mode can be accessed and, consequently, switching between modes changes the exposed values of properties.


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The adapter objects that possess such mode dependent properties are of course the ones that have something to do with lens settings, i.e. the ‘Illumination’ and the ‘Projection’ objects.

1.5 Remote Scripting Using TEM Scripting, it is possible to write applications that communicate with a microscope server that runs on another computer. Thus it is possible to control the microscope remotely. For this purpose, you have to have a version of TEM Scripting installed locally. This means that the files stdscript.dll (and for VBScript users also scriptevents.dll) plus all other TEM software files that these files depend on have to be installed on your local machine and registered. In order to be able to install the TEM Scripting adapter you have to install the regular TEM software on the same PC (many of the regular software files are used during the registration of the scripting adapter and without proper registration the software will not run). You can install the regular software on two ways: 1. Use the TEM software CD-ROM to install the software. In this case the software itself is not switched

to simulation and cannot be run (it expects the hardware to be present). 2. Request a simulation CD-ROM from FEI (contact Dr. Max T. Otten, FEI Electron Optics, P.O.Box

80066, 5600 KA Eindhoven, The Netherlands, Tel. +31-40-2356655, Fax +31-40-2356550, e-mail [email protected]). In this case the software will run in simulation, which allows you to test your script. Please specify the type of microscope you need the simulation for.

1.5.1 Specifying the microscope server To access the remote microscope computer, its name has to be known to TEM Scripting (and also possibly to other remote TEM tools that are available at the time of publishing). The name of the remote server is stored in the system's database, the so-called registry. You can do it yourself, by editing the system registry. (Be careful not to delete or change any other registry entries by accident!): 1. Login as administrator. 2. Open a registry editor: Click the Windows 'Start' button, choose 'run' and type 'regedit' or 'regedt32',

click 'ok'. 3. A registry editor should open. Under the key HKEY_LOCAL_MACHINE\SOFTWARE\FEI, add a new

sub-key named 'Scripting'. 4. Add a new string value named "RemoteHost" to this key and assign the name of the server to this

value (for example "\\ACHT405"). 5. Close the registry editor. 6. Logoff and login under your own user account. From now on, every application that uses TEM Scripting will connect to the remote microscope server. If you have a microscope server installed on your local machine as well, and you want to make contact to the latter again, then you have to set the name of the server in the registry value "RemoteHost" in the registry key HKEY_LOCAL_MACHINE\SOFTWARE\FEI\Scripting to an empty string or delete the whole key again.

1.5.2 Receiving events from a remote microscope server The only events that your application can receive from the microscope server are the events that are fired when the user buttons on the TEM hand panels are used. If you want to receive those events also from a remote microscope, then you may have to add some additional code to your application. The reason for this is security. The communication between application and microscope server uses COM (the Microsoft common object model). COM does a lot of things underwater for you, including initializing security. Now we have the situation that a system service (the microscope server probably runs as such) on a remote computer wants to call a function in your application. COM does not allow that by default. To


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allow it, you have to call a function named 'CoInitializeSecurity'. This function call has to be made, before COM does this itself underwater, because this function can only be called once per application. Since the details of how and when to call 'CoInitializeSecurity' has to be called are language dependent, we refer here to the chapter 'Scripting in various languages'. Applications that rely on a browser to run (for example scripts written in JScript or VBScript, or ActiveX components written in any language), cannot do that. They will probably work anyway (because we are able to handle security issues from the microscope server side in this case). Workaround: if 'temserver' service and remote application run under the same user account, then there is no problem. You would have to create a special account for remoting.


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2 The TEM Object Model

2.1 The microscope objects The following tree shows the main adapter object (‘Instrument’, the only microscope object to be created by your script) and all secondary objects that can be accessed through it. These secondary objects are modelled as read-only properties of the ‘Instrument’ object. They all relate to a specific part of the microscope and are directly connected to the running microscope. Thus, changes in microscope parameters (for example via the TEM user interface) will affect their properties and vice versa. One of the microscope objects (the ‘UserButtons’) in the object-tree appears in the plural form in the object-tree. In this case the returned object is a collection of 6 ‘UserButton’objects.

Instrument

Gun

InstrumentModeControl

Illumination

Projection

BlankerShutter

Vacuum

Stage

AutoLoader

TemperatureControl

(Plate) Camera

Acquisition

UserButtons


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2.2 Utility objects Besides the objects listed above, there are some utility objects that can contain more complex data, for example the stage position, the image shift etc. They are used to handle ‘compound’ or multi-dimensional properties. The utility objects ensure that pieces of information that belong together are read and set simultaneously. These objects are therefore local copies (they exist in your script only). In contrast to the microscope objects they have no connection to the TEM server.

Vector

SizeLong

StagePosition

SizeDouble

Gauge


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3 The Instrument object This is the main object of the scripting adapter. It is the only object that can be created directly by your script. Every other microscope object must be assessed as a property of the ‘Instrument’ object: var MyTem = new ActiveXObject("TEMScripting.Instrument") creates an instance of this object (in JScript syntax). Property Description AutoNormalizeEnabled [Boolean], read/write

Enables/disables the automatic normalization procedures performed by the TEM microscope. Normally they are active, but for scripting it can be convenient to disable them temporarily.

Gun [Object] Interface to the gun deflection and high tension functionality.

InstrumentModeControl [Object] Interface to the instrument control functions that allow checking whether the microscope has STEM and, if so, switch between TEM and STEM modes.

Illumination [Object] Interface to the ‘Illumination’ system that comprises the condenser lenses, the mini condenser lens, the beam deflection coils and the condenser stigmator. Responsible for beam (or ‘probe’) properties.

Projection [Object] Interface to the part of the instrument that forms the image, comprising the objective, diffraction and projection lenses, the corresponding stigmators and the image deflectors.

BlankerShutter [Object] Interface to the BlankerShutter ShutterOverrideOn property.

Vacuum [Object] Int erface to the vacuum system.

Stage [Object] Interface to the stage / goniometer functions.

AutoLoader [Object] Interface to the Autoloader.

TemperatureControl [Object] Interface to the temperature controlller.

Camera [Object] Interface to the camera module. Also gives access to control of the fluorescent screens.

Acquisition [Object] Interface to the acquisition of CCD and STEM images.

UserButtons [Collection of UserButton objects] UserButtons interface to the user buttons L1..L3, R1..R3 of the TEM hand panels.


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Methods Description NormalizeAll() [Void]

Normalizes all lenses. To normalize portions of the microscope, refer to the subsystems.


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4 The microscope objects These secondary microscope objects relate to specific parts of the microscope. Through these objects, most of the functionality of the microscope is accessible. They can (and have to) be retrieved as properties of the main ‘instrument’ object. For example var Proj = MyTem.Projection gives you an instance of the ‘Projection’ object (supposing your ‘Instrument’ is named ‘MyTem’). Object Description Gun Interfaces to the functionality of the gun and the high tension. InstrumentControlMode Interfaces to the TEM/STEM switch Illu mination Interfaces to the illumination system that forms and manipulates the beam Projection Interfaces to the imaging system BlankerShutter Interface for the shutter override function Vacuum Interfaces to the vacuum system Stage Interfaces to stage and specimen holder AutoLoader Interfaces to the Autoloader TemperatureControl Interfaces to the temperature controller Camera Interfaces to the plate camera and the fluorescent screens Acquisition Interfaces to the CCD and STEM acquisition UserButton(s) Interface(s) to the user button(s), can receive events


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5 The Gun object Property Description Tilt [Vector], read/write

The gun tilt alignment values. Range from -1.0 to +1.0 in x and y directions (logical units). The beamblanker changes the gun tilt. Therefore changing the gun tilt alignment is blocked as long as the beamblanker is active.

Shift [Vector], read/write The gunshift alignment values. Range from -1.0 to +1.0 in x and y directions (logical units).

HTState [HTState], read/write The state of the high tension. (The high tension can be on, off or disabled). Disabling/enabling can only be done via the button on the system on/off-panel, not via script. When switching on the high tension, this function cannot check if and when the set high tension value is actually reached.

HTValue [Double], read/write The value of the HT setting as displayed in the TEM user interface. Units: Volts.

HTMaxValue [Double], read only The maximum possible value of the HT on this microscope. Units: Volts.

5.1 Gun object constants

5.1.1 Enum HTState Value Description htOff The high tension is off htOn The high tension is on htDisabled The high tension is disabled, cannot be switched on by software command


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6 The InstrumentModeControl object Property Description StemAvailable [Boolean], read only

Returns whether themicroscope has a STEM system or not. InstrumentMode [InstrumentMode], read/write

Switches between TEM and STEM modes.

6.1 InstrumentModeControl object constants:

6.1.1 Enum InstrumentMode: Value Description InstrumentMode_TEM TEM mode InstrumentMode_STEM STEM mode


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7 The Illumination object Most of the properties of the illumination object are dependent on the microscope's mode. They distinguish between three optical modes: 1. the microscope is either at low ‘magnification’ (LM or LAD), 2. at higher ‘magnification’ (Mi, SA, Mh, D) in nanoprobe or 3. at higher ‘magnification’ (Mi, SA, Mh, D) in microprobe Note that the illumination system is independent of the projection system and thus insensitive to diffraction or imaging mode; hence the mixture of diffraction and imaging submodes. If a transition between two of these three modes occurs, then the properties will seem to have changed their values, which reflects their existence per mode. Property Description mode independent Mode [IlluminationMode], read/write

Mode of the illumination system (either nanoprobe or microprobe). (Nearly) no effect for low magnifications (LM).

DFMode [DarkFieldMode], read/write Holds information about whether microscope is in dark field mode and if so, which coordinates are used.

BeamBlanked [Boolean], read/write Activates/deactivates the beamblanker.

mode dependent CondenserStigmator [Vector], read/write

The condenser stigmator setting. Units: logical, range: -1.0 to +1.0. SpotsizeIndex [Long], read/write

The spot size index (usually ranging from 1 to 11). Intensity [Double], read/write

Intensity value of the current mode (typically ranging from 0 to 1.0, but on some microscopes the minimum may be higher.)

IntensityZoomEnabled [Boolean], read/write Activates/deactivates the intensity zoom in the current mode. This function only works, when it has been initialized by means of the microscope alignments (it needs to know at which intensity setting the spot is focused).

IntensityLimitEnabled [Boolean], read/write Activates/deactivates the intensity limit in the current mode. This function only works, when it has been initialized by means of the microscope alignments (it needs to know at which intensity setting the spot is focused).

Shift [Vector], read/write Beam shift relative to the origin stored at alignment time. Units: meters.

Tilt [Vector], read/write Dark field beam tilt relative to the origin stored at alignment time. Only operational, if dark field mode is active. Units: radians, either in Cartesian (x,y) or polar (conical) tilt angles. The accuracy of the beam tilt physical units depends on a calibration of the tilt angles.

RotationCenter [Vector], read/write


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Corresponds to the alignment beam tilt value. Units are radians, range is ± 0.2-0.3rad. Do not confuse RotationCenter with dark field (Tilt). Be aware that this is an alignment function.

StemMagnification [Double], read/write The magnification value in STEM mode. You can change the magnification only in discrete steps (the same as on the microscope). If you specify a value that is not one of those steps, the scripting will select the nearest available step.

StemRotation [Double], read/write The STEM rotation angle (in radians).

The following properties are specific to the Titan microscope. Property Description CondenserMode [CondenserMode] read/write

Mode of the illumination system, parallel or probe. IlluminatedArea [Double] read/write

The size of the illuminated area (in meters). Accessible only in Parallel mode.

ProbeDefocus [Double] read/write The amount of probe defocus (in meters). Accessible only in Probe mode.

ConvergenceAngle [Double] read/write The convergence angle (in radians). Accessible only in Probe mode.

Method Description Normalize ([in] IlluminationNormalization Norm)

[Void] Normalizes the condenser lenses and/or the minicondenser lens, dependent on the choice of ‘Norm’.

7.1 Illumination object constants

7.1.1 Enum IlluminationMode Value Description imMicroprobe Microprobe mode imNanoprobe Nanoprobe mode

7.1.2 Enum CondenserMode (Titan only) Value Description imParallelIllumination Parallel illumination mode imProbeIllumination Probe illumination mode


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7.1.3 Enum DarkFieldMode Value Description dfOff microscope is in bright field mode dfCartesian dark field mode, beam tilt angles are given in x and y tilt directions dfConical dark field mode, beam tilt angles are given as radial tilt and rotation angles

7.1.4 Enum IlluminationNormalization Value Description nmSpotsize normalize lens C1 (spotsize) nmIntensity normalize lens C2 (intensity) + C3 nmCondenser normalize C1 + C2 + C3 nmMiniCondenser normalize the minicondenser lens nmObjectivePole normalize minicondenser and objective nmAll normalize C1, C2, C3, minicondenser + objective


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8 The Projection object Some of the properties of the projection object are dependent on the microscope's optical mode. There are 5 types of mode dependencies (the two last of which are only introduced by functions created for easier handling of the modes) : Type A differentiates between 5 TEM modes for every lens program: • diffraction LAD (the mode is reached, when the projector is switched to diffraction from LM imaging) • diffraction D (the mode is reached, when the projector is switched to diffraction from imaging with

higher magnifications) • imaging with low magnifications (LM) • imaging with higher magnification and illumination system in microprobe mode • imaging with higher magnification and illumination system in nanoprobe mode Type B differentiates between 2 modes (irrespective, of whether microscope is switched into diffraction or imaging): • low ‘magnification’ (LM, LAD) • higher ‘magnification’ (Mi...Mh, D) Type C differentiates between 3 modes: • low ‘magnification’ (LM or LAD), • higher ‘magnification’ (Mi, SA, Mh, D) in nanoprobe • higher ‘magnification’ (Mi, SA, Mh, D) in microprobe Type D differentiates between 3 modes: • diffraction LAD • diffraction D • imaging Type E differentiates between 6 modes: • the four imaging submodes LM, Mi, SA, Mh • the two diffraction submodes LAD, D If a transition between modes occurs, then the properties will seem to have changed their values, which reflects their existence per mode. Property Description mode independent Mode [ProjectionMode], read/write

Main mode of the projection system (either imaging or diffraction). SubMode [ProjectionSubMode], read only

Submode of the projection system (either LM, Mi, ..., LAD or D). The imaging submode can change, when the magnification is changed.

