Nikon Live Cell 2 User manual

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 Zbutton 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
Nikon
Imaging Centre
King’s College London
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

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Nikon Imaging Centre @ King’s College London
Nikon
Imaging Centre
King’s College London
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

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Nikon Imaging Centre @ King’s College London
Incubation Chamber Control
Temperature and CO2control 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 CO2concentration (Fig. 12), the coloured
status indicator shows green when the settings are within the normal limits but will change to yellow if the
temperature/CO2concentration 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 CO2opens 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 CO2bottle (anticlockwise), then turn the regulator knob (clockwise) to achieve a pressure of
0.3 bar.
Nikon
Imaging Centre
King’s College London
Nikon
Imaging Centre
King’s College London
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

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Nikon Imaging Centre @ King’s College London
Fig. 11
Fig. 12
Nikon
Imaging Centre
King’s College London
Fig. 13
Press to set CO2
concentration

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Nikon Imaging Centre @ King’s College London
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.
Nikon
Imaging Centre
King’s College London
Field stop iris
diaphragm
Alignment screws (use
allen key if centering
required)
Condenser
focus
Fig. 13
Condenser
lens

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Nikon Imaging Centre @ King’s College London
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.
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Imaging Centre
King’s College London
Fig. 15
Fig. 16

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Nikon Imaging Centre @ King’s College London
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.
Nikon
Imaging Centre
King’s College London
Fig. 17
Fig. 18
Fig. 19
Fig. 20

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Nikon Imaging Centre @ King’s College London
Nikon
Imaging Centre
King’s College London
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

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Nikon Imaging Centre @ King’s College London
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.
Nikon
Imaging Centre
King’s College London
Fig. 24
Fig. 25