Photon Efficiency Optimization in Time-Correlated Single Photon Counting Technique for Fluorescence Lifetime Imaging Systems

In time-correlated single photon counting (TCSPC) systems, the maximum signal throughput is limited by the occurrence of pile-up and other effects. In many biological applications that exhibit high levels of fluorescence intensity (FI), pile-up-related distortions yield serious distortions in the fluorescence lifetime (FLT) calculation as well as significant decrease in the signal-to-noise ratio (SNR). Recent developments that allow the use of high-repetition-rate light sources (in the range of 50-100 MHz) in fluorescence lifetime imaging (FLIM) experiments enable minimization of pile-up-related distortions. However, modern TCSPC configurations that use high-repetition-rate excitation sources for FLIM suffer from dead-time-related distortions that cause unpredictable distortions of the FI signal. In this study, the loss of SNR is described by F- value as it is typically done in FLIM systems. This F-value describes the relation of the relative standard deviation in the estimated FLT to the relative standard deviation in FI measurements. Optimization of the F-value allows minimization of signal distortion, as well as shortening of the acquisition time for certain samples. We applied this method for Fluorescein, Rhodamine B, and Erythrosine fluorescent solutions that have different FLT values (4 ns, 1.67 ns, and 140 ps, respectively).