SubModeString [String], read only Submode of the projection system, given as a string. To be used as alternative to ‘SubMode’.

LensProgram [LensProg], read/write The lens program setting (currently EFTEM or Regular). This is the third property to characterize a mode of the projection system.


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Magnification [Double], read only The magnification value as seen by the plate camera. Use the ‘MagnificationIndex’ to change it, since the magnification can only be changed in discrete steps and the values may vary. 0, if microscope is in diffraction.

MagnificationIndex [Long], read/write The magnification values are indexed (from minimal to maximal magnification). Increasing or decreasing the index is what the magnification button on the hand panel does. In the process the imaging ‘SubMode’ may change. Note: On some microscopes the magnification ranges of LM and Ml submodes may overlap. Thus at this submode border the magnification value does not necessarily rise with increasing index. 0, if microscope is in diffraction.

ImageRotation [Double], read only The rotation of the image or diffraction pattern on the fluorescent screen with respect to the specimen. Units: radians.

DetectorShift [ProjectionDetectorShift], read/write Sets the extra shift that projects the image/diffraction pattern onto a detector.

DetectorShiftMode [ProjDetectorShiftMode], read/write This property determines, whether the chosen DetectorShift is changed when the fluorescent screen is moved down.

mode dependency type A Focus [Double], read/write

Focus setting of the currently active mode. Range: maximum between -1.0 (= underfocussed) and 1.0 (= overfocussed), but the exact limits are mode dependent and may be a lot lower.

Defocus [Double], read/write Defocus value of the currently active mode. Changing ‘Defocus’ will also change ‘Focus’ and vice versa. ‘Defocus’ is in physical units (meters) and measured with respect to a origin that can be set by using ‘ResetDefocus()’.

ObjectiveExcitation [Double], read only The excitation of the objective lens in percent.

mode dependency type B CameraLength [Double], read only

The camera length as seen by the plate camera. Use the ‘CameraLengthIndex’ to change it, since it can only be changed in discrete steps and the values may vary, for example with high tension. always 0, if microscope is in imaging.

CameraLengthIndex [Long], read/write The camera length values are indexed (from minimal to maximal camera length). Setting the Index thus is equivalent to setting the camera length. The series of camera length values for LAD and for D are not identical. always 0, if microscope is in imaging.

ObjectiveStigmator [Vector], read/write The objective stigmator setting. Range: -1.0 to +1.0 for x and y.

DiffractionStigmator [Vector], read/write The diffraction stigmator setting. Range: -1.0 to +1.0 for x and y.


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DiffractionShift [Vector], read/write The diffraction pattern shift with respect to the origin that is defined by alignment. Units: radians.

ImageShift [Vector], read/write The image shift with respect to the origin that is defined by alignment. Units: meters.

mode dependency type C ImageBeamShift [Vector], read/write

Image shift with respect to the origin that is defined by alignment. The apparent beam shift is compensated for, without affecting the Shift-property of the Illumination-object. Units: meters. Attention: Avoid intermixing ImageShift and ImageBeamShift, otherwise it would mess up the beam shift (=Illumination.Shift). If you want to use both alternately, then reset the other to zero first.

ImageBeamTilt [Vector], read/write Beam tilt with respect to the origin that is defined by alignment (rotation center). The resulting diffraction shift is compensated for, without affecting the DiffractionShift-property of the Projection object. For proper operation requires calibration (alignment) of the Beam Tilt - Diffraction Shift (for more information, see a0050100.htm on the TEM software installation CD under privada\beamtiltdiffshift). Units: radians. Attention: Avoid intermixing Tilt (of the beam in Illumination) and ImageBeamTilt. If you want to use both alternately, then reset the other to zero first.

mode dependency type D ProjectionIndex [Long], read/write

This index always contains a value. It corresponds to the camera length index or the magnification index, dependent on the microscope mode.

mode dependency type E SubModeMinIndex [Long], read only

The minimum ProjectionIndex of the current submode. Check this if you want to change the ProjectionIndex or MagnificationIndex but do not want to leave the submode.

SubModeMaxIndex [Long], read only The maximum ProjectionIndex of the current submode. Check this if you want to change the ProjectionIndex or MagnificationIndex but do not want to leave the submode.

Method Description ResetDefocus() [Void]

Resets the current ‘Defocus’ to 0 nm. This does not change the ‘Focus’ value (the focussing lens current). Use it when the image is properly focussed to adjust the ‘Defocus’ scale.

ChangeProjectionIndex ([in] Long Steps)

Changes the current Index by ‘Steps’.

Normalize ([in] ProjectionNormalization Norm)

[Void] Normalizes the objective lens or the projector lenses, dependent on the choice of ‘Norm’.


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8.1 Projection object constants

8.1.1 Enum ProjectionMode Value Description pmImaging Projector in imaging mode pmDiffraction Projector in diffraction mode

8.1.2 Enum ProjectionSubMode Value Description psmLM Imaging mode, low magnification psmMi Imaging mode, lower intermediate magnification range psmSA Imaging mode, high magnification psmMh Imaging mode, highest magnification range psmLAD Diffraction, LAD mode (the mode entered from LM imaging) psmD Diffraction mode as entered from higher magnification imaging modes

8.1.3 Enum LensProg Value Description lpRegular The default lens program lpEFTEM Lens program used for EFTEM (energy-filtered TEM)

8.1.4 Enum ProjectionDetectorShift Value Description pdsOnAxis Does not shift the image/diffraction pattern pdsNearAxis Shifts the image/diffraction pattern onto a near-axis detector/camera pdsOffAxis Shifts the image/diffraction pattern onto an off-axis detector/camera

8.1.5 Enum ProjDetectorShiftMode Value Description pdsmAutoIgnore The 'DetectorShift' is set to zero, when the fluorescent screen moves

down. When it moves up again, what happens depends on what detector TEM thinks is currently selected. Take care!.

pdsmManual The detectorshift is applied as it is chosen in the 'DetectorShift'-property pdsmAlignment The detector shift is (temporarily) controlled by an active alignment

procedure. Clients cannot set this value. Clients cannot set the 'DetectorShiftMode' to another value either, if this is the current value. They have to wait until the alignment is finished.


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8.1.6 Enum ProjectionNormalization Value Description pnmObjective Normalize objective lens pnmProjector Normalize Diffraction, Intermediate, P1 and P2 lenses pnmAll Normalize objective, diffraction, intermediate, P1 and P2 lenses


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9 The BlankerShutter object The BlankerShutter object has only one property, ShutterOverrideOn. This property can be used in cryo-electron microscopy to burn ice off the specimen while blocking the beam from hitting the CCD camera. When the shutter override is true, the CCD camera no longer has control over the microscope shutters. The shutter below the specimen is closed. Whether the beam is on the specimen is determined by the BeamBlanked property of the Illumination object. Property Description ShutterOverrideOn [Boolean], read/write

Determines the state of the shutter override function. WARNING: Do not leave the Shutter override on when stopping the script. The microscope operator will be unable to have a beam come down and has no separate way of seeing that it is blocked by the closed microscope shutter. Suggested procedure: 1. Blank the beam using the BeamBlanked property of the Illumination object. 2. Switch the shutter override on. 3. If necessary, wait for a short delay (one second) to allow the system to execute the shuttering. 4. Unblank the beam (the CCD no longer has control). 5. Wait for the time necessary to burn off the ice (sleep, Windows timer, ...) 6. Blank the beam. 7. Switch the shutter override off. 8. If necessary, wait for a short delay (one second) to allow the system to switch the shuttering back to

normal. 9. Unblank the beam (the CCD now has control again).


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10 The Vacuum object Property Description Status [VacuumStatus], read only

Status of the vacuum system (see below). ColumnValvesOpen [WordBool], read/write

The status of the column valves. PVPRunning [WordBool], read only

Checks whether the prevacuum pump is currently running (consequences: vibrations, exposure function blocked or should not be called).

Gauges Collection of gauge objects, giving information about the actual pressures. Method Description RunBufferCycle() [Void]

Runs a pumping cycle to empty the buffer. This function may take quite some time, so be careful when using it in a single-threaded application with user interface. (The program appears to be hanging while it waits for the function to return.)

10.1 Constants:

10.1.1 Enum VacuumStatus: Value Description vsUnknown Status of vacuum system is unknown vsOff Vac uum system is off vsCameraAir Camera (only) is aired vsBusy Vacuum system is busy, that is: on its way to ‘Ready’, ‘CameraAir’, etc. vsReady Vacuum system is ready vsElse Vacuum is in any other state (gun air, all air etc.), and will not come back

to ready without any further action of the user


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11 The Stage object Property Description Status [StageStatus], read only

The current state of the stage. Check this to determine the whether the stage is ready to perform a ‘GoTo’ or a ‘MoveTo’.

Position [StagePosition], read only The current position of the stage. (Changing the position can be done in various ways. Use the ‘GoTo’ and ‘MoveTo’ Methods described below.)

Holder [StageHolderType], read only The current specimen holder type.

AxisData ([in] mask StageAxes) [StageAxisData], read only Interface to a StageAxisData object that contains the minimum and maximum values available for the particular axis.

Method Description GoTo ([in] StagePosition NewPosition, [in] StageAxis AxesBits)

[Void] Makes the holder move to the position given by ‘NewPosition’. ‘AxesBits’ contains information about which axes are to be used to perform the movement. This is useful to suppress unnecessary movements of the holder due to fluctuations in position measurement. Also, you do not have to bother to give the correct parameters for axes-settings that you do not want to change. (In Delphi, this function is automatically renamed to GoTo_, because GoTo is a reserved keyword.)

MoveTo ([in] StagePosition NewPosition, [in] StageAxis AxesBits)

[Void] Makes the holder move in a way that all possible positions can be reached without touching the objective pole. Actually, the following sequence of movements is performed (a, b denote alpha and beta tilts, x,y,z the x,y and height positions, large letters the new position): b->0, a->0, z->Z, x,y->X,Y, 0->A, 0->B. If ‘AxesBits’ determines that a tilt axis (A or B) is not to be included into the movement, then it will still move, but return to its old setting after X,Y,Z have finished their movement.

11.1 StageAxisData object The StageAxisData object can be used to retrieve the minimum and maximum values allowed for a particular stage axis. This is really only useful for the dual-axis tomography holder as there the b tilt (which controls the flipflop motion) has hardware-dependent values. In all other cases the minimum and maximum values are as follows: • X and Y -1000 to +1000 (micrometers) • Z -375 to 375 (micrometers) • a -80 to +80 (degrees) • b -29.7 to +29.7 (degrees)


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Property Description MinPos [Double], read only

The lowermost value of the stage position allowed on the axis. MaxPos [Double], read only

The uppermost value of the stage position allowed on the axis. UnitType [MeasurementUnitType], read only

The unit (meters or radians) of the MinPos and MaxPos values

11.2 Stage object constants

11.2.1 Enum StageStatus Methods Description stReady The stage is ready (capable to perform all position management functions) stDisabled The stage has been disabled either by the user or due to an error. stNotReady The stage is not (yet) ready to perform position management functions for

reasons other than already accounted for by the other constants stGoing The stage is performing a ‘GoTo()’ stMoving The stage is performing a ‘MoveTo()’ stWobbling The stage is wobbling

11.2.2 Enum StageAxes The ‘GoTo’ and ‘MoveTo’ methods require a parameter (of type long) that contains bitwise information about which axis is to be involved in the movement. The bit order is BAZYX , so bit 0 contains the information about whether the X-axis is involved, bit 4 contains the information about the B axis. The members of the ‘StageAxes’ enumeration can be used instead of calculating with bits. You can combine them by bitwise ‘OR’s (i.e. in JScript: MyAxBits = (axisXY | axisA | axisB) to allow the X,Y,A,B axis to move, but leave the Z constant. Value Description axisX (= 1) Use X-axis axisY (= 2) Use Y-axis axisXY (= 3) Use X- and Y-axis axisZ (= 4) Use Z-axis axisA (= 8) Use alpha tilt-axis axisB (=16) Use B-axis, usually the beta tilt, but on Dual-Axis Tomography holders this

is the second (rotation or flip-flop) axis.


