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Live
Fig. 21
Fig. 23
Saturation indicator
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.
Capture
Mouse x/y
Nikon Imaging Centre @ King's College London
Nikon
Imaging Centre
8
King's College London
Fig. 22
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