Modeling and optimization of time correlated single photon counting accuracy in fluorescence lifetime measurements using delay control

An accurate determination of fluorescence lifetimes (FLs) provides valuable insights for various applications, from cell analysis to semiconductor characterization. While the FLs are commonly measured using time-correlated single photon counting (TCSPC), the accuracy is limited by pile-up deviation at high intensities and by statistical uncertainty at low count rates. To quantify the effects and identify the best compromise of low pile-up and high counting statistics, we present a theoretical model which analytically describes the TCSPC detection process and thus the accuracy of FL measurements. In addition, the theoretical model provides simple design rules for the maximum usable intensity and required photon counts. Furthermore, we utilize this findings in what we believe to be a novel method, which adjusts the signal intensity to the mentioned compromise by introducing a delay between excitation and detection. We applied and validated the method in single photon avalanche diode (SPAD) based FL measurements. In doing so, the delay method, applied with an algorithm, enables FL measurements with an accuracy of ±15<i>%</i> even at high detector saturations up to 99% with sub-ms measurement times. Unlike previous approaches, our model provides a detailed understanding of the TCSPC detection and the delay method enables robust and fast FL measurements.