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11.2.3 Enum StageHolderType Value Description hoInvalid The ‘invalid’ holder. No holder has been selected yet or the current

selection has become invalid hoSingleTilt Single tilt holder hoDoubleTilt Double tilt holder hoNone Holder is removed hoPolara Non-removable Polara holder hoDualAxis Dual-axis tomography holder

11.2.4 Enum MeasurementUnitType Value Description MeasurementUnitType_Unknown Unknown unit type MeasurementUnitType_Meters Unit type is meters (linear axes) MeasurementUnitType_Radians Unit type is radians (tilt axes)


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12 The AutoLoader object The AutoLoader is a system that allows specimen loading/unloading through cartridges from a cassette. Property Description AutoLoaderAvailable [Boolean], read only

Returns whether the AutoLoader is available on the microscope. Numb erOfCassetteSlots [Long], read only

The number of cassette slots in a cartridge. SlotStatus ([in] slot Long)

[CassetteSlotStatus], read only The status of the slot specified.

Method Description LoadCartridge ([in] fromSlot Long)

Loads the cartride in the given slot into the microscope.

UnloadCartridge() [Void] Unloads the cartridge currently in the microscope and puts it back into its slot in the cassette.

PerformCassetteInventory() [Void] Performs an inventory of the cassette (determines which slots are empty or occupied).

12.1 Cassette slot status constants

12.1.1 Enum CassetteSlotStatus Value Description CassetteSlotStatus_Unknown Cassette slot status has not been determined CassetteSlotStatus_Occupied Cassette slot contains a cartridge CassetteSlotStatus_Empty Cassette slot is empty CassetteSlotStatus_Error Cassette slot generated an error


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13 The TemperatureControl object The Temperature controller is a system that monitors temperatures and refrigerant levels for the AutoLoader and column dewars. Property Description TemperatureControlAvailable [Boolean], read only

Returns whether the Temperature controller is available on the microscope.

RefrigerantLevel([in] rl: RefrigerantLevel)

[Double], read only The level of coolant of the dewar specified.

Method Description ForceRefill [Void]

Forces a refill of the refrigerant. Notes: 1. This function takes considerable time to execute. 2. If the refrigerant level in the dewar specified is above 70% then ForceRefill will not do anything and return almost immediately.

13.1 Refigerant level constants

13.1.1 Enum RefrigerantLevel Value Description RefrigerantLevel_AutoLoaderDewar The dewar of the AutoLoader RefrigerantLevel_ColumnDewar The dewar on the microscope column RefrigerantLevel_HeliumDewar The liquid-Helium dewar


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14 The (Plate) Camera object Property Description MainScreen [ScreenPosition], read/write

The position of the main screen. Setting the main screen to the upward position also moves the small screen up (out).

IsSmallScreenDown [Boolean], read only The Position of the small screen. It cannot be moved down (in) by a software command. It moves up (out) with the main screen.

Stock [Long], read only The number of plates still on stock.

MeasuredExposureTime [Double], read only The measured exposure time (identical to the time displayed in the UI, given in seconds).

ManualExposureTime [Double], read/write The time setting used for ‘manual exposures’, in seconds.

ManualExposure [Boolean], read/write Flag that specifies whether ‘ManualExposureTime’ time will be the time used for an exposure (if set to "true") or the automatic time (if set to "false").

FilmText [String], read/write The text printed on the next exposure(s). Maximum 96 characters are accepted. If the string is longer, it is truncated.

ExposureNumber [Long], read/write Contains the sum of a five digit Long (accounting for a five digit image number) and 100000 times the ASCII code of one of the following characters: 0 to 9, A to Z (accounting for the first number or identifier). Use integer division and modulo to separate these components. Setting of of this property may be blocked by a supervisor.

UserCode [String], read/write A three-letter usercode that will be shown on the plate exposure.

PlateLabelDateType [PlateLabelDateFormat], read/write Specifies whether the date is shown on the plate exposure and which format will be used.

ScreenDimText [String], read/write The text shown on the screen when it is dimmed. The string may contain carriage return - linefeeds to distribute text over the three lines that are available for screendim text.

ScreenDim [Boolean], read/write Dims/undims the screen of the computer on which the TEM server is running (for example useful during exposures).

ScreenCurrent [Double], read only The current measured on the fluorescent screen (units: Amperes).


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Method Description TakeExposure() [Void]

Triggers a single plate exposure, using the actually selected time type. The function will start the same process as does the exposure button on the control pad. For example, if necessary it will move the screen up, make the exposure and move the screen down again.

14.1 Camera object constants

14.1.1 Enum ScreenPosition Value Description spUnknown Position of main screen is not known, for example during

movement spUp Main screen is up (in which case the small screen will also be up) spDown Main screen is down

14.1.2 Enum PlateLabelDateFormat Value Description dtNoDate No date is displayed dtDDMMYY The date is displayed as day-month-year (two numbers for each),

separated by hyphens dtMMDDYY The date is displayed as month/day/year, separated by slashes dtYYMMDD The date is displayed as year.month.day, separated by periods


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15 The Acquisition object The Acquisition object gives access to CCD and STEM image acquisition. Notes: • In order for acquisition to be available TIA (TEM Imaging and Acquisition) must be running

(even if you are using DigitalMicrograph as the CCD server). • If it is necessary to update the acquisition object (e.g. when the STEM detector selection on

the TEM UI has been changed), you have to release and recreate the main microscope object. If you do not do so, you keep accessing the same acquisition object which will not work properly anymore.

The Acquisition object exposes the CCDCameras and STEMDetectors together with a number of general methods. The main entry point to the acquisition functionality is the Acquisition interface. From this interface, it is possible to query all available (i.e. installed on the system) acquisition devices: CCD cameras and STEM detectors. For each of these devices it is possible to retrieve an information object, which tells something about (hardware) parameters of the device, for instance, the dimensions of the CCD chip (in pixels). For each of the CCD cameras, an acquisition parameters object can be retrieved to change the default acquisition settings (i.e. exposure time). In contrast, the STEM acquisition parameters are not device-specific but instead apply to all detectors.

Acquisition

Cameras

Detectors Info

AcqParams

Camera ...

Camera 1

Detector ...

Detector 1

Info

AcqParams


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In order to acquire an image from an acquisition device (CCD or STEM detector), the following steps need to be taken: • TIA (TEM Imaging and Acquisition) application must be running. • Get the Acquisition object from the main instrument interface of the Standard Scripting component. • Query available CCD cameras and STEM detectors, looking for the device from which you would like

to acquire an image. Note: in the current version of Standard Scripting only devices which are selected in the Microscope User Interface will be available in the query. In future versions of the Standard Scripting we envision the possibility to query and select any acquisition device available in the system, without the need for human interaction with the Microscope User Interface. The software interfaces of the Acquisition objects are already prepared for such extensions.

• Add the queried acquisition device to the list of devices in the Acquisition object. Internally, the Acquisition object maintains a list of acquisition devices on which is has to perform image acquisition when AcquireImages() method is called. You can manipulate this list of devices through Acquisition object interface.

• Acquire images by calling AcquireImages() method on the Acquisition object. The method will acquire the images from all the devices currently found in its internal list of acquisition devices (acquisition is performed sequentially). When acquisition is finished, the method returns an array of acquired images.

• Each of the acquired images has a name property which returns the device name the image was acquired from. The actual image data can be retrieved as a safe-array from the Image object.

15.1 Usage example in a pseudo-programming language // get Acquisition object Instrument instrObj = new TEMScripting.Instrument; Acquisition acqObj = instrObj.Acqusition(); // Query available acquisition devices CCDCameras ccdCollection = acqObj.CCDCameras; For (index = 0; index < ccdCollection.Count(); index++) { CCDCamera ccd = ccdCollection.At(index); Print (“found CCD camera: %s“, ccd.Name()); } STEMDetectors stemCollection = acqObj.STEMDetectors; For (index = 0; index < stemCollection.Count(); index++) { STEMDetector stem = stemCollection.At(index); Print (“found STEM Detector: %s”, stem.Name()); } // Acquire an image from “WAC CCD” camera acqObj.AddAcqDeviceByName(“WAC CCD”); AcqImages imageCollection = acqObj.AcquireImages(); // since we added only one acquisition device, there will be only // one image in the image collection AcqImage img = imageCollection.At(0); // get access to pixels Array imgData = img.AsSafeArray(); // do something with image data&ldots;


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Property Description Cameras [Object]

Interface to the CCD cameras. Detectors [Object]

Interface to the STEM detectors. Method Description AddAcqDevice ([in] pDevice )

[Void] Adds an acquisition device to the list of active acquisition devices.

AddAcqDeviceByname ([in] deviceName String)

[Void] Adds the acquisition device with the name specified to the list of active acquisition devices.

RemoveAcqDevice([in] pDevice)

[Void] Removes the acquisition device from the list of active acquisition devices.

RemoveAcqDeviceByName([in] deviceName String)

[Void] Removes the acquisition device with the name specified from the list of active acquisition devices.

RemoveAllAcqDevices [Void] Clears the list of active acquisition devices.

AcquireImages [AcqImages] Acquires the image or images using the currently set list of acquisition devices and returns an interface to the image collection.

15.2 CCDCameras CCDCameras contains all available CCD cameras on the microscope. Note: In order for a camera to be "available" it must be selected in the microscope user interface. Currently it is not possible to do this selection through scripting.

15.2.1 CCDCamera Property Description Info [Object]

Interface to the information on the CCD camera. AcqParams [Object]

Interface to the acquisition parameters of the CCD camera.


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15.2.1.1 CCDCameraInfo Property Description Name [String], read only

A string with the name of the CCD as it appears in the microscope's user interface.

Height [Long], read only The height of the CCD camera in pixels.

Width [Long], read only The width of the CCD camera in pixels.

PixelSize [Vector], read only The physical size in X and Y direction of the CCD pixels (that is, the size as they are on the CCD chip, typically a value in the range 15 to 30 micrometers).

Binnings [SafeArray], read only An array with the binning values supported by the CCD.

ShutterModes [SafeArray], read only An array with the shutter modes supported by the CCD.

ShutterMode [AcqShutterMode], read/write The currently selected shutter mode of the CCD.

Notes: • Generally the binning values and exposure time are related (quadratically). If you increase the

binning from 1 to 4, you normally have to decrease the exposure time by 4² in order to prevent CCD saturation. This relation does not exist for the Eagle CCD camera, which uses an optimised scheme where the ratios are as given in the table below.

• The ShutterMode of the CCD camera refers to the shutter(s) being used. The availability of different shutters is dependent on the type of CCD camera (e.g. SIS cameras have none, Gatan cameras typically have pre- and post-specimen, the Eagle has pre- and post-specimen and can also use both shutters simultaneously). The shutter mode is a global microscope setting, so if this is changed in a script, you will see the change back in the TEM User Interface (CCD/TV General flap-out). Make sure to retrieve the setting befor running a script and setting it back when closing down, so the user is not confronted with unintended changes to the microscope settings.

Eagle CCD type Binning values Exposure time 2k 1 : 2 : 4 1 : 0.5 : 0.125 4k 1 : 2 : 4 : 8 1 : 1 : 0.5 : 0.125


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15.2.1.2 CCDAcqParams Property Description ImageSize [AcqImageSize], read/write

The size of the image to be collected. ExposureTime [Double], read/write

The exposure time in seconds. Binning [Long], read/write

The binning value to be used for the image acquisition. Make sure the value is one of the supported binning values.

ImageCorrection [AcqImageCorrection], read/write The type of correction to be applied. Bias/gain correction can only be applied if this has been done in the CCD server prior to scripting.

ExposureMode [AcqExposureMode], read/write The currently selected exposure mode of the CCD.

MinPreExposureTime [Double], read only The minimum available pre-exposure time in seconds.

MaxPreExposureTime [Double], read only The maximum available pre-exposure time in seconds.

MinPreExposurePauseTime [Double], read only The minimum available pre-exposure pause time in seconds.

MaxPreExposurePauseTime [Double], read only The maximum available pre-exposure time in seconds.

PreExposureTime [Double], read/write The pre-exposure time in seconds.

PreExposurePauseTime [Double], read/write The pre-exposure pause time in seconds.

Note: Pre-exposures can only be be done when the shutter mode is set to "Both". This is only available for Eagle CCD cameras. When pre-exposure is used, the specimen is illuminated for the pre-exposure time defined. The pre-exposure is done by opening the pre-specimen shutter while the post-specimen shutter remains closed to prevent electrons from falling on the CCD. A pre-exposure may help to stabilize specimens (e.g. when charging). When a pre-exposure pause is used, there is a delay (of the defined pause time) inserted between the pre-exposure and the actual CCD exposure. During this delay both shutters are closed. The exposure mode is NOT a global microscope setting, so if this is changed in a script, you will NOT see the change back in the TEM User Interface (CCD/TV General flap-out).


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15.3 STEMDetectors STEMDetectors contains all available STEM detectors on the microscope.

15.3.1 STEMDetectors Property Description AcqParams [Object]

Interface to the acquisition parameters of the STEM detectors. Acquisition parameters for STEM are generic and not bound to a particular detector.

