Leica TCS SP8 SMD, User Manual

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Summarized manual for FLIM experiments
17.9 Selecting the Correct Laser Repetition Rate
For pulsed diode lasers from PicoQuant and for the white light laser with pulse picker, the
laser repetition rate can be determined in LAS AF (
Chapter 16.2.4 ). In general, the repetition
rate should be as high as possible to achieve the highest possible photon count rate.
However, the time window after a laser pulse should still be large enough to allow complete
fading of the fluorescence of the excited dye molecules before the next laser pulse.
For example, the duration between two laser pulses at a repetition rate of 80 MHz is 12.5 ns.
If a fluorophore with a lifetime of 6 ns is measured, due to the statistical process, more than
12 % of the photons are emitted after 12.5 ns. In this case, the repetition rate must be
decreased (
Figure 149
). At a laser repetition rate of 80 MHz, the fluorescence does not fade
completely before the end of the time window. Due to a "wrapping effect", the end of the
fading can be observed before the actual laser pulse (
Figure 149 A
). At 40 MHz the time
window is optimally adapted (
Figure 149 B
), while at 20 MHz the fluorescence has already
decayed at less than half of the detection window (
Figure 149 C
). Therefore, 40 MHz would
be the optimal repetition rate in this case.
Upper limit for the count rate
Unnecessarily low photon count rates lead to an extension the data
acquisition, because a certain number of photons is required for the FLIM
analysis. On the other hand, too high of photon count rates can lead to
artifacts and falsify the FLIM analysis due to the pile-up effect. The upper
limit for the count rate is given by:
• TCSPC principle: To prevent pile-up artifacts (pile-up effect:
overestimation of early photons), the identified photon count rate in
general should lie at around 5 % under the laser repetition rate.
When using a laser repetition rate of 20 MHz, this results in a photon
count rate of 1 MHz (1,000 counts/ms). This setting leads to a maximum
underestimation of the fluorescence lifetime of 1 % due to the pile-up
effect.
• TCSPC detector: Some detectors lead to occurrence of artifacts if they
are operated with a high count rate. The APD from type AQR by Perkin
Elmer shows a substantial IRF widening at a photon count rate above
1 MHz and a count rate-dependent IRF movement.
• TCSPC electronics: The acquired photon data are transmitted
continuously via a FIFO buffer from the TCSPC unit (PicoHarp) to the
workstation. If average photon count rates above approx. 3 MHz are
reached (depending on the workstation power), the data acquisition
stops and an error message is displayed: