Leica TCS SP8 SMD, User Manual

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Summarized manual for FLIM experiments
17.10 Starting FLIM Data Acquisition
1. Go to the Measurements step in the FLIM Wizard in LAS AF.
2. In the
Setup
tab, type in a file name that will be used in SPT (
Chapter 16.3.2
).
3. Specify the measurement time of the FLIM image ( Chapter 16.3.3 ).
4. If you wish, define a volume or lambda stack or a time series (
Chapter 16.3.4
to
Chapter 16.3.8 ).
5. Start the scan procedure in LAS AF by selecting the
Run FLIM
image acquisition button.
Normally, the SymPhoTime software will start the data acquisition automatically.
However, for a single FLIM measurement, you can also start the FLIM data acquisition
manually by selecting the
Acquire LSM Measurements button.
During the SP8 data acquisition, the preview window is displayed in the SymPhoTime
software (
Figure 148
), in which you can see the FLIM preview image. When scanning the
specimen several times, all frames are accumulated over time. This display allows you to
evaluate the quality of the FLIM data acquired. In doing so, note that the FLIM preview image
is accumulated for display only. Based on the stored raw data, separate individual images
can still be analyzed.
6. The SP8 scanning operation stops either after having scanned the chosen number of
frames or can be stopped manually by selecting
Stop FLIM
to interrupt the data
acquisition.
The recorded FLIM images will be stored in the SymPhoTime software and are accessible
for further data analysis from the workspace.
17.11 Resulting Raw Data File and Documentation
The resulting raw data file and documentation is shown in the SymPhoTime software.
The measurement result is an FLIM image with a *.PT3 raw data file, which contains the
greatest available data volume for each photon detected (temporal and spatial information
related to the detection channel).
Each raw data file is saved with an annotation file. It is characterized in the working area
overview by a symbol in the shape of a red dot (
Figure 150
). The raw data file contains all
measurement parameters transferred to SymPhoTime software and contains a preview
image (bitmap) which provides a first impression of the image scanned.
Prevent light incidence during MP FLIM measurement
During an MP FLIM measurement, the room light 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.