Note: In order for a detector to be "available" it must be selected in the microscope user interface. Currently it is not possible to do this selection through scripting.

15.3.2 STEMDetector Property Description Info [Object]

Interface to the information on the STEM detector.

15.3.2.1 STEMDetectorInfo Note: The maximum size of the unbinned STEM image is 2048² pixels. Property Description Name [String], read only

A string with the name of the STEM detector as it appears in the microscope's user interface.

Brightness [Double], read/write The brightness setting of the STEM detector.

Contrast [Double], read/write The contrast setting of the STEM detector.

Binnings [SafeArray], read only An array with the binning values supported by the STEM detector. Technically speaking these are "pixel skipping" values, since in STEM we do not combine pixels as a CCD does, but other than that these values work the same way as in CCD acquisition (e.g. half frame with binning 4 gives a 256² image on a 2k CCD as well as on the STEM).


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15.3.2.2 STEMAcqParams Property Description ImageSize [AcqImageSize]

The size of the image to be collected. DwellTime [Double]

The pixel dwell time in seconds. The frame time equals the dwell time times the number of pixels plus some overhead (typically 20%, used for the line flyback).

Binning [Long] The binning value to be used for the image acquisition. Make sure the value is one of the supported binning values.

15.4 AcqImages The AcqImages contains all images acquired (through the AcquireImages; it does not contain a backlist of all previous recordings).

15.4.1 AcqImage Property Description Name [String], read only

The name of the detector (CCD camera or STEM detector) used to acquire the image.

Width [Long], read only The width (in pixels) of the image.

Height [Long], read only The height (in pixels) of the image.

Depth [Long], read only The maximum number of bits in the image. The image as retrieved always has 16 bits, but the original - CCD - image may have had less depth, dependent on the CCD camera used.

AsSafeArray [SafeArray] read only A SafeArray with the pixel values (Long) of the image.

15.5 Acquisition Constants

15.5.1 Enum AcqImageSize The image sizes supported by the CCD and STEM acquisition are a subset of what is usually available on the microscope. They have been chosen so they are supported by all CCD cameras. Value Description AcqImageSize_Full Image size covers the whole CCD or STEM range AcqImageSize_Half Image size covers half of the CCD or STEM range, centered AcqImageSize_Quarter Image size covers one quarter of the CCD or STEM range,

centered


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15.5.2 Enum AcqImageCorrection Property Description AcqImageCorrection_Unprocessed CCD images are uncorrected AcqImageCorrection_Default CCD images are bias- and gain-corrected

15.5.3 Enum AcqShutterMode Property Description AcqShutterMode_PreSpecimen The pre-specimen shutter (blanking before the specimen) is used) AcqShutterMode_PostSpecimen The post-specimen shutter (blanking after the specimen) is used AcqShutterMode_Both Both pre- and post-specimen shutters are used together

15.5.4 Enum AcqExposureMode Property Description AcqExposureMode_None Default setting AcqExposureMode_Simultaneous The pre- and post-specimen shutter are used together AcqExposureMode_PreExposure Same as previous but before the actual CCD exposure the

specimen is illuminated for the duration of the pre-exposure time set

AcqExposureMode_PreExposurePause Same as previous but after the pre-exposure and before the actual CCD exposure is a pause fro the duration of the pre-exposure pause time set.

15.6 SafeArray handling SafeArrays (or variant arrays) are the standard way of marshaling data arrays through COM. In general you should look up the documentation provided with the programming language you are using, but below is some additional information. For using SafeArrays int Delphi, see the code of the example program provided. C++ Very important: Make sure the declaration for the image array is _variant_t, otherwise you run the risk that C++ clears the data before you can access them. The code snippet below gives an example on how you can access the pixels. if (m_pCamServer) { HRESULT hr = m_pCamServer->AcquireImages(&m_pAcquisition); if (SUCCEEDED(hr)) { int n = m_pAcquisition->Count; if (n == 0) ShowMessage(_T("No images here")); else { m_pAcqImage = m_pAcquisition->Item[0]; if (m_pAcqImage) { int h = m_pAcqImage->get_Height(); int w = m_pAcqImage->get_Width();


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_variant_t img = m_pAcqImage->AsSafeArray(); CComSafeArray<short> data; data.Attach(img.Detach().parray); ATL::CWindow pWindow = NULL; pWindow = GetDlgItem(IDC_EDIT1); CString txt,txt2; txt = ""; for (int i = 0; i<25; i++){ short pixel = data[i]; txt2.Format(_T("%s,%i"),txt,pixel); txt = txt2; } pWindow.SetWindowText(txt); } } else ShowMessage(_T("Cannot acquire image(s)")); }


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16 The UserButton object ‘UserButton’ objects are members of the ‘UserButtons’ collection: var MyL1 = MyTem.UserButtons("L1") retrieves an object that represents -and references to- the user button L1 (supposing that ‘MyTem’ is an instance of an ‘Instrument’ object). Property Description Name [String], read only

The name of the button (i.e. "L1", "L2", etc.). Label [String], read only

The current label that is assigned to the button (not necessarily assigned by your script)

Assignment [String], read/write The script-assigned label of the button. Setting this property directs the events generated by use of the button to the script. If the script has made an assignment, then ‘Assignment’ will equal ‘Label’, if not temporarily overwritten by somebody else, for example an alignment procedure. (In that case, no events will be received.) Default value = "". Setting this property to an empty string or a string consisting only of blanks (that would be invisible in the UI) restores the situation that was there, before the script took control over the button.

Event Description Pressed() Event that is raised when the button is pressed.


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17 The utility objects ‘Utility’ objects are used to handle compound (or multi-dimensional) properties. They can be created by your script (except for the ‘gauge’ that is ‘read only’), but since they are specific TEM objects, this creation is also handled by the main ‘Instrument’ object. In JScript this may look as follows (supposed that your ‘Instrument’ is named ‘MyTem’: var MyVector = new MyTem.Vector(2.0,3.0) This code creates a TEM vector object MyVector that is initialized with (2.0,3.0). Of course, reading a compound property, for example var MyOtherVector = MyTem.Gun.Shift will also return an instance of an utility object, in this case a vector containing the actual gun shift. Utility object Description Vector Object used to access 2-dimensional compound properties StagePosition Object used to read and set the position of the stage Gauge Object used to read information about pressures and the status of the

corresponding measurement devices

17.1 The Gauge (utility object) The gauge objects are used to retrieve information about the vacuum system measurement devices and the actual pressures measured with them. Since this a ‘read only’ task, you cannot create instances of this utility object yourself. ‘Gauge’ objects are always members of the vacuum systems ‘Gauges’ collection: var MyGauge = MyTem.Vacuum.Gauges("P1") retrieves the latest pressure and status information for the vacuum buffer. (‘P1’ is the name of the measurement device associated with the buffer, as you can see from the TEM vacuum overview. ) Note: On the Tecnai pressures that are read out through IGPcurrents are also accessible through ‘virtual’ gauge-elements named ‘Pxx’, where xx represents a number. Currently the following assignments are used on Tecnai: IGP1=P4; IGP2=P6, IGP4=P8. On the Titan the gauges have the same name as the pump itself. Property Description Name [String], read only

Name of the gauge. Status [GaugeStatus], read only

The status of the gauge. It is important for the interpretation of the pressure values.

Pressure [Double], read only Last measured pressure for this gauge. Units: Pascal

PressureLevel [GaugePressureLevel], read only Indicates, in which range the pressure lies. Actions of the vacuum system depend on this property.


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Method Description Read [Void]

Forces a read of the pressure level. Execute before retrieving the pressure, otherwise the pressure may not be up-to-date.

17.1.1 Gauge object constants

17.1.1.1 Enum GaugeStatus: Value Description gsUndefined No information on the gauge available gsUnderflow Underflow, pressure is lower than can be measured with this gauge, the

measurement is not interpretable gsOverflow Overflow, pressure is higher than can be measured with this gauge, the

measurement is not interpretable gsValid Valid, the pressure measurement is valid and interpretable gsInvalid Invalid, the pressure measurement is invalid and not interpretable

17.1.1.2 Enum GaugePressureLevel The pressure range for a every gauge is divided into four regions (low.....high). Depending on the gauge, a change in pressure - and thus a switching from one level to another - may result in an action of the vacuum system. (Example: On the Tecnai a buffer cycle will start, when the gauge ‘P1’ reaches plGaugePressurelevelMediumHigh.) Value Description plGaugePressurelevelUndefined Gauge is not active plGaugePressurelevelLow Lowest pressure range plGaugePressurelevelLowMedium Low to medium pressure range plGaugePressurelevelMediumHigh Medium to high pressure range plGaugePressurelevelHigh Highest range


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17.2 The StagePosition (utility object) Property Description X [Double], read/write

Position of X-axis, in meters. Y [Double], read/write

Position of Y-axis, in meters. Z [Double], read/write

Position of Z-axis, in meters. A [Double], read/write

Position of A-axis (alpha-tilt), in radians B [Double], read/write

Position of B-axis (beta-tilt), in radians Method Description GetAsArray ([out] Double[5] Position)

[void] Returns the properties of the StagePosition as an array of 5 doubles. The ordering is [X,Y,Z,A,B].

SetAsArray ([in] Double[5] Position)

[void] Sets the properties of the StagePosition from an array of 5 doubles. The ordering is [X,Y,Z,A,B].

17.3 The Vector (utility object) This is a general object used to retrieve and set two-dimensional properties, the components of which have to be read or set simultaneously. The vector object must be used for tilt, shift, and stigmator properties. X and Y must then be given in the appropriate units (ie radians, meters or logical). Property Description X [Double], read/write;

The x value. Y [Double], read/write;

The y value.


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18 TEM constants TEM-specific constants are defined in the following enumerations that are used by the objects in the right column of the table:

enum object AcqImageCorrection CCDAcqParams object AcqImageSize CCDAcqParams object,

STEMAcqParams object CassetteSlotStatus AutoLoader object DarkFieldMode Illumination object GaugeStatus Gauge object GaugePressureLevel Gauge object HightensionState Gun object IlluminationMode Illumination object IlluminationNormalization Illumination object InstrumentMode InstrumentModeControl object LensProg Projection object MeasurementUnitType StageAxisData object MinicondenserMode Illumination object PlateLabelDateFormat Camera object ProjDetectorShiftMode Projection object ProjectionDetectorshift Projection object ProjectionMode Projection object ProjectionNormalization Projection object ProjectionSubMode Projection object RefrigerantLevel TemperatureControl object ScreenPosition Camera object StageHolderType Stage object StageStatus Stage object StageAxes Stage object TEMScriptingError All VacuumStatus Vacuum object


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19 TEM error codes There are TEM-specific error codes available for errors that are due to microscope specific error conditions and specific errors raised by the TEM software. There will also be support for retrieving more information about the error that occurred (descriptive string, source etc.). How this can be done is described in the chapter "Scripting in various languages". These error codes are the following:

19.1 Enum TEMScriptingError: Value Description E_VALUE_CLIP a parameter exceeded its possible range, it is clipped to its highest or

lowest allowed value E_OUT_OF_RANGE a parameter exceeded its possible range. The command is ignored. E_NOT_OK another error occured. Query the error object for more information. Only a few functions return these error codes. Mostly, standard Microsoft OLE-error codes are returned. However, they are used in a specific way. These standard codes are displayed below in hexadecimal format. Scripting languages may translate the standard error values into others, as indicated in the table below. In Delphi apparently the error codes cannot easily be retrieved, but the standard describing string is well available. These descriptions are pretty much the same for all languages and close to what the meaning of the error in the TEM-context is. Only for E_UNEXPECTED you will get a message like "catastrophic failure", which does not quite fit to the error description given in the table. Error C++ JScript VBScript VB Description E_UNEXPECTED 8000FFFF 8000FFFF 8000FFFF 8000FFFF This error is generally raised,

if you do a function call that is not allowed in the current state of the microscope, and you could in principle have known about this because the information was available.

E_NOTIMPL 80004001 800A01BD

1BD 1BD Function not yet implemented (Should not happen for TEM scripting functions).

E_INVALIDARG 80070057 800A0005 5 5 The arguments you supplied for a function call are of the wrong type or out of range

E_ABORT 80004004 80004004 80004004 11F An action was started (i.e. an exposure), but then aborted

E_FAIL 80004005 80004005 80004005 80004005 Unspecified failure. E_ACCESSDENIED 80070005 800A0046 46 46 If this error is raised a TEM

component cannot be accessed, either because it is not there or because a hardware or software of this component is in an error state.


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20 TEM-specific issues When writing scripts for the TEM, it is of course important to keep in mind that there is a physical microscope involved. Changing a property of an adapter object mostly results in a physical action on the actual TEM. This also means that no matter how many objects of one type are created, be it in one or also in several scripts running in parallel, they finally all correspond to the same thing. And if somebody sitting at the microscope or another script decides to change microscope parameters, the properties of your objects may change without you knowing. Apart from this perhaps trivial -but easily forgotten- aspect there are some details about the TEM's inner structure that may be necessary to know when programming. We already introduced the

• Microscope modes. This concept had to be known in order to understand the behavior of some objects properties. Occasionally you may run into trouble, because the TEM microscope does some things for you that you are not aware of:

• Normalizations • TEM sessions

It may also be worthwhile to have a look at the notion of • Synchronous functions

Finally, consider • Setting parameters out of range

20.1 Synchronous functions Although adapter-functions are programmed to be synchronous, the action of setting a property to a certain value may in some special cases trigger further reactions of the microscope. These cases are:

20.1.1 Automatic normalization Certain changes in lens excitations trigger a normalization procedure that can take up to two seconds.

