3
•
Make sure Köhler illumination is set properly if you intend to use the ESID detector
(for bright field images)!
•
Press the “AF” (Autofocus) button to find your focus, alternatively go to “Live” acquisition
modus and find it manually
•
Set pinhole to Airy Unit (AU) 1 in the channel dialog to achieve confocality. Note, that activating
the ‘1AU’ button by clicking on it once (gets light blue background color) fixes the pinhole to
1AU. In this case, the match pinhole function in the Z-stack setup will not change pinhole sizes.
•
In the ‘Channels’ dialog you can adjust the pinhole, gain and laser power manually
•
Press the “Auto exposure” button and the software will adjust the gain of the PMTs but not the
laser power, according to the brightness of your dyes.
•
What Auto exposure does, is to increase the PMT voltages until about 1% of all pixels are
overexposed. If the resulting gain is below 400 V, decrease the laser power. If the gain is above
900 V, increase the laser power.
•
To zoom in your image, go to ‘Acquisition mode’ and agjust the slider accordingly. With “Crop”,
you can select the desired area of your image, then press “Live” or “Snap” to apply. “Reset all”
will remove the zoom again. Depending on the scan speed, the lowest zoom is 0.5 and not 1.0.
•
Before taking an image, press ‘optimal’ in the ‘acquisition mode’ tab to adjust optimal number
of pixels and the pixel size according to Nyquist sampling criteria.
Note: More pixel than suggested by the software as ‘optimal’ almost never make sense. On the
other hand, feel free to decrease the pixel number to increase acquisition speed at the cost of
resolution.
•
To capture single images, click on ‘Snap’
