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What is the pile-up effect?

In TCSPC the ‘pile-up’ effect occurs when within one laser pulse period a second fluorescence photon is excited. This cannot be detected since the TCSPC electronics is still busy analyzing the first photon event within the same period. In this case, the recorded fluorescence decay curve does not match the real fluorescence decay. For a thought experiment, please imagine a typical multiphoton FLIM (MP-FLIM) setup with Ti:Sapphire laser usually firing pulses with a repetition rate of 80 MHz. An emission of two photons per pulse would result in a count rate of 160 MHz which is extremely unlikely. Not worth mentioning, this high detection rate would saturate or damage any known single photon detector.

Interestingly, it is often assumed that the photon rate must be < 0.1% of the excitation pulse rate in order to avoid any ‘pile-up’ effects. But this assumption is not correct. It might originate from a misunderstanding of the “duty cycle” value of “<0.1” reported in [1]. There some values were reported with unit “%” and others not. So, the value of “<0.1” should be rather read as “<10 %” and not as “<0.1 %”. In a MP-FLIM setup with a 80 MHz laser this results in 8 MHz count rate. Under these conditions, a lifetime error of less than only < 2.5 % occurs. This means, FLIM techniques can record 100 times faster than commonly believed, and ‘pile-up’ is not a severe problem.

A derivation of the correct size of the pile-up error and the conclusion of its irrelevance for TCSPC FLIM can be found in [2]. How deeply the misconception of pile-up is fixed in the minds of the users becomes evident from the fact that [2] is often cited as a proof of the allegedly devastation effect of pile-up on TCSPC FLIM. In fact, it proves the opposite. Therefore, please make sure that you cite the reference in the correct context.

[1] Pawley, James B., ed. 2006. Handbook Of Biological Confocal Microscopy. Boston, MA: Springer US. https://doi.org/10.1007/978-0-387-45524-2.
[2] Becker, Wolfgang, Advanced Time-Correlated Single Photon Counting Techniques. Springer 2005

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