20.1.2 Pole touch During movement of the stage (especially with a microscope equipped with UTWIN objective lens) a pole touch may be detected. The stage will then try to solve this problem. These are situations in which the microscope, or rather parts of it, sometimes behave as independent users themselves. This hampers the use of synchronous functions. But there are remedies. For the first situation they are explained in the section on Normalizations, the second situation can often be avoided by using ‘MoveTo’ in place of ‘GoTo’ in case you want to reach extreme positions of the stage.

20.1.3 Setting the high tension Setting the high tension or switching it is considered done, when the command is delivered to the electronics (as is in fact also the case with most other commands). However, switching and setting the high voltage may need a considerable additional time. It also has to be checked whether the action was successful.

20.2 Normalizations Many actions that change excitations of optical elements trigger normalization procedures to ensure reproducibility of the settings (since the optics contain magnetic lenses). You can examine the ‘Normalizations’ control panel in the TEM user interface to get information about when normalization procedures are automatically initiated (see image below).


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On the Tecnai these settings are user-dependent (note the checkboxes in the control cluster that allow individual settings to be made). The example in the image shows that -for this user- automatic normalizations are invoked for C1 and C2 condenser lenses, if the spotsize is changed. The automatic normalization procedures are done independently from any other action. They take about two seconds on Tecnai and five seconds on Titan. This disturbs the adapter's concept of using synchronous functions in order to allow a simple order of commands. Remedy: In scripting applications it can be helpful to disable automatic normalizations and invoke the necessary normalizations via the script, using the (synchronous) ‘Normalize()’ methods of the ‘Illumination’ and ‘Projection’ objects. Do not forget to enable autonormalizations again before ending the application! Example: MyTem.AutoNormalizeEnabled = FALSE var Illum = MyTem.Illumination Illum.SpotsizeIndex = 3 Illum.Normalize(nmCondenser) MyTem.AutoNormalizeEnabled = TRUE Please note that entering the alignment procedures via the TEM user interface overrides the setting of the ‘AutoNormalizeEnabled’-property.

20.3 TEM sessions The TEM software architecture supports the concept of sessions and user levels. If a user is logging on (starting a session), user-specific data (preferences, alignment settings etc.) are read from the registry. User rights are determined as well. In turn, some data are updated when the user logs off again. Logging on/off here means, that somebody is actually starting up/closing down the user interface of the TEM microscope, and is not to be confused with logging on/off in Windows NT. You may have noticed, that already without a session started, that is without the TEM user interface running, your script can communicate with the TEM server. But because of the facts mentioned above, you may run into problems. So, for every application that does more than passive protocolling tasks, it is strongly recommended that a TEM session is initialized before the script is executed!


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20.4 Setting parameters out of range Generally, if you set a certain property to a value that is valid by type (i.e. a double), but exceeds the physically allowed limits (i.e. setting the magnification to 2x) this property should take its maximal or minimal value. Exceptions from this rule can be expected if the action takes a lot of time and is probably erroneous anyway. In these cases an error will be raised. The stage, for example will not react on setting its x-position to +100mm, but will raise an error to indicate that something is wrong. This behavior is not yet implemented consistently in the microscope server. You will probably find properties that will not react at all, if you feed out of range parameters. To make things a little more difficult, it is not always easy to tell what the allowed range of values for a property is, because it may depend on the alignment settings of the microscope. One of the more complicated cases is the beam-deflection setting, for example. One single set of coils executes two different kinds of actions: beam shift and beam tilt. Both shift and tilt are further divided into an alignment-value (shift not accessible through the TEM scripting adapter; tilt = rotation center) and a user value (also accessible via the ‘Illumination’ object). Additionally the beam-deflection coils are affected by the alignment of the condenser stigmator. Shifts, tilts and stigmator adjustments are translated through pivot points into settings on the upper and lower deflection coils. These coils ultimately trigger an out-of-range response. This complexity makes it very difficult to predict what the actual limit is for a specific value. To determine the actual limit for a parameter that does not change on an attempt to set it to an out-of-range value, the following procedure can be used (in JScript code): //Applies to setting between -1 and +1 //moving in positive direction var step = 0.1; var CurrentValue = ...... ; // get the current setting do var CheckValue = CurrentValue; //try increase: Currentvalue = CurrentValue + step; if (Math.abs(Checkvalue - CurrentValue) < 1E-06) { //nothing happened - reduce stepsize step = step/2; } else { step = step*2; } while (Math.abs(step)< 1E-04); //value arbitrarily chosen It is advisable to rather check a property's value before relying on the success of an attempt to set it. Anyway, another script or human user could also in principle interfere at all times, so checking can never harm.


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21 Property- and function- types Throughout this help, the types of properties (and also the return types of functions) have been described in a more or less symbolic way (for example as ‘Boolean’, ‘String’, ..). These descriptions differ from programming language to programming language and may thus not be clear to everybody. The following table holds a description and a ‘translation’ to common scripting and programming languages, that use typing (JScript and VBScript doe not declare the types of variables). ‘Symbolic’ type used here

Visual Basic

Delphi C++

Description

Boolean Boolean boolean BOOL a Boolean type (having values "TRUE" and "FALSE" that are actually transmitted as -1 and 0)

String String widestring BSTR (or _bstr_t)

a wide, double-byte (Unicode) string on 32-bit Windows platforms

Long Long long long a 32 bit integer Double Double double double a 64 bit real Void -

(sub) - (procedure) void keyword specifying that there is no return-

type Object Object ComObject IDispatch* a variable that refers to an object that has

its own properties and methods. (Under water it contains a pointer to the default interface of something called a COM-object, where COM = component object model.)


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22 TEM scripting in C++ For the explanations in this chapter, it is assumed that you are going to create a scripting client using Microsoft Developer Studio (MSDEV) and the Microsoft Foundation Classes (MFC), as it is done in the example program delivered with the adapter.

22.1 Import the dynamic link library In order to be able to compile your project (at least if you use the hard types defined in the adapter) you have to include the typelib (or the DLL): #import "stdscript.tlb" named_guids using namespace TEMScripting; The above statement assumes that the typelib is in the same directory as your source files. From the typelib, the compiler will generate the stdscript.tlh and .tli files that contain all necessary definitions and a number of wrapper-functions. You will then also get the full support of the ClassView and the IntelliSense mechanism. In C++, the names of the ‘objects’ can be different from what was written in the remainder of this help, because unlike most scripting languages, C++ knows the difference between objects and interfaces implemented by them. Due to details in the adapter's implementation that are meant to make its use consistent in scripting languages, you see that most interfaces are named as are the ‘objects’ in the remainder of this help file, but that, as a C++ user, you have to replace ‘Instrument’ by ‘InstrumentInterface’ and Userbutton by ‘IUserButton’. Your application must also initialize the COM-libraries. Using MFC, this is typically done by the following code if (!AfxOleInit()) { AfxMessageBox(IDP_OLE_INIT_FAILED); return FALSE; } that you have to add to your Applications ::InitInstance() if you did not check the ‘automation’-check box, when creating it.

22.2 Create an ‘Instrument’ and get the secondary microscope object that you need The following piece of code shows one possible way to get access to the projection system and the stage: InstrumentInterfacePtr MyInstrument; ProjectionPtr MyProjection; StagePtr MyStage; MyInstrument.CreateInstance(_T("TEMScripting.Instrument.1")); MyProjection = MyInstrument->Projection; MyStage = MyInstrument->Stage; Here we used smart, _com_ptr_t-derived pointers that are defined in the stscript.tlh and stdscript.tli files. (Their names all end with the postfix ‘Ptr’.) These pointers will do the interface reference-counting for you. We also made use of the ‘property-way’ of writing, as do most scripting languages. A ‘property’ appears in the ClassView as a public member variable. In effect it can be used as such, except that for example it may be ‘read only’, i.e. setting may not be permitted.


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The above code is entirely equivalent to the use of the wrapper functions that is shown in the following piece of code: MyProjection = MyInstrument->GetProjection(); MyStage = MyInstrument->GetStage(); In fact these functions are also called under water in the first example. The advantage of the wrapper functions is a convenient treatment of errors. If an error occurs, these functions will raise an exception (of type _com_error) that you can catch (see below) and that allows easy access to detailed error information. The wrapper functions also encapsulate BSTR (and VARIANT) types into _bstr_t-(and _variant_t-)objects that are easier to handle. You can of course also choose to go the hard way and use the ‘raw’ functions, that finally implement the properties: HRESULT hr; hr = MyInstrument->get_Projection(&MyProjection); hr = MyInstrument->get_Stage(&MyStage); For handling of errors you would then have to check the value of hr. If there is extended error information, you could query it via the IErrorInfo-interface. (See any book on COM on how to do this). In this last example, it does not matter, whether you use intelligent pointers or not, while in the first two examples you have to use them, otherwise you will experience difficulties with the reference count. Your objects might be destroyed due to the fact that the wrapper functions are designed in a way that results in a hidden Release()-call for the interface in question. This call is used to compensate for the AddRef() that is connected to the use of the ‘=’-operator for intelligent pointers. (This also implies, that you have to use ‘=’ and not the .Attach() method of the smart pointer class).

22.3 Manipulate and read microscope parameters, invoke microscope actions To manipulate microscope parameters, you can in principle use all of the three different ways of writing assignment statements that are already described above (only that for simple properties you do not retrieve interface pointers here). In the following examples, we choose the way of writing that stresses the notion of ‘properties’, because it is the easiest to write and because it is most consistent with the remainder of this help. An example: MyProjection->MagnificationIndex = 5; This statement will set the magnification index to a new value (=5). long MyLong; MyLong = MyProjection->MagnificationIndex; reads the current value of the magnification index into the variable MyLong. ‘Compound’ properties are read and set by using the TEM scripting adapter's ‘utility objects’. Thus IlluminationPtr MyIllumination; VectorPtr MyShift; double MyShiftX; MyIllumination = MyInstrument->Illumination; MyShift = MyIllumination->Shift; MyShiftX = MyShift->X; would read the full 2D-beam shift first and then store its x-component into the variable MyShiftX. Be aware that MyShift contains a copy of the actual beam shift. Thus, if you want to get the new actual


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beam shift at a later moment in time, you have to ask for MyIllumination->Shift again! Setting compound parameters then works as follows: VectorPtr MyNewShift; MyNewShift = MyIllumination->Shift; MyNewShift->X = 0; MyNewShift->Y = 0; MyIllumination->Shift = MyNewShift; The codes first gets a Vector-object by reading a 2D-property, assigns new values (0,0) and then copies it back, thus setting the beam shift to zero.

22.4 Use the collections The adapter contains several collections, such as the ‘Gauges’ and the ‘UserButtons’ collections. Collection interfaces were originally designed for Visual Basic and are very convenient to use in scripting languages. The collections that come with the adapter are of fixed size, the script cannot add items. Reading items can be done as follows: GaugesPtr MyGauges; GaugePtr g; CString sMsg; MyGauges = MyInstrument->Vacuum->Gauges; for (long i; i<MyGauges->Count; i++) { g = MyGauges->GetItem(i); CString sGauge; sGauge.Format(_T("%s : %d\n"), g->Name, g->Pressure); sMsg = sMsg + sGauge; } AfxMessageBox(sMsg); loops over all gauges of the vacuum system and shows for each gauge the name and the pressure measured with it on a message box. In C++, you cannot use Item as a property (although other languages use it as such), i.e. g = MyGauges->Item(i); //does not work! does not work. Some scripting languages allow the use of the handy abbreviation MyGauges(i), because ‘Item’ is the default property of a collection. This is also not allowed in C++. Probably you will often not know the index of the gauge, but its name, that you can read from the vacuum display of the microscope. GetItem therefore also accepts the gauge name as identifier, for example it is possible to write g = MyGauges->GetItem(_T("P1")); g now contains the information about the buffer tank pressure. Remember that the ‘gauge’ objects are utility objects, so to get the new actual values, you have to ask for a new collection again, thus to repeat: MyGauges = MyInstrument->Vacuum->Gauges;


