Developing and Testing a Bayesian Analysis of Fluorescence Lifetime Measurements

TL;DR

This paper introduces a Bayesian inference method for analyzing FLIM data to accurately measure FRET efficiencies, even with limited photon counts or low FRET fractions.

Contribution

It presents a novel Bayesian analysis approach that accounts for experimental complexities in FLIM measurements, improving parameter extraction accuracy.

Findings

Accurately extracts FRET parameters from low-photon data.

Verifies robustness in low-fraction regimes.

Demonstrates improved precision over traditional methods.

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.