4
•
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 adjust 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’ rarely 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’ (Snapped images are overwritten if you press
Live
before saving them)
1.
Setting up Z-stack:
Enable Z-Stack and the Z-Stack dialog will appear
Press “Live” and set your first and last slice
The software will suggest optimal slice number and interval which you can change manually
Activate the
‘Optimize sectioning and step’ checkbox to see slice overlapping. Here you can
again set an optimal interval to ensure ~50% overlap between sections.
If you wish to use the match pinhole function, make sure that the ‘1AU’ button in the
‘Channels’ menu is deactivated on all tracks (no blue background of button). This function will
match pinhole sizes to the optimal pinhole size of the lowest wavelength of your imaging setup
and ensure that all sections have the same thickness.
Press “Start experiment”
2.
Setting up time series:
Enable “time series”
Time series dialog will open
Set cycle number and interval time
Press “start experiment”
3.
Tile Scan (in this mode you have: Tiles, Multipositions):
Enable “tile scan”
