Leica TCS SP8 SMD User Manual page 167

4. Make the settings in the Measurement Preview window (such as the selection of the
displayed channels, the resolution of the time trace etc.) by selecting the Show
Measurement Preview... button in SymPhoTime while no measurement is running.
Defaults can be saved using the Set Defaults button.
5. Adjust the photon count rate by changing the intensity of the diode laser at the laser
coupling unit (LCU) (Chapter 9.6) or, if you do not have an LCU, but rather a direct fiber
coupled laser, in a similar way at the laser scaffold with the intensity regulation knob.
Information on attenuation by modifying the laser current can be found in the manual on
the laser driver (PDL or SEPIA). Keep in mind that changing the laser intensity via the
laser current does alter the pulse shape of the laser.
6. Control the white light or MP laser intensity directly in LAS AF.
7. For adjustment use the peak count rate in the preview window in SymPhoTime, since the
average count rate does not give you information about the count rate in the brightest
parts of the image.
Regions of the image recorded with photon count rates above the limit (Table 21) can
lead to inaccurate fluorescence lifetimes being calculated.
8. To attain the shortest possible FLIM data acquisition time, we recommend increasing the
photon count rate wherever possible to the TCSPC limit listed in Table 21. Of course, for
weak fluorescent specimens, this upper limit cannot be attained, even at maximum laser
power.
Using the Image tab in the Measurement Preview window in SymPhoTime, you can see a
preview of the FLIM image. The lifetime displayed in the image by the color code is
calculated by the mean arrival time of the fluorescence photons after the last laser pulse.
The image is updated almost once each second if the scanning speed exceeds one frame
per second. It will be slower at lower scanning frequencies.
9. Make sure that even in group measurements, these high count rates are not reached, as
otherwise the group measurement will stop.
10. In order to reach even higher detection count rates for FLIM, the fast FLIM preview can
be switched off using the check boxes under Previews in the FLIM preview window
(Figure 148).
Summarized manual for FLIM experiments
167