live cell 2 guide - king's college london | homenikon imaging centre @ kings college london...

Live Cell 2 guide NIKON IMAGING CENTRE @ KING’S COLLEGE LONDON

King’s College London Nikon Imaging Centre

1 Nikon Imaging Centre @ King’s College London

Start-up

1. Switch on the numbered components in order and refer to figures 5-10 for details of the controls.

2. Using the joystick, move the stage (fig. 9) so that the objective is centrally positioned. The speed of the XY-

stage can be varied via the XY button above the joystick. A focus wheel can be found on the side of the joystick

unit and the speed of focussing can be changed by pressing Z button on the microscope or joystick. The speed

of movement is indicated on the joystick display panel - ^ denoting slow movement.

3. Objective Lenses on the live cell system are 4x, 10x, 20x and 40x dry objectives. Check the correction collar is

set to 0.17 on the 20x and 40x objective lenses for imaging through standard #1.5 coverglass; the collar can

be adjusted for thicker samples. 60x and 100x objectives can be added to the system but are not permanently

installed.

Nikon Imaging Centre



King’s College London

Fig. 1

sCMOS

(monochrome)

camera

Colour

camera

Fig. 2

Fig. 3

Do not

switch off

Fig. 4

Microscope

Switch




Objective

selector

Light path

Fig. 5

Intensity

dial Transmitted

light on/off

Condenser

element

selector

Fig. 6

Fig. 7

Epi-fluorescence

shutter

Epi-fluorescence filter turret

Fig. 8

PFS

on/off

z-speed


Incubation Chamber Control

Temperature and CO2 control is facilitated through the external Oko-lab Touch controller (Fig. 11) which in turn allows

for monitoring of the incubator status.

1. The home screen shows a readout of the current temperature and CO2 concentration (Fig. 12), the coloured

status indicator shows green when the settings are within the normal limits but will change to yellow if the

temperature/CO2 concentration drops too low for a significant amount of time. This yellow indicator is

accompanied by an audible alarm which can be silenced by pressing the alarm indicator on the touch screen.

2. Selecting temperature or CO2 opens a second window where the set point can be adjusted (Fig. 13) The values

can be changed using the +/- buttons. Confirm the settings by pressing set.

3. For CO2, the valve on the tank needs to be adjusted to ensure that it is entering the system correctly: open the

top valve on the CO2 bottle (anticlockwise), then turn the regulator knob (clockwise) to achieve a pressure of

0̴.3 bar.




Fig. 10

Fig. 1

Fig. 9

4-way display selector

(access to x,y,z

information)

XY speed

PFS on/off

z-speed

LED lamp

switch

Lamp

intensity

controls

Fig. 10


Fig. 11

Fig. 12



Fig. 13

Press to set CO2

concentration


Adjusting Kӧhler illumination (Transmitted light)

For transmitted light imaging it is essential to optimise the condenser alignment to ensure that the full numerical

aperture of the microscope system can be realised (to provide an even illumination of the sample). It is therefore

important to ensure that the light passes through the condenser optimally to allow for high quality imaging.

N.B. Kӧhler Illumination adjustment should only be conducted through the eyepiece.

1. Through the eyepiece, focus on your. Fully close the field iris by turning it anticlockwise. (Fig. 14)

2. Using the adjustment knob, move the condenser whilst looking through the eyepiece until you see the

octagonal field iris. Turning the knob towards you to bring the condenser lens close to the sample. Adjust the

condenser focus so that the outline of the field iris is sharp and in focus. (Fig. 15)

3. If required, centre the field iris in the eyepiece using the adjustment screws on the condenser unit.

4. Once centred, open the field iris again so that the field iris edges are just outside of the field of view.



Field stop iris

diaphragm

Alignment screws (use

allen key if centering

required)

Condenser

focus

Fig. 13

Condenser

lens


Viewing your sample through the eyepiece:

The light-path and filter settings are stored in Elements using so-called optical

configurations. These are accessed via the OC Panel (Fig.16). The optical

configurations are grouped into categories to control viewing through the eye

port, viewing with the DS-Qi2 (sCMOS) camera (used for epi-fluorescence and

transmitted light imaging) and with the colour DS-U3 camera (e.g. for

histological stainings).

To view your sample through the eye-port, select one of the configurations

from the ‘eyes’ group of configurations. These configurations:

1. Set the light path to EYE

2. Set the filter in the turret to that relevant to the dye being used.

3. Make sure that the fluorescence lamp is turned on and use the

microscope base for control of the fluorescence shutter.