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22.5 Receive events from the user buttons To receive the events connected with pressing user buttons of the TEM control pads (L1,..,L3 and R1,..,R3), you have to do some work in C++. First, the class that implements the event interface should support OLE-automation. Thus the constructor should include the following statement: EnableAutomation(); Also you should use the following macros to your classes header file: DECLARE_DISPATCH_MAP() DECLARE_INTERFACE_MAP() The class wizard will generally add these for you. In the corresponding macros that actually define the interface and dispatch maps in your classes *.cpp-file, you have to make some adjustments, however: a) In the dispatch map macro, you have to assign the button event Pressed to a function (called OnPressed in this example) of your CCmdTarget-derived, event- receiving class (which is called CMyButton here). BEGIN_DISPATCH_MAP(CMyButton, CCmdTarget) DISP_FUNCTION(CMyButton, "Pressed", OnPressed, VT_EMPTY, VTS_NONE) END_DISPATCH_MAP() VT_EMPTY indicates that the function has no return parameter and VTS_NONE specifies that it does not take any data. b) The interface that contains the user button event has to be included into the INTERFACE_MAP macro: BEGIN_INTERFACE_MAP(CMyButton, CCmdTarget) INTERFACE_PART(CMyButton, DIID_UserButtonEvent, Dispatch) END_INTERFACE_MAP() Furthermore you have to include code that handles the connection point mechanism used to receive COM-events. This might look as follows: //Assume, we alreay have a pointer pMyButton,pointing to a UserButton interface IConnectionPointPtr MyConnectionPoint; IConnectionPointContainerPtr CPContainer = pMyButton; DWORD MyCookie; HRESULT hr = CPContainer->FindConnectionPoint (DIID_UserButtonEvent, &MyConnectionPoint); if (hr == S_OK) //pass pointer of eventsink interface for callback by server hr = MyConnectionPoint->Advise(GetIDispatch( TRUE), &MyCookie); And, in the end (for example in the destructor of your event-receiving class), you have to disregister with the connection point again. (For this reason you had to remember MyCookie): MyConnectionPoint->UnAdvise(MyCookie);


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In order to make the above code work, you further have to overwrite the connection point's GetIID() function to retrieve the ID of the user button event-interface: IID CMyButton::GetIID () { return DIID_UserButtonEvent; } Having done all this, void CMyButton::OnPressed() { // Do whatever you want } will be called, whenever the corresponding button (the button "pointed to" by pMyButton) is pressed, provided you have activated it. Activation is done by assigning a string that will then also be displayed in the TEM UI: _bstr_t bstrNewAssignment(_T("any new assignment")); pMyButton->Assignment = bstrNewAssignment;

22.6 Receive Events from a remote microscope server If you want your application to connect to a remote microscope server and also receive the userbutton events, you have to add a call to the function CoInitializeSecurity, that opens your application for calls from a remote system service. Just place the following code directly after the call to AfxOleInit (see above), because you have to call CoInitializeSecurity, before COM calls it under water for you: HRESULT sc = ::CoInitializeSecurity( NULL, -1, NULL, NULL, RPC_C_AUTHN_LEVEL_NONE, RPC_C_IMP_LEVEL_IMPERSONATE, NULL, EOAC_NONE, NULL); if (FAILED(sc)) { TRACE1("CoInitializeSecurity failed! (returned 0x%X)\nno remote events will be received", sc); } Also, the compiler switch _WIN32_DCOM has to be defined. Otherwise the relevant function declarations may not be included into your project. Thus if you get a compiler error, you might have to add the following line of code, preferably in stdafx.h: #define _WIN32_DCOM

22.7 Errors and error handling Some of the microscopes functions may return (raise) an error. This can happen due to a physical reason, i.e. if the requested action is not possible at that moment. For example, you cannot ask the stage to perform a GoTo(), when it is wobbling or already moving. The high tension may not be switched on or raised under certain conditions and so forth. You may also have given parameters that


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are out of range (in that case the stage for example would not move either). Generally, calls to another process (i.e. from your script to the TEM server) may fail occasionally. So be aware and do the error handling - otherwise your application might die! If an error is raised, then some global object may be filled with detailed information about the error. (In case of TEM errors that will be done for versions >1.0). The minimal information, that is always available, consists of an HRESULT error code and a system generated standard error message. Usually you will get an error code, a textual description and some more information as described below. The easiest way to obtain this information is to use the smart pointer classes and the wrapper functions that are defined in the stdscript.tli/tlh-files and that are automatically generated when the typelib or the dll is imported into your project. You can then use the C++ try/catch mechanism to catch the errors, as shown below. Suppose you want to move the stage to a new position that is calculated from the old one -just as an example, you could invent some code here: try { StagePositionPtr OldPos; StagePositionPtr NewPos; StagePtr MyStage; MyStage = MyInstrument->Stage; OldPos = MyStage->Position; CalculateNewPosition(OldPos, &NewPos) // some function MyStage->Goto(NewPos, axisXY); } catch ( _com_error E) { CString sDescription; if (E.Description().length() > 0) { sDescription.Format(E.Description()); } else if (E.ErrorMessage() != NULL) { sDescription.Format(E.ErrorMessage()); } AfxMessageBox(Description); } In case of an error, a message-box will show a description of the error that ocurred. This will be either the application-delivered one, if available, or the standard message for the error code in question. Among others, the exception object supports the following handy methods: HRESULT Error( ) // returns the HRESULT _bstr_t Description( ) // server generated description _bstr_t Source( ) // name of the source const TCHAR * ErrorMessage( ) // the standard message For more information check the _com_error's member functions.


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23 TEM scripting in Delphi

23.1 Introduction and package installation The Delphi version used for the example program was the Professional edition of Delphi 4.0, but the other variants (including Desktop) will work as well. Delphi 3.0 (in all variants, including Desktop) will work as well, but when the example program is opened in Delphi 3.0 it will complain about some (in Delphi 3.0) unsupported properties. Look for the complaints from Delphi 3.0, then load the frm file as text (e.g. in Notepad) and remove the offending statements.

23.2 Events Delphi (at least up to version 4.0) provides no direct handling of events. Instead you either have to write your own handler or (to keep it simple) use the EventSinkImport utility written by Binh Ly, which was used here (can be found on the world wide web). The Event Sink Import utility creates two files, a standard TLB (as obtained when using the Delphi Import Type Library function) and a separate Events unit. The Events unit for TEM Scripting was compiled into a package that can be installed, creating a non-visual component under ActiveX. Important note: The declaration of the Events component under the uses clause must always precede the declaration to the TLB itself. To install the Scripting Events component for Delphi 4.0: • Copy the TemScripting.bpl file to the Imports folder of the Borland\Delphi 4 folder. • Start Delphi. • Select Component, Install Packages. • Press the Add button and select the TemScripting.bpl file. • In the ActiveX tab of Delphi you will now find the UserButtonEvent component. To install the Scripting Events component for Delphi 3.0 (or Delphi 5.0) and higher: • Copy the TemScriptingEvents.pas and TemScriptingEvents.dcr file to the Imports folder of the

Borland\Delphi 3 or 5 folder. • Start Delphi. • Select Component, Install Component. • Select the Into new package tab. • For unit file name, Browse to TemScriptingEvents.pas. • For Package file name Browse to the Imports folder and enter TemScripting.dpk. • For Package description enter Tem Userbutton Events • Press OK. The package will be compiled and installed. Save the package. • In the ActiveX tab of Delphi you will now find the UserButtonEvent component.

23.3 Using the dynamic link library To use the type library, simply copy the TemScripting_TLB.pas file to the Imports folder of the Borland\Delphi folder. Then use Project, Add to project and select the TemScripting_TLB.pas.

23.4 Example program The program has a form with a PageControl containing a number of TabSheets, each of which contains a logical group of TEM scripting functions (Optics, Stage, Vacuum, Camera). Since some of these groups do not have very many functions, their TabSheets are rather empty, but the overall size is determined by the fullest TabSheet.


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The connection with the microscope is done in FormCreate for instrument and then per tab (checked against previous connection through the nil). This method makes startup faster and eliminates unnecessary connections. Some units, such as GetVersion are very small and can easily be combined into a program itself. Since they may be used repeatedly for more than one application, they are separated off for easy importing.

23.4.1 Large fonts The TEM microscopes often have Windows set to Large Fonts. When this is the case, the size of all controls changes (if your program was designed with the PC set on Small Fonts) but typically not the form size (and controls will often be partially hidden). To correct for this, you can change the settings dependent on the font setting read at start up. The font size can be determined at run time by inspecting the PixelsPerInch property. It is 96 for Small Fonts and 120 for Large Fonts. Note: The font in a statusbar may come out bold on Large Fonts when compiled in Small Fonts. Make sure you have the ParentFont property of the statusbar set to true and it will come out normal.

23.4.2 About box and Version number The About box for the Exampler is located in the System Menu (under the icon of the Window). For displaying the version number, these settings are read from the program file itself (to make it only necessary to update the Version Info of the program and not have to keep two settings in sync. The About Box is called through the Windows function WMSysCommand. The Version number is read through the GetVersion unit.

23.5 Create an ‘Instrument’ and get secondary microscope objects The following piece of code shows how to get access to the projection system and the stage: In the declaration section (under var) FTem : Instrument; FProj : Projection; FStage : Stage; In a section accessed before any calls to the microscope are made (e.g. the FormCreate). In the example program the subunits (Projection, Stage, etc.) are connected per tab of the PageControl (the connections take time, so this reduces the number of connections to those necessary and reduces start-up time). try if FTem = nil then FTem := CoInstrument.Create; try { no sense in trying this if the previous didn't work } if FProj = nil then FProj := FTem.Projection; except on E : Exception do Application.Messagebox( pchar('Error - No connection to projection - '+E.Message), 'Error',mb_OK); end; try if FStage = nil then FStage := FTem.Stage; except on E : Exception do Application.Messagebox(


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pchar('Error - No connection to stage - '+E.Message), 'Error',mb_OK); end; except on E : Exception do Application.Messagebox( pchar('Error - No connection to instrument - '+E.Message), 'Error',mb_OK); end;

23.6 Manipulate and read microscope parameters, invoke microscope actions Many of the values of the microscope can manipulated in one of two ways, using properties or using functions/procedures. If you use Delphi, you will be quite familiar with properties. Nevertheless, here is an example: FTem.MagnificationIndex := 5; ‘Compound’ properties are read and set by using the TEM scripting adapter's ‘utility objects’. Thus var FShift : Vector; FX : double; In the code: FShift := FProj.ImageShift; FX := FPos.X; would read the full 2D-image shift and then store the x-component in the variable FX. Be aware that FShift contains a copy of the image shift. If you want to get the new actual image shift at a later moment in time, you have to ask for FProj.ImageShift again! For setting the multidimensional properties, you have to create an instance of the utility object first (by reading it, as above), then put the right coordinate values into it and then assign it to the property. Example: var FShift : Vector; In the code: FShift := FStage.ImageShift; FShift.X := 1e-6; { image shift is in meters } FShift.Y := -1e-6; FProj.ImageShift := FShift; creates a new Vector object that is initialized with the coordinates as given and is then used to set the image shift to these values. (An exception here is the position of the stage, that is read only, because for moving the stage you have to use the Goto_ and MoveTo functions. The underscore on Goto is added by Delphi automatically because Goto is a reserved word).

23.7 Use the collections The adapter contains several collections, such as the ‘Gauges’ and the ‘UserButtons’ collections. A collection is a convenient way to store things of the same kind. The collections that come with the adapter are of fixed size, the script cannot add items. Reading items can be done in several ways:


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var FTem : Instrument; FVacuum : Vacuum; FGauges : Gauges; FGauge : Gauge; indx : integer; td : double; OleVar1 : OleVariant; In the code: try if FTem = nil then FTem := CoInstrument.Create; try { no sense in trying this if the previous didn't work } if FVacuum = nil then FVacuum := FTem.Vacuum; if FGauges = nil then FGauges := FTem.Vacuum.Gauges; for indx := 0 to FGauges.count-1 do { fill labels, see example program - better } begin OleVar1 := indx; FGauge := FGauges.Item[OleVar1]; Label1.caption := FGauge.Name; td := FGauge.Pressure; with Label2 do if td > 100 then caption := RealToStr(td,0) { for RealToStr see example program } else if td > 10 then caption := RealToStr(td,1) else if td > 1 then caption := RealToStr(td,2) else if td > 0.001 then caption := RealToStr(td,4) else caption := RealToStr(td,8); with Label3 do case FGauge.Status of gsUndefined : caption := 'Undefined'; gsUnderflow : caption := 'Underflow'; gsOverflow : caption := 'Overflow'; gsInvalid : caption := 'Invalid'; gsValid : caption := 'Valid'; end; end; except on E : Exception do Application.Messagebox( pchar('Error - No connection to vacuum - '+E.Message),'Error',mb_OK); end; except on E : Exception do Application.Messagebox( pchar('Error - No connection to instrument - '+E.Message),'Error',mb_OK); end; loops over all gauges of the vacuum system. You can access single gauges via the ‘Item’-property of the collection (thus as MyGauges.Item[3] for example). Probably often you will not know the index of the gauge, but you can find out its name from the vacuum display of the microscope.


