Abstract
New imaging methodologies in quantitative fluorescence microscopy and nanoscopy have been developed in the last few years and are beginning to be extensively applied to biological problems, such as the localization and quantification of protein interactions. Fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) is currently employed not only in biophysics or chemistry but also in bio-medicine, thanks to new advancements in technology and also new developments in data treatment. FRET-FLIM can be a very useful tool to ascertain protein interactions occurring in single living cells. In this review, we stress the importance of increasing the acquisition speed when working in vivo employing Time-Domain FLIM. The development of the new mathematical-based non-fitting methods allows the determining of the fraction of interacting donor without the requirement of high count statistics, and thus allows the performing of high speed acquisitions in FRET-FLIM to still be quantitative.