4. Transmitted light imaging can be achieved using the relevant OC and

using the lamp on/off but on the left hand side of the microscope;

intensity can be adjusted using the dial. For transmitted light imaging

you can select from a number of configurations: phase imaging 1 for

objectives up to 20X, phase imaging 2 for the 40X, DIC for 60X and

100X and open for standard brightfield.



Fig. 15

Fig. 16


Image Acquisition

As with the settings for viewing the sample through the eye-port, optical

configurations are employed to make changes to the selected filters and the

light path. Further to this, the optical configurations also save the detector

settings which may vary between configurations on multichannel samples.

When starting Elements software the correct camera driver needs to be

selected (note that it is possible to select both). After logging in it is possible to

select the required driver from the dropdown menu: Nikon DS-Qi2 for

brightfield/fluorescence imaging, Nikon DS-U3 for colour imaging (e.g.

histological stainings). (Fig. 17)

Acquiring a colour image – DS-U3 camera.

1. Select the colour camera BF optical configuration from the OC

Panel (Fig. 16 )

2. Focus on the sample and adjust the detector settings so that you

can see the surface.

3. Set the transmitted light to 100%.

4. Move to a blank region of the sample (Fig.19) and select Auto

White in the DS-Fi2 setting window (Fig. 18). This will ensure that

your images have the correct white balance (Fig.20).

5. Move back to your sample to image it with the correct colour

representation. If your Kohler illumination is set correctly

acquisition time for the detector should be <100ms and the gain

should be <10x. Ensure that high quality capture is selected for

your images. (It can be useful to use the Auto Exposure option to

set the exposure time.)

6. Click the capture button or run relevant ND acquisition.



Fig. 17

Fig. 18

Fig. 19 Fig. 20




Channel Settings

1. Select the optical configuration for transmitted light or the fluorescent marker that you want to optimise

settings for (this needs to be done for all channels).

2. Set camera settings to around 100ms exposure and 2X gain then begin live mode.

3. Optimise the exposure settings, keeping the exposure time below 1 second and adjusting the gain so that the

sample is visible and that the background contribution isn’t too high. Ensure that there are no oversaturated

pixels in the image. Oversaturation is indicated through the presence of a dot in the top right-hand side of the

histogram (indicating pixels with 65535 value) or through the saturation indicator (Fig.20). Note that the DS-

Qi2 camera is a low noise sCMOS camera and therefore it is not necessary to use the full 16bit range, especially

if these means setting a very long exposure time.

4. The PE-pad can be used to adjust the intensity of the LED lamp. This defaults to 100% but can be reduced if

the sample is prone to photobleaching or phototoxicity.

5. The camera used for fluorescence imaging has two resolution options which vary the number of pixels in the

image. This can be changed with the channel settings however if a multicolour image is being acquired the

resolution needs to be the same for every optical configuration. Note – higher resolution images will lead to

much larger file sizes.

6. Press capture to take a single channel image. Note - Single channel images are not automatically saved and

will require saving as ND2 or TIFF files.

7. Multichannel acquisition requires the use of the ND acquisition panel. Select the tab and add the number

of channels you require. For each channel select the optical configuration from the drop-down list, ordered so

that the configurations go from longest wavelength to shortest. Press Run Now acquire the multichannel

image.

Live Capture

Fig. 21

Fig. 22 Fig. 23 Saturation indicator Mouse x/y


Multi-dimensional acquisitions

For timelapse, large images, Z-stacks and multipoint imaging, the ND acquisition window is used. This allows files to

be saved whilst image acquisition is running.

Timelapse imaging

1. For timelapse imaging, select the Time tab in ND

acquisition and add a time point into the ND

acquisition menu.

2. Set a frame interval and duration that are

suitable for your samples. From the drop-down

menu in each of these sections the time units can

be adjusted from msec to hours. The interval can

be set to ‘no delay’ which will begin a time loop

immediately after the previous. On the duration

box, the time can also be set to continuous which

will require the user to stop the acquisition after

the desired period.

3. Other ND acquisition modes can be added to the

timelapse (e.g. z-stacks, multipoints) which when

checked and set up in the ND acquisition window

are added to the image sequence. NOTE –

multidimensional parameters will affect the

achievable frame interval of the timelapse.

Clicking ‘timing’ will allow you to see if the

interval is set at an achievable rate.

4. To begin the time lapse image press ‘Run Now’

Multi-points

1. To image multi-points, select the XY menu in ND

acquisition.

