Quantitative comparison of polar approach versus fitting method in time domain FLIM image analysis

Abstract

We calculate here analytically the performance of the polar approach (or phasor) in terms of signal-to-noise ratio and F values when performing time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) to determine the minimal number of photons necessary for FLIM measurements (which is directly related to the F value), and compare them to those obtained from a well-known fitting strategy using the Least Square Method (LSM). The importance of the fluorescence background on the lifetime measurement precision is also investigated. We demonstrate here that the LSM does not provide the best estimator of the lifetime parameter for fluorophores exhibiting mono-exponential intensity decays as soon as fluorescence background is superior to 5%. The polar approach enables indeed to determine more precisely the lifetime values for a limited range corresponding to usually encountered fluorescence lifetime values. These theoretical results are corroborated with Monte Carlo simulations. We finally demonstrate experimentally that the polar approach allows distinguishing in living cells two fluorophores undetectable with usual time-domain LSM fitting software.