4.
Setting up Fluorescent light imaging
~ In software, navigate to
“Acquisition” tab (G). Select
required Image Format and Live
Format (binning 1x1, 2x2 etc)
and “Black & White mode” for
the camera (H).
~ Then, on “Acquire”
tab
(I), set
up
light path for imaging of
fluorophores of interest (J)
.
Press “+” or “-” button to achieve
necessary number of channels
(one for each fluorophore to be
imaged). The selected channel is
colored red. For each channel,
select from preset settings (dropdown list) or manually set by selecting “FLUO”, then selecting the
appropriate filter set (hover over filter for details) and set LUT by colored bar at top of channel.
~ Set image acquisition parameters for each fluorophore, as appropriate. To do that, select the one to be
adjusted. Then click the “Live” button to check settings.
~ View image as intensity values by selecting LUT (Look Up Table) tool with the QLUT button.
~ Adjust Exposure and Gain (K) so that there are mostly orange and red pixels and no blue (saturated)
pixels. Click “Stop”. Gain should be below 4 for highest quality image. Switch to another channel and
repeat adjustments.
~ When all settings are set, click "Start" to start the acquisition.
5.
Acquiring a Z-series
~ In the Acquisition tab (G), be sure X, Y & Z are selected under Acquisition Mode.
~ Click “Live”. Move to the top of your sample with the focusing knob and set the position by clicking the
“Begin” arrowhead (L).
~ Move to the bottom of your sample with the focusing knob and set the position by clicking on the “End”
arrowhead (L). Click “Stop”.
~ Set the desired number/size of z steps.
~ Ensure imaging settings are correct, then hit “Start” to collect z-stack.
6.
Save images
~ In the “Open projects” tab, click “Save All”. Select the appropriate
directory and change the Experiment file name, if desired.
C
lick OK to save.
7.
Shut Down
~ Remove sample. Clean oil off objectives with lens paper. Return objective turret to 10x.
~ Transfer files to flash drive or network storage. Close software.
~ If there is another user coming within 1 h, stop here, leaving microscope ON.
~ If not, proceed with following steps for complete shutdown:
~ Turn OFF the microscope controller box (A) and LED box (B). Cover microscope.
~ End reservation in NUCore.
If help is needed, please see BIF staff
