2
III.
Visual examination of the sample
1.
Press “Locate” in the main microscope menu and “Online”
2.
You can change objectives automatically on the small touch screen attached on the right side
of the microscope by selecting “microscope” menu and “objectives”
3.
Please make sure the chosen objective corresponds to the one shown in the software. If this is
not the case please restart the system. (Otherwise your pinhole and pixel sizes as well as the
sectioning thickness are all incorrect.)
4.
If you wish to view your sample in brightfield, press the TL button in the “Locate” menu
5.
For visual examination of fluorescence, press the buttons DAPI, GFP, RFP or FarRed
6.
If sample appears too dim, increase light intensity by pressing the ‘Colibri 5/7’ button and
adjust the LED intensity slider.
7.
Before switching to acquisition mode, close transmitted and reflected light shutters by
pressing ‘off’ and turn off the LED light source by clicking on ‘Colibri 5/7’ and pressing
‘Deactivate all LEDs’.
IV.
Acquiring confocal images
•
Select the ‘Acquisition’ tab in the main menu of Zen blue
•
Go to “Smart Setup” choose your dyes (set the LUT) and configuration, you can choose
between simultaneous (fastest), Best compromise line scanning mode (Smart) and sequential
(best signal) scan, press ”Apply”.
•
If you use ‘Smart setup’, please make sure that you DO NOT have any track using “Ch2”,
which is the Airyscan GaASP detector! You’ll have to manually switch the channels that are
assigned to it to a different detector: low wavelengths to ‘Ch1’ and high wavelengths (from
mCherry on) to ‘Ch3’. Due to the arrangement of dichroic mirrors in the scanhead, it won’t
work the other way round!
•
If you are using the detector for transmitted light (ESID): activate (ESID) represented in the
light path scheme. Adjust intensity in the ESID Gain, then adjust digital offset and digital gain
of the ESID detector in order to “stretch” the histogram and use the whole dynamic range of
the detector.
