e lectron m icroscopy advantages and disadvantages of tem advantages and disadvantages of sem...
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EELECTRONLECTRON
MMICROSCOPYICROSCOPY
Advantages and disadvantages of TEMAdvantages and disadvantages of TEM
Advantages and disadvantages of SEMAdvantages and disadvantages of SEM
IntroductionIntroduction
Principles of operation of TEMPrinciples of operation of TEM
Principles of operation of Principles of operation of SEMSEM
Sample preparation for Sample preparation for TEMTEM
Sample preparation for SEMSample preparation for SEM
Investigating micrographsInvestigating micrographsSchool of
BiologicalSciences
INTRODUCTION
With the invention of the light microscope it was discoveredthat plant and animal tissues were made up of aggregates ofindividual cells. However, light microscopes are limited to approximately x1000 magnification and have poor resolutionresolution..Therefore not all the internal structures of a cell can be seenwith a light microscope.
In 1924 a French physicist by the name of de Broglie stated that a beam of electrons should behave in a similar way to a beam of
light i.e with wave properties the wavelength should be shorter. Therefore an electron
beam should give better resolution.
RESOLUTIONWhen there is sufficient light, two points 0.2mm apart or
morecan be distinguished with the naked eye as being separate points. When this distance is less than 0.2mm, only one
point is seen. This distance is called the resolving power (or
resolution) of the eye.In other words resolution is the closeness two objects can be
inproximity and still be perceived as two separate objects.
Can’t see two separate objects
Can see two separate objects
The invention of the electron gunled to the development of theelectron microscope.
The metal tungsten filament is heated to about 2500oC which causes it to release electrons.
Due to the large voltage difference between the
filament and the anode plate the electrons are forced to flow in the direction of the arrow.
The cathode shield increases the electron flow further and concentrates the electrons into a narrow beam. Anode
Electron beam
Cathode
Filament
High voltage generator
Back to principles of operation
TEM Transmission SEM Scanning
TEM produces a high resolution image of the internal structures of cells. TEM uses the electrons that have passed through the specimen to form an image.
Two main types of electron microscopes;
SEM produces a three dimensional image of the specimen surface. A beam of electrons scans the whole specimen which then emits low energy, secondary electrons. This technique can be used to study whole cells.
When an electron beam strikes a specimen a number of events occur. Electrons are scattered depending on the nature of the material.
If the electrons hit a dense array they are scattered out of the main beam and fewer electrons will reach the viewing screen. There is no fluorescence and that area appears dark.
If the electrons pass a scarcity of atoms they travel straight through, hitting theviewing screen and causing fluorescence. That area will appear light. The image comes from the arrangement of light and dark patches on the screen.
PRINCIPLES OF OPERATION OF TEM
Specimen holder
Electron gun
Projection chamber
THE ELECTRON GUN – producesan electron beam.
THE COLUMN – uses electromagnetic
lenses to control the beam and produce a magnified image
IMAGE VIEWING AND RECORDING
The image is produced on afluorescent screen below which a shutter and camera are located.
Condenser lens
The main components of a TEM are:
Objective lens
Projector lens
Specimen
Fluorescent screen
Vacuum
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Electrons only behave like light when they are manipulated in vacuum. Therefore the whole column is evacuated since atoms such as O2 and CO2 scatter the electrons.
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SAMPLE PREPARATION FOR TEM
The aims of sample preparation are as follows:
To preserve the material in its natural state
To ensure that the material withstands changes which might occur on exposure to atmosphere, vacuum and electron beam.
Fixation for TEM
The tissue is cut into tiny pieces
It is then placed into fixing solution
Dehydration and embedding of TEM
Tissue is dehydrated in alcohol
It is then placed in a dilute solution of resin embedding media
Specimen vials
Tissue is placed in final embedding mixture and the resin is polymerised in the oven
Section cutting of TEM
Sections are cut on an ultramicrotome with a glass or diamond knife. The sections are floated off the edge of the knife onto the surface of a water trough.
The colour of the sections vary with thickness. When the sections are gold they are picked off the surface with a copper grid.
The section on the copper grid is now ready for staining and viewing in the electron microscope.
