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this case, the detector must be switched on again on the DSN.
• Check whether the correct SMD filter cube emission filters are placed in the X1 port
adapter. If you have a 2 MPD detector unit from PicoQuant, make sure that the beam
splitter is set to the correct position.
• When all detectors are on and background counts are displayed in the oscillator
window, place a fluorescent specimen in the Leica microscope, configure all the
settings for FLIM in LAS AF and start the scanning operation while the time trace
measurement is active in the Preview window. The count rate should increase. If the
count rate does not increase, make sure that the shutters in front of the detectors are not
closed (the filter should sit in the filter holder correctly). Shutters should click quietly
when opening and closing.
• Make sure that the X1 port setting of the Leica TCS SP8 is correct. In LAS AF, select the
following in the beam path window:
• The --- position if you use external detectors
• The Mirror position if you use internal SP FLIM detectors.
• If the shutter is not the problem, take a FLIM image as described in Chapter 16.3.
Although no counts are displayed, a PT3 file is generated.
Calculate a time trace from your FLIM *.pt3 file, the same as if it were a point
measurement, by choosing the MCS Trace button (only available if you have a software
license for point analysis). The displayed time trace should contain the photon count.
Select marker 1 - 3 to show line start, line stop, and frame markers that are transferred
from the instrument. You may have to select a suited display range in order to display the
marker signals.
If there are no marker signals, check the cable connection between the Leica TCS SP8
and the SMD upgrade. If there are no marker signals even though the connection is
intact, contact PicoQuant.
Troubleshooting
251
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