Leica TCS SP8 SMD, User Manual

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179
F(L)CS Data Acquisition
7. Select one (or more) suitable laser line(s) (
Figure 154, item 3
) and a suitable detection
range on the detector ( Figure 154, item 5 ),
8. Then specify the
xyz
mode and a scan speed of 400 Hz for an image.
9. Select Glow (O&U) in the color look-up table (LUT).
10. Click
Calibration
in the pull-down menu (
Figure 154, item 4
).
A window appears for the calibration procedure (
Figure 155
). The system automatically
determines the following values: Zoom 32 and pixel format 1024x1024.
11. Start a continuous scan by clicking the Live button ( Figure 155, item 1 ) and manipulate
the
Gain
and
Offset
on the control panel to obtain a well-illuminated image.
Figure 155: Procedure for calibrating the FCS measurement position – Definition of instrument
parameter settings
12. With
Define bleach time
, enter the bleaching period; The default value is 100 ms
( Figure 155, item 2 ). For thin and easily bleached specimens, it may be more useful to
select a bleaching period of 500 ms or shorter.
13. Now select the Calibrate button ( Figure 155, item 3 ).
An image will be acquired automatically. Then, the previously selected laser lines will
automatically be set to 100 % AOTF transmission for photobleaching, and the laser will
bleach in the middle of the image for the set time. Then, another image will be acquired with
the settings made previously.
14. Now position the crosshair in the middle of the bleach point (
Figure 156, item 1
) and