Zeiss LSM 880 Operating Manual page 622

ZEISS Level-adjustable Sample Holder with Insert for Piezo Stage (ELYRA)
2. Draw fiber plug of X-Cite completely out from the X-Cite casing. Start acquisition. The image will be
dark since fiber was pulled out. Put fiber back and slowly move in until you will see a coarsely
adjusted image to have sufficiently reduced light levels you can work with (Fig. 8/A). (Note: with the
fiber all in, the lamp is simply to bright leading to oversaturation of the camera). If the illumination is
far from being centric, the sample is already strongly tilted and it is recommended to do a pre-
adjustment. To this end adjust the edges of the clefts viewed from the right side (Fig. 7/csv) and
back side (Fig. 7/cbv) to be parallel using screws sy and sx, respectively (Fig. 7). The cleft's width
should be approximately 2 mm as preset by the factory, but can be adjusted with screw sz if needed.
3. Now pull (or push) fiber on the ELYRA site until the dark ring, which is already faintly visible in its
contours, appears focused (Fig. 8/B). Dependent on the sample the focus could look worse,
especially when the reflecting planes are wavy or you get reflection from different planes (Fig. 8/C).
4. Move in 10x objective and focus the already visible ring with the z-drive of the stand. The ring
appears now well focused as long as the sample is not completely unsuitable (Fig. 8/D)
Mark the Ring with a ROI from the Graphics tool (3 point-circles will work best) (Fig. 8/E).
5.
6. Mirror the circle on the center of the image. (Note: If the circle is already centered in the middle that
step is not required; deviations <20 pixel SIM camera or 10 pixel Palm camera are no problem and
can be tolerated). To mirror the coordinates of the ROI X, Y (visible in the Graphics tab), calculate
the new values as follows: X' = Xmax-X & Y' = Ymax-Y with Xmax and Ymax representing the
number of pixels of the camera in X and Y-direction. E.g. if the coordinates X and Y would have
been 502 and 512, respectively, the new coordinates X' and Y' would be calculated using the SIM
camera to 1004-502=502 and 1002-512=490.
7. Draw a new circle with the same diameter in the original image with the new coordinates (Fig. 8/F).
The old circle is not necessary anymore and can be switched off or deleted (Fig. 8/G).
8. Turn nosepiece back to free position. Than level stage by turning the screws of the sample holder
(Fig. 7/sx & sy) until the more or less focused circle of the fiber overlaps with the ROI-circle (Fig. 8/H).
20
CHAPTER 4 - ANNEX
000000-2071-464
LSM 880
10/2014 V_01