Fluorescence decay data analysis correcting for detector pulse pile-up at very high count rates

Abstract. Using time-correlated single photon counting for the purpose of fluorescence lifetime measurements is usually limited in speed due to pile-up. With modern instrumentation, this limitation can be lifted significantly, but some artifacts due to frequent merging of closely spaced detector pulses (detector pulse pile-up) remain an issue to be addressed. We propose a data analysis method correcting for this type of artifact and the resulting systematic errors. It physically models the photon losses due to detector pulse pile-up and incorporates the loss in the decay fit model employed to obtain fluorescence lifetimes and relative amplitudes of the decay components. Comparison of results with and without this correction shows a significant reduction of systematic errors at count rates approaching the excitation rate. This allows quantitatively accurate fluorescence lifetime imaging at very high frame rates.