OPERATING INSTRUCTIONS
ZEISS
System Operation
Lattice Lightsheet 7
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04/2021 V_01
3.2.4
Lightsheet Focusing
1.
Switch of TL LED.
2.
Choose appropriate laser line e.g. 488 and set to
approximately 2%
3.
Choose 1000 x 300 px
4.
Choose camera integration time of 30 – 50 ms.
5.
Choose appropriate emission filter (default is
LBF-Quad that will work with all laser lines)
6.
Switch
Dither
off.
7.
Start
Continuous
. A fluorescence image should
be visible that might be rather blurred.
8.
Zoom in and use
Focus Y
to bring the Lightsheet
into the focus plane of the detection objective
(Fig. 64). The intrinsic structuring of the
Lightsheet (“lattice”) should become visible
(Fig. 63). Maximize the contrast of this lattice.
It might be useful to first coarsely align to
determine the region of focus and then fine
focus by smaller steps. Focus is where the line
patten is sharpest.
You might also perform this step with
Dither
On
and optimize for the sharpest image.
9.
Position the beam waist of the illumination in the
Field of view (FoV) by moving the
Focus illumination
to minimize out-of-focus
blur.
It might be useful to first coarsely align to
determine the region of focus and then fine
focus by smaller steps. Focus is where the line
patten is sharpest.
You might also perform this step with
Dither
On
and optimize for the sharpest image.
10.
Depending on the sample it can be necessary to
repeat steps 8. & 9. in an iterative way. Once this
is done, the system is focused (Fig. 65).
11.
Switch on
dithering
. The lattice is smeared out
to the actual light sheet.
12.
Move the sample via the Ergopointer in X and Y
to bring areas of interest into the focus/FoV.
Fig. 62
Lightsheet tilt correction:
uncorrected (upper panel) and X-tilt
corrected (lower panel)
Fig. 63
Light sheet geometry. Movement of
sheet by Focus Y and Focus
Illumination is indicated
Fig. 64
Lightsheet focusing by Focus Y.
Upper panel unfocused, lower panel
focused using Focus Y
Fig. 65
Lightsheet focusing by Focus
Illumination. Upper panel
unfocused; lower panel focused
using Focus Illumination
