Zeiss LSM 880 Operating Manual page 625

LSM 880
3. Start a reflection bidi line bidi z scan at zoom 1.0x and focus on the border between coverslip and
sample, which can be identified by the brightest reflection line. A high pixel resolution is not
important (use e.g. the 256x1line format). Adjust the detector gain and laser power (the laser power
likely will be below 1%). The maximum intensity should be between 1,000 and 3,000 when using a
12Bit data depth. If very thin samples are used, be cautious to get the reflection of the sample-
coverslip border and not e.g. the sample-slide border.
4. Activate the Z-Stack acquisition checkbox. Switch to center mode and set up a Z-Stack with about 80
slices and an interval of 0.2µm. Activate z-Piezo. Click on the 'Center' button and the start the Z-
Stack with a long time series. Use Corr X, Corr Y and Corr Z to remove jitter as much as possible.
5. Check for the tilt of the coverslip. If the reflection line is horizontal the coverslip in not tilted in the x
direction (Fig. 12/A). Any tilt will appear as a sloping line (Fig. 12/B). We recommend choosing a z/x
ratio of 10 to make the tilt more evident in the image.
Adjust the adjustment screw sx of the sample holder until the reflection line is horizontal (Fig. 11).
6.
You might need to re-adjust the z-range when turning the screw.
Rotate image clockwise by 90 degrees. Adjust the adjustment screw sy of the sample holder until
7.
the line reflection is horizontal. You might need to re-adjust the z-range when turning the screw.
Screw sz will only have to be adjusted in case the range of screws sx and sy is not sufficient.
10/2014 V_01
CHAPTER 4 - ANNEX
Level-adjustable Sample Holder for LSM
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ZEISS
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