Leica TCS SP8 SMD, User Manual

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138
FLIM Data Acquisition
cube and mount it in the X1 port adapter (
Chapter 23
).
In LAS AF, a simulator photomultiplier is activated. This means that no image from the
external photomultiplier will be displayed in LAS AF.
3. Internal photomultipliers cannot be used for image acquisition simultaneously with
external PMT detection. Deactivate the internal PMTs in LAS AF.
4. If no FLIM image is displayed in the SymPhoTime software, check whether the
---
option
is set at X1 Port ( Figure 121, item 3 ).
Activating the external PMT
Since the signal from the external FLIM PMT is not transferred to the TCS
SP8, only a black intensity image is displayed in LAS AF. Despite this, let
the external detector remain activated. If no detector is selected at all, it
will not be possible to start a scanning operation.
16.2.1.4 FLIM Data Acquisition with Detectors at NDD Position (HyD RLD)
HyD RLD can only be used if the excitation occurs via an MP laser. They can be used for the
intensity image acquisition in LAS AF as well as for the FLIM data acquisition in
SymPhoTime.
Follow these steps for FLIM data acquisition:
1. Switch the HyD RLD on as described in
Chapter 13.3
before starting the scanner and
LAS AF. Observe this sequence, since otherwise the cooling for the detectors is not
initialized.
2. Select a suitable spectral range for detection by choosing the appropriate NDD filter
cube and mounting it in the NDD detection unit.
3. Go to the Setup tab in the Setup FLIM step.
4. In the
FLIM Detector
dialog, select the
Non-descanned (MP FLIM)
option. In
Beam Path
Settings , the HyD RLD should be selected automatically. If that is not the case, activate
the detectors in
Beam Path Settings
with
ON
(
Figure 122
).
Dependency of the spectral detection range of the photomultiplier
The spectral detection range of the photomultiplier is determined by the
SMD filter cube used, not by the slider settings for the photomultipliers.
Prevent light incidence during MP FLIM measurement
During an MP FLIM measurement (particularly with HyD RLD), the light in
the room should be switched off and the shutter of the fluorescence lamp
should be closed. Furthermore, the iris diaphragm on the condenser
should be completely closed.