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The ‘Item’ property therefore also accepts the gauge name as identifier, for example it is possible to write OleVar1 := 'P1'; FGauge := FGauges.Item[OleVar1]; { in Delphi you cannot use the name as in VB or JScript- thus you cannot write FGauges[OleVar1] }

23.8 Receive events from the user buttons To receive the events connected with pressing user buttons of the TEM Control pads (L1,..,L3 and R1,..,R3), you have to do the following in addition to the code for connecting to the instrument. First put a UserButtonEvent control on the form (as many as you need, so if you want L1..L3 you'll need three): FTem : Instrument; FUserButtons : UserButtons; F_UB_L1 : UserButton; try if FUserButtons = nil then begin FUserButtons := FTem.UserButtons; F_UB_L1 := FUserButtons.Item[1]; UserButtonEvent1.Connect(IUnknown (F_UB_L1) ); end; except ... end; When you double-click on the UserButtonEvent control, the following procedure will be added to the code: procedure TForm1.UserButtonEvent1Pressed (Sender: TObject; newval: Integer); begin { whatever } end; The procedure UserButtonEvent1Pressed will be invoked when User button "L1" is pressed, provided you have activated the events by using the assignment-property of F_UB_L1.

23.9 Receive events from a remote microscope server If you want your application to connect to a remote microscope server and also receive the userbutton events from that remote machine, you have to change the standard COM security setting. This has to be done, before COM automatically initializes security. The hard-work approach (doing everything yourself): Immediately after the first CoInitialize call you must call a function named CoInitializeSecurity as follows: OleCheck (CoInitializeSecurity (nil, -1, nil, nil, RPC_C_AUTHN_LEVEL_NONE, RPC_C_IMP_LEVEL_IMPERSONATE, nil, EOAC_NONE, nil)); where


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RPC_C_AUTHN_LEVEL_NONE = 1 RPC_C_IMP_LEVEL_IMPERSONATE = 3 EOAC_NONE = 0 When you are using the functionality from Binh Ly (comlib) you simply call InitializeComSecurity (alNone,ilImpersonate); In the examples, this call is made immediately in the FormCreate procedure.

23.10 Errors and error handling Some of the microscopes functions may return (raise) an error. This can happen due to a physical reason, i.e. if the requested action is not possible at that moment. For example, you cannot ask the stage to perform a GoTo_, when it is wobbling or already moving. The high tension may not be switched on or raised under certain conditions, and so forth. You may also have given parameters that are out of range (in that case the stage for example would not move either). Generally, calls to another process (from your script to the TEM server) may fail occasionally. So be aware and do the error handling, typically by a try except end; If an error is raised, then Exception.Message will be filled with detailed information about the error, if available. You can use this information for your error handling. See the error handling in the code examples above.

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24 TEM scripting in JScript This chapter explains how run a script in an HTML-page, as is also demonstrated in the example program. This way you can easily build a graphical user interface, using HTML input tags. An alternative approach would be to run a script using the windows scripting host.

24.1 Create an ‘Instrument’ and get the secondary microscope object that you need The following code demonstrates how to get access to the projection system and the stage. In this example, the code is included into the HTML page (you could also specify the source-file using the SRC attribute of the <SCRIPT>-tag) and will be triggered, when the body of the HTML page is loaded: <HTML> ... <BODY ONLOAD="Connect()"> ... <SCRIPT LANGUAGE="JavaScript">  </SCRIPT> </BODY> </HTML>

24.2 Manipulate and read microscope parameters, invoke microscope actions If you use JScript, you will be quite familiar with properties. Nevertheless, here is an example: MyProjection.MagnificationIndex = 5; This statement will set the magnification index to a new value (=5). var MyLong;MyLong = MyProjection.MagnificationIndex; reads the current value of the magnification index into the variable MyLong. Compound properties are read and set by using the TEM scripting adapters ‘utility objects’. Thus var MyIllumination;var MyShiftX; var MyShift; MyIllumination = MyTem.Illumination; MyShift = MyIllumination.Shift; MyShiftX = MyShift.X;


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would read the full 2D-beam shift first and then store its x-component in the variable MyShiftX. Be aware that MyShift contains a copy of the actual beam shift. Thus, if you want to get the new actual beam shift at a later moment in time, you have to ask for MyIllumination.Shift again! Setting compound parameters then works as follows: var MyNewShift; var MyVector; yVector = MyTem.Vector; MyNewShift = new MyVector(0,0); // or alternatively: // MyNewShift = new MyTem.Vector(0,0); MyIllumination.Shift = MyNewShift; The above code is specific for JScript and works in the same fashion for the ‘StagePosition’ object. It first defines a ‘(My)Vector’ type by retrieving a ‘Vector’ constructor function object, then actually constructs a new ‘(My)Vector’-object, that is initialized with (0,0) and then uses this newly created object to set the beam shift to zero. MyNewShift = MyIllumination.Shift; MyNewShift.X = 0; MyNewShift.Y = 0; MyIllumination.Shift = MyNewShift; would do the same, using the ‘Vector’-object retrieved by reading the beam shift first.

24.3 Use the collections The adapter contains several collections, such as the ‘Gauges’ and the ‘UserButtons’ collections. A collection is a convenient way to store things. The collections that come with the adapter are of fixed size, the script cannot add items. Reading items can be done in several ways: var MyGauges; var g; var sMsg = "";MyGauges = MyTem.Vacuum.Gauges; for (i=0; i < MyGauges.Count; i++) { g = MyGauges(i); sMsg = sMsg + g.Name + ": " + g.Pressure; } alert(sMsg); loops over all gauges of the vacuum system and then shows a message box containing the names of all gauges and the pressures measured with them. You can also access single gauges via the ‘Item’-property of the collection (thus as MyGauges.Item(3)) for example), but since ‘Item’ is the default property, you can use the array-type syntax (MyGauges(i)) as demonstrated in the above example. Probably often you will not know the index of the gauge, but its name, that you can read from the vacuum display of the microscope. The ‘Item’ property therefore also accepts the gauge name as identifier, for example it is thus possible to write g = MyGauges("P1"); g now contains the information about the buffer tank pressure. Remember that the ‘Gauge’ objects are utility objects, so to get the new actual values, you have to ask for a new collection again:


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MyGauges = MyTem.Vacuum.Gauges; does the job.

24.4 Receive events from the user buttons The Connect() function above is an example of how easy it is to define event handlers that react on events that originate from HTML elements. To define event handlers for TEM user button-events is a bit more involved. Following code shows how to do it (for one button): // Variables used throughout the script ... var MyL1; // ... function Connect() { ... MyL1 = MyTem.UserButtons("L1"); ... DefineEventHandlers(); ... ... } function DefineEventHandlers() { function MyL1::Pressed() { //Do what you want to do here ... } } This maybe strange looking code is necessary to convince the script-interpreter that MyL1 is indeed a ‘UserButton’ object and that the subsequent definition of MyL1::Pressed() makes sense and implements the ‘UserButton’-event. The main point is, that after assigning a ‘UserButton’ object to MyL1 a function (here named DefineEventHandlers()) is called that then contains the definition of the event handler. Simply defining MyL1::Pressed() inside or outside the Connect() function would yield an error ("MyL1 not an object" or "MyL1 undefined"). MyL1::Pressed() will be called, whenever the corresponding TEM user button L1 is pressed, provided you have activated it for the script. Activation is done by assigning a string that will then also be displayed int the TEM UI, as it is done in: MyL1.Assignment = "my new assignment" ;

24.5 Errors and error handling Some of the microscopes functions may return (raise) an error. This can happen due to a physical reason, i.e. if the requested action is not possible at that moment. For example, you cannot ask the stage to perform a GoTo(), when it is wobbling or already moving. The high tension may not be switched on or raised under certain conditions and so forth. You may also have given parameters that are out of range (in that case the stage for example would not move either). Generally, calls to another process (ie from your script to the TEM server) may fail occasionally. So be aware and do the error handling - otherwise your application might die! If an error occurs an ‘Error’ object will be thrown. It contains an error code (a number) and a textual description. You can then use the JScript try/catch mechanism to catch the errors, as shown below.


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Suppose your HTML page contains a form with a button named ‘cmdGoto’, and a text area named ‘TextErrors’. Pressing the button triggers movement of the stage to a new position that is calculated from the old one -just as an example, you could invent some code here: <FORM ID="theForm"> ... <INPUT TYPE=BUTTON NAME="cmdGoto" VALUE="Go" ONCLICK="OnGoto()"> ... <TEXTAREA NAME="TextErrors" COLS=.. ROWS=.. READONLY> </TEXTAREA> ... </FORM> ... ... function OnGoto() { try { var NewPos; var OldPos; var MyStage; MyStage = MyTem.Stage; OldPos = Mystage.Position; NewPos = new MyTem.StagePosition(0,0,0,0,0); CalculateNewPosition(OldPos, NewPos) // some function MyStage.Goto(NewPos, axisXY); } catch (Err) { var n; var sn; //transform error code into hexadecimal string representation n = new Number(Err.number); sn = (n + Math.pow (2,32)).toString(16); with (theForm.TextErrors) { value = value + sn + "\n"; value = value + Err.description + "\n"; } } } In case of an error, the error code (in hexadezimal format) and the error description will be added to the text in the text area. In some cases, for example when the creation of the ‘Instrument’ object fails, JScript insists on its own error code and error message, overwriting any description given by for example by TEM scripting. Note: For example JScript will miss the description "TEM scripting: Protection key missing!", that indicates that the protection dongle is missing.

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25 TEM scripting in VBScript This chapter explains how to run a script in an HTML-page, as is also demonstrated in the example program. This way you can easily build a graphical user interface, using HTML input tags. An alternative approach would be to run a script using the windows scripting host.

25.1 Create an ‘Instrument’ and get the secondary microscope object that you need The following code demonstrates how to get access to the projection system and the stage. In this example, the code is included into the HTML page (you could also specify the source-file using the SRC attribute of the <SCRIPT>-tag) and will be triggered, when the body of the HTML page is loaded: <HTML> ... <BODY ONLOAD="Connect()"> ... <SCRIPT LANGUAGE="VBScript">  </SCRIPT> </BODY> </HTML>

25.2 Manipulate and read microscope parameters, invoke microscope actions If you use VBScript, you will be quite familiar with properties. Nevertheless, here is an example: MyProjection.MagnificationIndex = 5 This statement will set the magnification index to a new value (=5). Dim MyLong;MyLong = MyProjection.MagnificationIndex reads the current value of the magnification index into the variable MyLong. Compound properties are read and set by using the TEM scripting adapters ‘utility objects’. Thus Dim MyIlluminationDim MyShiftXDim MyShift Set MyIllumination = MyTem.Illumination Set MyShift = MyIllumination.Shift MyShiftX = MyShift.X would read the full 2D-beam shift first and then store its x-component in the variable MyShiftX. Be aware that MyShift contains a copy of the actual beam shift, although it seems to be passed as a reference (using "Set"). The reason is, that the copy is made by the adapter and is then passed. Thus, if


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you want to get the new actual beam shift at a later moment in time, you have to ask for MyIllumination.Shift again! Setting compound parameters then works as follows: Dim MyNewShift as Tecnai.Vector Set MyNewShift = MyIllumination.Shift MyNewShift.X = 0 MyNewShift.Y = 0 MyIllumination.Shift = MyNewShift The code first gets a ‘Vector’ object by reading a 2D-property, then assigns new values (0,0) and then copies it back, thus setting the beam shift to zero (you cannot use "Set" here).

25.3 Use the collections The adapter contains several collections, such as the ‘Gauges’ and the ‘UserButtons’ collections. A collection is a convenient way to store things of the same kind. The collections that come with the adapter are of fixed size, the script cannot add items. Reading items can be done in several ways: Dim MyGauges Dim g Dim sMsgs Msg = ""Set MyGauges = MyTem.Vacuum.Gauges For Each g In MyGauges sMsg = sMsg & g.Name & ": " & g.Pressure & vbcrlf next Msgbox sMsg loops over all gauges of the vacuum system and then shows a message box containing the names of all gauges and the pressures measured with them. You can access single gauges via the ‘Item’-property of the collection (thus as MyGauges.Item(3) for example), but since ‘Item’ is the default property, you can more easily use the array-type syntax Set g = MyGauges(3) Probably often you will not know the index of the gauge, but its name, that you can read from the vacuum display of the microscope. The ‘Item’ property therefore also accepts the gauge name as identifier, for example it is possible to write Set g = MyGauges("P1") g now contains the information about the buffer tank pressure. Remember that the ‘Gauge’ objects are utility objects, so to get the new actual values, you have to ask for a new collection again: Set MyGauges = MyTem.Vacuum.Gauges does the job.

25.4 Receive events from the user buttons Since we did not succeed in recieving events in VBScript from objects that cannot be created directly, an additional DLL is delivered for VBScript. Its name is scriptevents.dll. It only delivers one object, the ‘TEMScriptingEvents’-object. Attention: Creating this object using CreateObject("TEMScripting.TEMScriptingEvents")


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does not work! This function does not connect to the events. You have to use the <object>-tag in your HTML-code: <object id="MyTemEvents" classid="clsid:5E896A91-A0BF-11d3-A688-00C04F9D480A" > </object> The name of the id-parameter can be choosen arbitrarily and is the name under which you later address the ‘TEMScriptingEvents’-object in your script. The ‘TEMScriptingEvents’-object does not have any properties or methods, it only supplies one event: Pressed(ButtonName). This function has to be implemented in your script and will be called, whenever one of the User Buttons on the TEM handpanels is pressed. In contrast to the ‘normal’ way of receiving the events via the ‘UserButton’-objects of TEM scripting, the VBScript-client will have to check whether the event is actually meant for him. He cannot make use of the intelligence that is built into the ‘UserButton’-objects of TEM scripting. The following example shows how to do it: Dim MyButtons 'need a "global" UserButtons-collection ... 'initialize collection in your "connect()" function Set MyButtons = MyTem.UserButtons ... 'implement the event: Sub MyTemEvents_Pressed(ButtonName) 'In VBScript you have to check whether the event is meant for you Dim Button Set Button = MyButtons("" & Name) 'Using "" & makes clear that we deal with a string if ( (Button.Label <> "") and (Button.Assignment = Button.Label) ) then ' we indeed control the button .... end if End Sub Remember: The ‘UserButton’-Label is the Label of the Button as it is shown in the TEM user interface. If your script did an assignment to a button, then assignment and label coincide. Your assignment can (hopefully temporarily) be overwritten by another application (for example by the alignment procedures). You would then probably not want to react on the events.