2. To add image coordinates click add and click the

arrow point at the coordinates to capture them.

Check the include z box to allow capture of z in

the same way (if PFS is used this can also be set

in the same way).

3. The arrows, shown in red boxes in Fig. 25, are

used to update stored positions.



Fig. 24

Fig. 25


Large images

1. Tiled images can be captured by selecting large

image in ND Acquisition. (Fig. 26)

2. Set the number of fields for the large image,

select stitch and set overlap to 15%.

3. Click ‘run now’ to acquire. If selected alongside

timelapse, make sure that large image

acquisition loop fits into frame interval via the

timing button.

4. A more advanced method of tiling can be

accessed through Acquire>Scan Large image on

the upper menu bar.

5. Select the objective for capturing the image, set

the number of fields in XY and whether the tiling

should scan around the current position of the

stage or with the stage position being the top

left. Set the overlap to 15% and select blending

(making sure image registration is checked). (Fig

27)

6. Large image files can either be saved as the

whole large image, the single frames (as TIFFs)

or both. Set your folder for saving and file name

for the save option required.

7. Z-stacks may also be applied to the image as

well as automatic refocusing throughout the

acquisition. Once all options have been

adjusted, click scan.

Fig. 26




Z-stacks

Although not an ideal imaging mode for wide-field techniques due

to the presence of out-of-focus light, it is possible to perform Z-

stacks which may be used for 3D visualisation or for maximum

intensity projections. Select the Z-stack tab in the ND Acquisition

window (Fig. 28).

1. Press reset to remove any previously saved z-positions.

2. Three options are available for setting Z-stack limits: Top

and Bottom (mark the limits of the stack), Symmetric

(mark the centre of the stack) and Asymmetric (set the

number of slices above and below the centre).

3. For the top-bottom option, whilst live imaging, adjust the

focus to find the top of your sample (use the Z value as a

guide) and click Top. Focus back through the sample to do

the same for the bottom plane of the sample. Stop imaging

to avoid sample bleaching.



Fig. 28

Fig. 27


4. If you want to conduct Z-stacks alongside multipoint imaging it is better to use relative Z-stacks in the

symmetric and asymmetric options.

5. Select either the optimal suggested step size or define the number of steps.

6. Press Run now to acquire.

Saving Data

Whenever you use the Capture button you will need to manually save each acquired image (ND acquisition can

autosave).

1. Got to the File menu and save the image in ND2 format. This will store the complete setup of the microscope

in the metadata of the image. Using the ND2 format also means that you can reuse the acquisition/camera/ND

acquisition settings at a later date (see below for details).

2. Save your data to a folder on Datadrive E:/ and at the end of the session transfer your files to the network

storage drive (N:/LiveCellData). Data is kept for a maximum of 1 week in the documents folders and 1 month

on the network drive.

3. Instructions on how to connect the network drive to your King’s computer can be found when you login to

your PPMS account, go to documents and select the N drive access document.

Reusing settings

1. When comparing images; such as for fluorescent intensity it is

important to keep the settings the same to enable a fair comparison. To

re-apply the same settings from an image, open image in the NIS

software, right click and select reuse camera settings and reuse device

settings.

2. You can also reuse ND setup for keeping the same settings in the ND

acquisition window.

3. When reusing settings check the Ti2 pad and the camera settings to

ensure that they have been applied correctly.



Fig. 29

Fig. 30


Troubleshooting

1. If NIS elements or the microscope stops working or crashes, try to restart the software.

2. If this doesn’t solve the problem, restart all microscope components and the computer.

3. If you are still having difficulties, notify a member of NIS staff immediately. In the event that you cannot find

a member of staff you should send an email describing the problem or use the incident reporting system in

PPMS.

Switching off and tidying.

1. Check the PPMS booking system and if another user is booked within the next hour, leave all the components

on (except for the CO2, if you have used it) and just exit the software.

2. Remember to transfer your data from the datadrive to livecelldata.

3. Remove your sample from the microscope stage.

4. Lower the objective fully using the manual focus wheel and move the stage to the central position.

5. If no user is due on the system after you shut down all components in reverse order to the system start up.

6. Clean oil objectives with the provided lens tissue and ethanol (if you have used oil)

7. Return any oil bottle or slide cleaner used to the image analysis room (shelf above the safe)

8. Dispose of any used tissue in the bins, slides in the sharps bin and clinical waste in the yellow bin in the TC

room.



live cell 2 guide - king's college london | homenikon imaging centre @ kings college london...

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