Stage Leveling Using X-Cite In Reflection - Zeiss LSM 880 Operating Manual

LSM 880 Level-adjustable Sample Holder with Insert for Piezo Stage (ELYRA)
e) Press the lower pointing arrow of the X input box of the Stage tool to return to the initial
position. If the image is in focus (Fig. 6/F) X leveling is complete; otherwise repeat steps 3a) - e)
iteratively. If applicable, the multiplication factor of 3c) can be adjusted.
4. Adjust leveling in Y in the following way:
a) Enter in the Stage tool in the Step [µm] input box "1000". Press the upper or lower pointing
arrow of the Y input box, whatever is required to step 1000 µm upwards in the Y direction
(Fig. 6/A).
b) If the sample is out of focus (Fig. 6/B) refocus manually by using the stand's focus drive
(Fig. 6/C); otherwise no further leveling is necessary. Record the relative change in the focus
position by reading the value in the Z-Position input box of the Focus tool.
c) Multiply that value with 40 and manually set the focus onto this value using the stand's focus
drive (Fig. 6/D). (Note: The factor 40 is only accurate, if the sample was positioned in the center.)
d) Use the front right screw (Fig. 5/sy) of the sample holder to readjust the focus (Fig. 6/E). Turn
clockwise if the focus position was positive, counter clockwise if it was negative. (Note that one
turn corresponds to approximately 125 µm).
e) Press the lower pointing arrow of the Y input box of the Stage tool to return to the initial
position. If the image is in focus (Fig. 6/F) Y leveling is complete; otherwise repeat steps 4a) - e)
iteratively. If applicable, the multiplication factor of 4c) can be adjusted.
8.2

Stage Leveling Using X-Cite in Reflection

Adjustable sample holder –
Fig. 7
schematic view
1. Assemble a fluorescent sample you want to level with sample bottom downwards into the sample
holder. The sample site should be clean and free of any immersion oil. Position the sample such that
the objective lens is in the middle of the sample. Use WF illumination with the X-Cite at 12 % and
filter for excitation with the 561 nm line (optimal would be a small band pass to avoid too much
light). Swing nosepiece to empty position (make sure no cover is in the empty position) and use
preferentially Andor SIM camera, albeit the Andor PALM camera would also work. Take Optovar 1.0
and set EM gain to zero.
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CHAPTER 4 - ANNEX
Fig. 8
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Focus control in reflection
ZEISS
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