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Live Cell 1 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.
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Objective
selector
Light path
Fig. 4
Fig. 3
Do not
switch off
Fig. 1
Fluorescence
camera
Fig. 2
Microscope
Switch
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Nikon Imaging Centre
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Fig. 7
PFS
on/off
z-speed
Intensity
dial Transmitted
light on/off
Condenser
element
selector
Fig. 5 Fig. 6
Epi-fluorescence
shutter
Epi-fluorescence filter turret
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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.
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Fig. 8
4-way display selector
(access to x,y,z
information)
XY speed
PFS on/off
z-speed
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LED lamp
switch
Lamp
intensity
controls
Fig. 9
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Fig. 10
Fig. 11
Fig. 12
Press to set CO2
concentration
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Adjusting Kӧhler illumination (Brightfield)
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.
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Field stop iris
diaphragm
Alignment screws (use
allen key if centering
required)
Condenser
focus
Fig. 13
Condenser
lens
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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.
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.
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.
Fig. 16
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Live Capture
Fig. 17
Fig. 15
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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.
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Fig. 18
Fig. 19 Saturation indicator Mouse x/y
Fig.20
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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. 22
Fig. 21
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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. 23
Fig. 24
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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).
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.
Saving Data
Whenever you use the Capture button you will need to manually save each acquired image.
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.
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Fig. 25
Fig. 24
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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 (Fig. 25).
3. When reusing settings check the Ti2 pad and the camera settings to
ensure that they have been applied correctly.
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.
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Fig. 26
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