QUANTIFYING THE SHORT LIFETIME WITH TCSPC-FLIM: FIRST MOMENT VERSUS FITTING METHODS

Abstract

Combing the time-correlated single photon counting (TCSPC) with fluorescence lifetime imaging microscopy (FLIM) provides promising opportunities in revealing important information on the microenvironment of cells and tissues, but the applications are thus far mainly limited by the accuracy and precision of the TCSPC-FLIM technique. Here we present a comprehensive investigation on the performance of two data analysis methods, the first moment (M1) method and the conventional least squares (Fitting) method, in quantifying fluorescence lifetime. We found that the M1 method is more superior than the Fitting method when the lifetime is short (70 ~ 400 ps) or the signal intensity is weak (<103 photons).

Keywords

• Fluorescence lifetime imaging
• time-correlated single photon counting
• the distribution of the first moment (M1)