Fluorescence Lifetime Imaging Microscopy (FLIM) combines time-resolved fluorescence spectroscopy to imaging microscopy and aims to analyze quantitative parameters of fluorescence at a cell or tissue level. The fluorescence lifetime (() of a fluorescent molecule species may be defined as the average time that this molecule remains in its excited state prior to returning to its ground state. For fluorescent molecules commonly used in biology, this phenomenon typically ranges in the nanosecond timescale. Unlike intensity based measurements of fluorescence, the fluorescence lifetime is independent of the local probe concentration. In contrast, variations in the local environment of the molecule induce changes in its fluorescence lifetime. This parameter is thus a characteristic of a given fluorescent molecule in a given environment, and a change in the fluorescence lifetime of a fluorophore reflects a change in its local environment.
Among numerous applications, FLIM measurements of lifetime values of fluorescent probes have been used to detect molecular interactions (Lakowicz, 1999; Hink et al., 2002; Duncan, 2006). In this case, transfer of energy from the donor to the acceptor decreases the fluorescence lifetime of the donor fluorophore, and the relative difference of lifetime is a measure of energy transfert efficiency. FLIM is thus particularly well adapted to detect protein interactions in live cells. In spite of several available commercial instrumentations, FLIM is still an emerging technology in biology laboratories. This is likely due to the high level of technicity and expertise required to obtain reliable measurements.
We developped in our institute (FRAIB - CNRS-UPS) a FLIM apparatus coupling ultrafast infrared laser for multiphoton excitation and a streak camera for lifetime measurements. Briefly, the streak camera (Streakscope C4334, Hamamatsu Photonics, Japan) consists of a photocathode surface, a sweep electrode to deflect the photoelectrons according to their arrival time, a microchannel plate-photomultiplier tube (MCP-PMT) to amplify photoelectrons and a phosphor screen to detect the amplified photoelectrons. The images on the phosphor screen are read-out by the CCD camera. This system is unique in Europe and technological developments are performed in collaboration with the Hamamatsu company. In our platform, we obtained results in the domain of protein interactions for different resarch teams (see references).
References: Applications realized in the lab 1- Plant microbe interactions - Maud Bernoux et al., Plant Cell, 2008. Solène Froidure et al., PNAS, 2010. Céline Tasset, et al, PlosPathogens. 2010. Joanne Canonne et al., Plant Cell, 2011. Katharina Heidrich et al., Science 2011. 2- Human cancer cells -Rose Boutros et al., Biol Cell., 2011.