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136
FLIM Data Acquisition
acquisition is applied to the detectors. In other words, the gain cannot be changed as long
as
FLIM
is selected.
3. Select a suitable range for spectral detection by adjusting the spectral sliders of the SP
FLIM detectors accordingly (
Figure 120, item 2 and 3
).
At the same time, additional detectors can be activated and used for intensity image
acquisition. The data of these additional detectors is used for the intensity image
acquisition, but not for FLIM.
4. If you do not obtain an image, check that the X1 port is in the Mirror position.
5. Check to ensure that the fluorifier disc is in a suitable position that blocks excitation light
(see Chapter 16.2.3 ).
16.2.1.2 FLIM Data Acquisition with External MPD APDs
Proceed as follows to prepare the TCS SP8 for collecting FLIM data using external APDs:
1. Switch the APDs on electrically.
2. Select a suitable spectral range for the detection by choosing the appropriate SMD filter
cube and mount it in the X1 port adapter (see Chapter 23 ).
3. Open the
Setup
tab.
4. In the FLIM detector pull-down menu, select the External option ( Figure 121, item 1 ). In
Beam Path Settings
, the APDs should be selected automatically (
Figure 121, item 2
).
Overload protection for internal SP FLIM detectors
If too much light strikes a FLIM detector, it is temporarily switched off to
prevent damage and automatically switched back on after a few
seconds. If the detector count rate is not reduced, the detectors will
continuously switch off and back on, resulting in horizontal streaks in the
image.
The overload protection of the internal FLIM detectors is active in FLIM
mode and image acquisition mode. To avoid streaking, reduce the
detector count rate. This can be done in a number of ways:
• Reduce the laser intensity
• Reduce the Gain (possible in image acquisition mode only)
• Reduce the
Pinhole
• Use specimens with less intensive fluorescent dye