High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean–Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donor–acceptor distances. Our results for this simple control system indicate that the in vivo FLIM–FRET studies of more complex protein–protein interactions would benefit greatly from such quantitative measurements.