Stage Leveling Using Lsm In Reflection Mode And Z-Piezo - Zeiss LSM 880 Operating Manual

ZEISS
3. Start a reflection scan at zoom 1.0x and focus on the border between coverslip and sample, which
can be identified by the brightest reflection. A high pixel resolution is not important (use e.g. the
256x256 image format). The border will appear as a very bright uniform signal when focusing.
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 0.2 µm. Click on the 'Center' button and the start the Z-Stack.
Alternatively focus continuously up and down manually using the focus drive without using a Z-
Stack in Continuous mode.
5. Check for the tilt of the coverslip border. If the picture gets uniformly brighter and darker when
focusing through the coverslip border, the stage is leveled. Any tilt will appear as a brightness
gradient or even a bright zone or line that wanders through the slices (Fig. 10).
6. Adjust the adjustment screws of the sample holder. Turn screw sx for the tilt along the x-axis and
screw sy for the tilt along the y-axis until you observe visually an even reflection image (Fig. 9). You
might need to re-adjust the z-range when turning the screws. Screw sz will only have to be
adjusted in case the range of screws sx and sy is not sufficient.
7. If you use a defined z-scan repeat steps 4. through 6. until you achieve a good result. If you focus
manually, turn screws until reflection looks even (Fig. 10/B).
9.2

Stage Leveling Using LSM in Reflection Mode and Z-Piezo

Adjustable sample holder – schematic
Fig. 11
view
0. Adjust the edges of the clefts viewed from the right side (Fig. 11/csv) and back side (Fig. 11, cbv) to
be parallel by using screws sy and sx, respectively (Fig. 11). The cleft's width should be
approximately 2 mm as preset by the factory, but can be adjusted with screw sz if needed.
1. First locate the area of interest of your sample. It is important to level the sample in the same region
as the actual measurements, as the coverslip may be bent or tilted at other locations.
2. Prepare a LSM reflection image with the pinhole diameter to 1 AU.
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CHAPTER 4 - ANNEX
Level-adjustable Sample Holder for LSM
000000-2071-464
Fig. 12 Focus control with z-Piezo. Reflections
on tilted (A) and even (B) coverslip
LSM 880
10/2014 V_01