Enhanced FRET contrast in lifetime imaging

In combination with two photon excitation, FLIM is currently one of the best techniques to quantitatively study the subcellular localization of protein–protein interactions in living cells. An appropriate analysis procedure is crucial to obtain reliable results. TCSPC is an accurate method to measure FLIM. It is however an indirect process that requires photon decay curve fitting, using an exponential decay equation. Although choosing the number of exponential terms is essential, it is labor‐intensive and time consuming. Therefore, a mono‐model is usually applied to a whole image. Here we propose an algorithm, named Liχ, allowing pixel by pixel analysis based on the Δχ2 value. Liχ was validated using simulated photon decay curves with known lifetimes and proportions. It showed a high robustness for decay curves with more than 103 photons. When applied to lifetime images acquired from living cells, it resulted in a more realistic representation of the interaction maps. We developed an easy‐to‐use procedure for multi‐model FLIM analysis, which enables optimized FRET quantification for all interaction texture studies, and is especially suitable to avoid the classical misinterpretation of heterogeneous samples. © 2008 International Society for Advancement of Cytometry