3.05mm
Advantages Disadvantages
Thin sections are effectively two dimensional slices of tissue and do not convey the three dimensional arrangement of cellular components
Very good resolution
Can see sub-cellular components and measure them
magnification
Artefacts may be created
TEM
PRINCIPLES OF PRINCIPLES OF OPERATION OF SEMOPERATION OF SEM
Control panel
Image viewing
Specimen chamber
Electron gun
DetectorDetector
SEM uses electrons that are emitted from the specimensurface.
The specimen is scanned with a very fine beam of electrons.
These are scattered as they hit high and low points in the specimen.
The scattered electrons are measured by a detector and used to control a second beam which forms an image on a TV screen
Specimen
Vacuum
Detector
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Electron source
Electron beam
T.V. Monitor
Sample preparation for SEM
The preservation used will usually determine which
drying process to use.
There are two basic methods of drying the specimen:
Critical point drying – used after chemical fixation and dehydration
Freeze-drying – used after freezing
Freeze drying for SEM
Then placed in liquid nitrogen to allow easy handling
Sample placed in nitrogen slush to maintain it’s structure
Sample is mounted on a stub
Sample placed in copper holder and……….
…..placed in freeze drier
Critical point drying for SEM
Sample is chemically fixed
Then dehydrated with alcohol
Sample is placed in critical point drier. Here the sample is flushed several times with liquid CO2. The pressure andtemperature is then raised which converts the liquid CO2 to gas. The gas is then vented off slowly.
The sample is removed and mounted on a stub
Coating the specimen for SEM
Most biological specimens are poor conductors and poor
emitters of secondary electrons therefore the surface of the
sample needs to be coated with a thin layer of a conducting material.
There are two ways to do this:
Sputter coating
Evaporation of carbon
Sputter coatingSputter coating for SEMWhen power passes to the anode, the noble metal evaporates (called the plasma effect) and the metal falls onto and coats the specimen.METAMETA
LL
ANODE
CATHODE
A sputter coater
Evaporation of carbon for SEM
Two carbon rods are placed end to end. One of the rods is
sharpened to a point. These are placed in a vacuum and the
specimen is placed below them. When electricity passes
through the carbon rods, the carbon tip evaporates and the
carbon falls onto and coats the specimen.
CARBON RODS
CARBON RODS
Advantages Disadvantages
Provides great depth of focus
Micrographs show a 3D image of specimen
Smaller and simpler in comparison to TEM
Only surface features seen
Resolution attainable is not very high (approx 10nmn)
SEM
Investigating MicrographsTransmission Electron Micrographs Scanning Electron Micrographs
Can you spot the differences between the two types of electron microscopy?
Transmission Electron Micrographs
Mitochondrion
Plasma membrane
Endoplasmic reticulum
Golgi membranes
Virus particles
Section of mammalian cell
Scanning Electron Micrographs
Sample of geranium petal showing the cone shaped projections and the internal structure.
Measuring MicrographsMagnification of micrograph is X100000 How to work out the size
of an organelle?
Measured size Magnification
80000 m 100000
= 0.8m or 800nm
Measured size = 80mm
Convert to m = 80000
for using this programme. We hope that it has been useful!
THANKS TO:Chris Gilpin, Ian MillerLes Lockey, Samantha Newby
This programme was developed as part of a work placement project by Sumerah Khan and Sheerin Dariani
References
1) B. Schotanus (1980) Electron microscopy, what is it ? Marketing electron optics. Philips Export B.V. Eindhoven.
2) Dr Yvonne Miller (1998) Preparation of specimens for TEM and SEM.
3) Mike Mahon, Chris Gilipin, Ian Miller (2000) Microscopy and analysis University of Manchester - School of Biological Sciences.
4) Sam Newby (2000) Freeze drying and critical point drying EMPGU.
5) Specimen preparation (1991) (21/1/00) http://www.lifelong.com/lifelong-universe/Academic world/SEM/ specimenprep.html pages 1-2.
6) Dr. Ron Butler (1980) Transmission electron microscopy, What an SEM is ?, Aims of specimen preparation and Electron microscopy unit. EMPGU