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Quantitative analysis in Förster resonance energy transfer (FRET) imaging studies of protein–protein interactions within live cells is still a challenging issue. Many cellular biology applications aim at the determination of the space and time variations of the relative amount of interacting fluorescently tagged proteins occurring in cells. This relevant quantitative parameter can be, at least partially, obtained at a pixel-level resolution by using fluorescence lifetime imaging microscopy (FLIM). Indeed, fluorescence decay analysis of a two-component system (FRET and no FRET donor species), leads to the intrinsic FRET efficiency value ( E ) and the fraction of the donor-tagged protein that undergoes FRET ( f  D ). To simultaneously obtain  f  D  and  E  values from a two-exponential fit, data must be acquired with a high number of photons, so that the statistics are robust enough to reduce fitting ambiguities. This is a time-consuming procedure. However, when fast-FLIM acquisitions are used to monitor dynamic changes in protein–protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are unreliable, even for single lifetime donors. We introduce the concept of a minimal fraction of donor molecules involved in FRET ( mf  D ), obtained from the mathematical minimization of  f  D . Here, we discuss different FLIM techniques and the compromises that must be made between precision and time invested in acquiring FLIM measurements. We show that  mf  D  constitutes an interesting quantitative parameter for fast FLIM because it gives quantitative information about transient interactions in live cells.

Time-Domain Fluorescence Lifetime Imaging Microscopy: A Quantitative Method to Follow Transient Protein–Protein Interactions in Living Cells