7.
Login: LSMuser (password: Useme!11)
8.
Open
ZEN Blue
Software and press “ZEN System”
9.
“Image Processing” is for offline analysis of your images, please do not use it here
III.
Visual examination of the sample
1.
Put mechanical switch on the right hand side of the microscope to the ”Eye” position.
2.
Press “Locate” in the main microscope menu.
3.
Since this is an upright system, changing objectives can only be manually done by rotating the objective
revolver. Rotation should work very easily when objectives are in the “up” position.
4.
For imaging or visual inspection, the objectives must be lowered entirely with the black wheel in front of
the microscope stand.
5.
Please make sure that 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.)
6.
If you wish to view your sample in Brightfield mode, press the “TL” button
7.
For visual examination of fluorescence, press the buttons DAPI, GFP or RFP
IV.
Acquiring confocal images
1.
Set mechanical switch on the right hand side of the microscope to the “Camera” position.
2.
Select “Acquisition” in the main menu of Zen. (Software will ask you about the mechanical switch,
Press”OK”.)
3.
Go to “Smart Setup” add your dyes pressing “+” button (set the LUT) and configuration, you can choose
between simultaneous (fastest) and sequential (best signal) scan, press ”Apply” after choosing the
appropriate setup.
4.
If you are using the detector for transmitted light: activate the ESID Detector represented in the light
path scheme. Make sure Köhler illumination is set properly if you are getting bad bright field images!
5.
Go to “Live” acquisition modus and find the focus manually.
6.
Set pinhole to Airy 1 in the channel dialog to achieve confocality.
7.
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.
7.1.
What Auto exposure does is to increase the PMT voltages until about 1% of all pixels are
overexposed. Gain cannot go below 500 V and above 900V.
8.
In the channel dialog you can adjust the pinhole, gain and laser power manually to have the best image.
You can increase laser power by default only till 4,5%. If you need more power you need to enable
“Higher Intensity Laser Range”.
Be aware that GaAsP PMT are very sensitive and if you will put to much
power, they shut down for the course of imaging (& do not turn on automatically). In that case you need
to stop acquisition and restart the imaging manually!
9.
To zoom in your image, go to “Acquisition parameteres” at the drop down menu. With “Crop” you can
select the desired area of your image, then go to “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” to adjust the optimal number of pixels and the pixel size
according to Nyquist sampling criteria.
