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

TL;DR

This paper introduces a data analysis method that corrects for detector pulse pile-up artifacts in fluorescence lifetime measurements, enabling accurate FLIM at very high count rates.

Contribution

It presents a physically motivated correction technique for detector pulse pile-up in TCSPC, improving accuracy at high count rates.

Findings

Significant reduction of systematic errors with correction

Enables accurate fluorescence lifetime imaging at high frame rates

Improves reliability of measurements near excitation rate

Abstract

Using Time-Correlated Single Photon Counting (TCSPC) 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 artefacts due to frequent merging of closely spaced detector pulses (detector pulse pile-up) remains an issue to be addressed. We propose here a data analysis method correcting for this type of artefact 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 show a significant reduction of systematic errors at count rates approaching the excitation rate. This allows quantitatively accurate fluorescense lifetime imaging (FLIM) at very high frame rates.