Leica TCS SP8 SMD, User Manual

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Summarized manual for FLIM experiments
17 Summarized Manual for FLIM Experiments
17.1 Prerequisites
The following description assumes that the following prerequisites are met:
• SymPhoTime has been started and a workspace has been loaded.
• LAS AF was started.
• All components (lasers, detectors, software) of the PicoQuant SMD upgrade are
switched on. Detailed information on starting the components can be found in
Chapter 13.4
.
• The right objective is chosen with high numerical aperture and - if present - the
correction ring is adjusted to the specimen used (
Chapter 18.1.1
and
Chapter 18.1.4
).
• Your specimen is already placed onto the microscope and brought into focus.
• The selected excitation lasers are switched on and activated (
Chapter 15
).
• The correct emission filter(s) was placed in front of the detectors. For external FLIM
detectors, the correct SMD filter cube has to be set in the X1 port adapter (
Chapter 23
);
for internal SP FLIM detection, the barrier filter in the fluorifier disc is used
(
Chapter 16.2.3
). With HyD RLD, the correct barrier filter and a suitable filter cube must
be used in the detector unit.
• For beam splitter systems: The correct beam splitter for your wavelength has been
selected in LAS AF. If pulsed diode lasers are used, the beam splitter is to be in the
Substrate
position.
• With AOBS systems: The acousto-optical beam splitter is in fluorescence mode.
17.2 Selecting Position for the FLIM Measurement
1. Start the Wizard in LAS AF by selecting the
FLIM
operating mode.
2. Scan an image (for this, use the Setup Imaging step in the FLIM Wizard). To be able to
get an initial overview, you can use continuous wave lasers and internal detectors.
3. Select the correct sample region at which the FLIM measurement is to be carried out.
17.3 Changing from Continuous to Pulsed Excitation
1. Make sure that the PicoQuant laser driver is set to external trigger.
2. In the FLIM Wizard in LAS AF, go to the Setup FLIM step.
3. Select a suitable laser wavelength for pulsed excitation (
Chapter 16.2.2
).
4. Check whether the selectable beam splitter for MFP is in the correct position
( Chapter 16.2.2 ).
5. Select the laser pulse frequency (repetition rate) in LAS AF ( Chapter 16.2.4 ). In general,
40 MHz is the best frequency to start with. Lower the frequency to 20 MHz only if you
determine that your lifetime decay curve does not fit in the TCSPC histogram. This