ZEISS
Level-adjustable Sample Holder with Insert for Piezo Stage
ELYRA 7
20
000000-2262-999
03/2019 V_02
5.4
Stage leveling using X-cite in reflection and two glas plates
1.
Swing nosepiece to empty position (make sure
no cover is in the empty position). Place a
glass plate evenly on the opening. Do not use
coverslips as they tend to bend. Use a glass
piece the thickness of a slide. You can also use
a slide, though its big size makes placing it
more difficult. Note that systems with
Automated Component Recognition (ACR)
will have a slightly elevated pin at the rim of
the opening. Avoid placing the glass plate on
top of it as it will cause the glass surface to be
tilted.
2.
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 opening is fairly 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). Use preferentially sCMOS pco.edge camera, albeit the Andor PALM camera would also work.
Take Optovar 1.0 and set EM gain to zero.
3.
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. (Note: with the fiber all
in, the lamp is simply to bright leading to oversaturation of the camera). You might see already two
unfocused rings; but there is also a chance that you cannot see the fiber outline at al. Pull the fiber
on the ELYRA site until the two rings become clearly visible and are in focus (Fig. 8,
A
). 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. 4,
csv
) and back side (Fig. 4,
cbv
) to be parallel using screws
sy
and
sx
, respectively (Fig. 4). The cleft’s
width should be approximately 2 mm as preset by the factory, but can be adjusted with screw sz if
needed.
4.
Level stage by turning the screws of the sample holder (Fig. 3,
sx
&
sy
) until the more or less focused
circles overlap (Fig. 8,
B
).
Fig. 14
Focus control in reflection using
two glass plates before and after
stage leveling