Developing and Testing a Bayesian Analysis of Fluorescence Lifetime Measurements.

Abstract

FRET measurements can provide dynamic spatial information on length scales smaller than the diffraction limit of light. Several methods exist to measure FRET between fluorophores, including Fluorescence Lifetime Imaging Microscopy (FLIM), which relies on the reduction of fluorescence lifetime when a fluorophore is undergoing FRET. FLIM measurements take the form of histograms of photon arrival times, containing contributions from a mixed population of fluorophores both undergoing and not undergoing FRET, with the measured distribution being a mixture of exponentials of different lifetimes. Here, we present an analysis method based on Bayesian inference that rigorously takes into account several experimental complications. We test the precision and accuracy of our analysis on controlled experimental data and verify that we can faithfully extract model parameters, both in the low-photon and low-fraction regimes.