Leica TCS SP8 SMD, User Manual

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FLIM Data Acquisition
Figure 134: Selecting the white light laser pulse frequency in Beam Path Settings
If the repetition rate is changed, it can become necessary to adapt the resolution in
SymPhoTime (see
Table 20
).
16.2.7 Setting the Pinhole
On the Acquisition tab in LAS AF, set the pinhole diameter ( Pinhole ) to 1 Airy for FLIM
measurements with VIS and UV excitation and to the maximum value for MP excitation.
16.2.8 Optimizing FLIM Settings
1. First, find the position of your specimen you want to use for FLIM measurement.
2. Click on the image acquisition button
Run FLIM Test
to start the scanning operation. You
can view and optimize the fluorescence intensity in the Count Rate Monitor on the Setup
tab (
Figure 135
).
3. Laser line and intensity can be modified during the test measurement until you attain the
count rate desired.
4. When you start the test measurement, an online FLIM image is displayed in
SymPhoTime.
Do not select other frequencies on the laser
If you select manual in the pull-down menu, you can select additional
frequencies on the laser yourself. This is, however, not recommended
because the cable lengths are not calibrated for these frequencies.
Scan speeds 100 to 200 Hz recommended
Do not use scan speeds that are too high because these can lead to
longer analysis times in SymPhoTime. Scan speeds at 100 to 200 Hz are
recommended. Avoid the use of the resonant scanner for FLIM data
acquisition as much as possible.
The online FLIM image is not saved.
This image is not saved. To save your data, you have to work in the
Measurements step.