25.5 Errors and error handling Some of the microscopes functions may return (raise) an error. This can happen due to a physical reason, i.e. if the requested action is not possible at that moment. For example, you cannot ask the stage to perform a GoTo(), when it is wobbling or already moving. The high tension may not be switched on or raised under certain conditions and so forth. You may also have given parameters that are out of range (in that case the stage for example would not move either). Generally, calls to another process (ie from your script to the TEM server) may fail occasionally. So be aware and do the error handling - otherwise your application might die! If an error is raised, then a global Error object (Err) will be filled with detailed information about the error, if available. You can use this information for your error handling. Usually you will get an error code (a number), a textual description and maybe a text string with the name of the source that raised the error. If you want to do the error handling, you have to insert the following statement into the code: On Error Resume Next


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This statement makes VBScript ignoring errors and is valid for the code to follow until the end of the function or subroutine. You then got the chance to ask the error object for its content. An example: Suppose your HTML page contains a form with a button named ‘cmdGoto’, and a text area named ‘TextErrors’. Pressing the button triggers movement of the stage to a new position that is calculated from the old one -just as an example, you could invent some code here: <FORM ID="theForm"> ... <INPUT TYPE=BUTTON NAME="cmdGoto" VALUE="Go" ONCLICK="OnGoto()"> ... <TEXTAREA NAME="TextErrors" COLS=.. ROWS=.. READONLY> </TEXTAREA> ... </FORM> ... ... Sub OnGoto() Dim NewPos Dim OldPos Dim MyStage On Error Resume Next Set MyStage = MyTem.Stage CheckError Set OldPos = MyStage.Position CheckError CalculateNewPosition(OldPos, NewPos) // some function MyStage.Goto(NewPos, axisXY) CheckError End Sub Sub CheckError() If (Err.Number <> 0) Then With theForm.TextErrors .Value = .Value + "Error:" & Hex(Error.Number) & vbCrLf _ & "Source: " & Error.Source & _ vbCrLf & Error.Description & vbCrLf End With End If Err.Clear 'Clears the error object End Sub In case of an error, the error code (in hexadezimal format), its source (if available) and the error description will be added to the text in the text area. If we would not clear the error object by using Err.Clear, we would handle the same error with the next check again. In some cases, for example when the creation of the ‘Instrument’ object fails, VBcript insists on its own error code and error message, overwriting any description given by TEM scripting. Note: For example you will miss the description "Tecnai scripting: Protection key missing!", that indicates that the protection dongle is missing.


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26 TEM scripting in Visual Basic

26.1 Import the dynamic link library To get the support of Visual Basics IntelliSense mechanism and to be able to declare variables of the types that the adapter delivers (ie to use hard typing and early binding), you have to import the adapters dynamic link library into your project. This is easily achieved. • create a VB Project if that is not already done • reference the adapter : Click on the menu item Project – References. The following window will pop up (the actual contents will vary):

Find the TEMScripting and check it. Now that the adapter is included into the project, you will see its objects, their properties and their methods appear in the object browser (click View – Object Browser or press F2):


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For every item you will find a small description in the lower gray part of this window. Context sensitive help (try pressing F1) is also available, if you are working with VB6.0

26.2 Create an ‘Instrument’ and get the secondary microscope object that you need The following piece of code shows how to get access to the projection system and the stage: Dim MyTem as TEMScripting.Instrument Dim MyProjection as TEMScripting.Projection Dim MyStage as TEMScripting.Stage Set MyTem = new TEMScripting.Instrument Set MyProjection = MyTem.Projection Set MyStage = MyTem.Stage

26.3 Manipulate and read microscope parameters, invoke microscope actions If you use VB, you will be quite familiar with properties. Nevertheless, here is an example: MyProjection.MagnificationIndex = 5 This statement will set the magnification index to a new value (=5). Dim MyLong as Long MyLong = MyProjection.MagnificationIndex


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reads the current value of the magnification index into the variable MyLong. ‘Compound’ properties are read and set by using The TEM scripting adapters ‘utility objects’. Thus Dim MyIl as TEMScripting.Illumination Dim MyShiftX as Double Dim MyShift as TEMScripting.Vector Set MyIl = MyTem.Illumination Set MyShift = MyIL.Shift MyShiftX = MyShift.X would read the full 2D-beam shift first and then store its x-component into the variable MyShiftX. Be aware that MyShift contains a copy of the actual beam shift, although it seems to be passed as a reference (using "Set"). The reason is, that the copy is made by the adapter and is then passed. Thus, if you want to get the new actual beam shift at a later moment in time, you have to ask for MyIl.Shift again! Setting compound parameters then works as follows: Dim MyNewShift as TEMScripting.Vector Set MyNewShift = MyIl.Shift MyNewShift.X = 0 MyNewShift.Y = 0 MyIl.Shift = MyNewShift The codes first gets a ‘Vector’ object by reading a 2D-property, then assigns new values (0,0) and then copies it back, thus setting the beam shift to zero (you cannot use "Set" here).

26.4 Use the collections The adapter contains several collections, such as the ‘Gauges’ and the ‘UserButtons’ collections. A collection is a convenient way to store things of the same kind. The collections that come with the adapter are of fixed size, the script cannot add items. Reading items can be done in several ways: Dim MyTem as TEMScripting.Instrument Dim MyGauges as TEMScripting.Gauges Dim g as TEMScripting.Gauge Open "TESTFILE" For Output As #1 Set MyGauges = MyTem.Vacuum.Gauges For Each g In MyGauges Print #1, g.name & ": " & g.Pressure next Close #1 loops over all gauges of the vacuum system and prints their names and the pressures measured with them to a file named "TESTFILE". You can access single gauges via the ‘Item’-property of the collection (thus as MyGauges.Item(3) for example), but since ‘Item’ is the default property, you can more easily use the array-type syntax Set g = MyGauges(3) Probably often you will not know the index of the gauge, but its name, that you can read from the vacuum display of the microscope. The ‘Item’ property therefore also accepts the gauge name as identifier, for example it is possible to write Set g = MyGauges("P1")


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g now contains the information about the buffer tank pressure. Remember that the ‘Gauge’ objects are utility objects, so to get the new actual values, you have to ask for a new collection again: Set MyGauges = MyTem.Vacuum.Gauges does the job.

26.5 Receive events from the user buttons To receive the events connected with pressing user buttons of the TEM control pads (L1,..,L3 and R1,..,R3), you have to do the following (the example uses the user button "L1"): Private WithEvents MyButton as TEMScripting.UserButton Set MyButton = MyTem.UserButtons("L1") ... 'Activate events'and let a label be displayed in the TEM UI MyButton.Assignment = "my new label" ... Private Sub MyButton_Pressed() 'Do what you want here End Sub... The procedure MyButton_Pressed will be invoked whenever the user button "L1" is pressed.

26.6 Receive events from a remote microscope server If you want your application to connect to a remote microscope server and also receive the userbutton events from that remote machine, you have to add a call to the function CoInitializeSecurity that opens your application for calls from a remote system service. You have to do that, before VisualBasic does this for you under water, because this function call succeeds only once per application. What you have to do is to add a module to your application (Menue--Project--AddModule). In this module, add the following code: Option Explicit 'see example VB application for more constants Private Const RPC_C_AUTHN_NONE As Long = 0 Private Const RPC_C_AUTHN_LEVEL_NONE As Long = 1 Private Const RPC_C_IMP_LEVEL_IMPERSONATE As Long = 3 Private Const EOAC_NONE As Long = &H0 ' Import function from ole32.dll Private Declare Function CoInitializeSecurity Lib "OLE32.DLL" ( _ pSD As Any, _ ByVal cAuthSvc As Long, _ asAuthSvc As Long, _ pReserved1 As Any, _ ByVal dwAuthnlevel As Long, _ ByVal dwImpLevel As Long, _ ByVal pAuthInfo As Long, _ ByVal dwCapabilities As Long, _ pvReserved2 As Any _) As Long


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Sub Main() ' following code will fail in the IDE (debug mode). Dim lngHr As Long Dim lngAuthn As Long lngAuthn = RPC_C_AUTHN_NONE lngHr = CoInitializeSecurity(ByVal API_NULL, _ -1, _ lngAuthn, _ ByVal API_NULL, _ RPC_C_AUTHN_LEVEL_NONE, _ RPC_C_IMP_LEVEL_IMPERSONATE, _ API_NULL, _ EOAC_NONE, _ ByVal API_NULL) If (S_OK <>lngHr) Then MsgBox "CoInitializeSecurity failed with error code: 0x" _ & Trim$(Str$(Hex(lngHr))) & vbCrLf & _ "Ignore, if running in IDE - but you will not receive events " & _ "from remote microscope server", _ vbCritical, _ "Application Initialization Failure" Exit Sub End If ' Any additional code you need here. frmYourframe.Show ' showing your starting form End Sub Now, in Project--Properties--General tab, change the startup object to Sub Main. Note that Sub Main has to have code to make your forms visible (such as frmYourframe.Show). The call to CoInitializeSecurity will fail, when you are working in the VB development environment (IDE), debugging your application. In that case, the IDE has already initialized COM security. This means, you will receive events only, if you run the compiled executable.

26.7 Errors and error handling Some of the microscopes functions may return (raise) an error. This can happen due to a physical reason, i.e. if the requested action is not possible at that moment. For example, you cannot ask the stage to perform a GoTo(), when it is wobbling or already moving. The high tension may not be switched on or raised under certain conditions and so forth. You may also have given parameters that are out of range (in that case the stage for example would not move either). Generally, calls to another process (from your script to the TEM server) may fail occasionally. There are also some failures that may be VisualBasic-specific (see below). So be aware and do the error handling - otherwise your script might die! If an error is raised, then a global Error object (Err) will be filled with detailed information about the error, if available. You can use this information for your error handling. Usually you will get an error code (a number), a textual description and maybe a text string with the name of the source that raised the error. The following piece of code gives an example: (Suppose you have a form with at least a button named ‘cmdGoto’ and a textbox named ‘txtDisplay’. As reaction on a button-click a new stage position is calculated -just as an example, you could invent some


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code here- and the stage is requested to move to that position. In case of an error the content of the error object is shown in the textbox): Private Sub cmdGoto_Click() Dim NewPos as TEMScripting.StagePosition Dim OldPos as TEMScripting.Stageposition Dim MyStage as TEMScripting.Stage On Error GoTo ComError: Set MyStage = MyTem.Stage Set OldPos = MyStage.Position CalculateNewPosition OldPos, NewPos MyStage.Goto NewPos, axisXY Exit Sub ComError: txtDisplay.Text = _ txtDisplay.Text & "Error # " & Hex(Err.Number) & _ " was generated by " & Err.Source & vbCrLf _ & Err.Description & vbCrLf txtDisplay.SelStart = Len(txtDisplay.Text) End Sub At the end of the error handling you can either do nothing (as in the above example), that is leave the function or add either Resume which jumps back to the line in your code where the error occurred or Resume Next which jumps back to the next line in your code Goto MyLabel jump to a specified label, here named MyLabel. Another possibility is to use the On Error Resume Next ... On Error Goto 0 statements. This allows you either to ignore errors or ask for the contents of the Err object after each relevant statement and do the error handling ‘in place’. If you handled an error you should then clear the content of the error object, using Err.Clear. For more information see the Visual Basic documentation.

26.8 Visual Basic-specific Errors At last, two examples of errors that you might encounter and which may be specific to Visual Basic 5.0 (but we have not yet checked in other scripting languages and the 6.0 version): 1) From a VB application it is not allowed to do more than one out-of-process call at a time. You might think that this is a rare situation, but you can easily encounter this case. Imagine the following: You have a timer running in your application that polls for certain microscope parameters of interest in regular time intervals. (Maybe you want to protocol the vacuum pressures or the stage position.) At the same time


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your application has a user interface, through which the user can request actions by pressing a button (maybe start a pumping cycle or an exposure of some kind). Then, especially when you use functions that take a long time to complete, there is a chance that your application tries to issue a call from your timer loop while a first one (issued via a button command) did not yet return. This second call will then fail (error description: "illegal to call out while inside message filter"). The reason is the way the apartment threading and message loop are implemented in VB. 2) A right click on the applications button in the Windows button list will also cause the out of process calls to fail, as long as the small "Restore, Move, Size etc."-menu is open (see image below). We did not do any further research why this happens, but it may be related to 1).

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