Quantitative analysis of fluorescence lifetime imaging made easy

Fluorescence lifetime imaging is a valuable and versatile tool for the investigation of the molecular environment of fluorophores in living cells. It is ideally suited— and is therefore increasingly used—for the quantification of the occurrence of Förster Resonance Energy Transfer, a powerful microscopy method for the detection of subnanometer conformational changes, protein‐protein interactions, and protein biochemical status. However, careful quantitative analysis is required for the correct and meaningful interpretation of fluorescence lifetime data. This can be a daunting task to the nonexpert user, and is the source for many avoidable errors and unsound interpretations. Digman and colleagues (Digman et al., 2007, Biophys. J. 94, L14–6) present an analysis technique that avoids data fitting in favor of a simple graphical polar data representation. In this “phasor” space, the physics of lifetime imaging becomes more intuitive and accessible also to the inexperienced user. The cumulated information from image pixels, even over different cells, describes patterns and trajectories that can be visually interpreted in physically meaningful ways. Its usefulness is demonstrated in the study of the dimerization of the uPAR receptor.