FRET-FLIM Microscopy

摘要

Visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The current advances in fluorescence microscopy coupled with the development of new fluorescent probes provide the tools to study protein interactions in living specimens. Spectral bleed-through or cross talk is a problem in one- and two-photon microscopy to recognize whether one is observing the sensitized emission or the bleed-through signals. In contrast, FLIM (fluorescence lifetime imaging microscopy) or lifetime measurements are independent of excitation intensity or fluorophore concentration. The combination of FLIM and FRET will provide high spatial (nanometer) and temporal (nanoseconds) resolution when compared to steady state FRET imaging. Importantly, spectral bleed-through is not an issue in FLIM imaging because only the donor fluorophore lifetime is measured. The presence of acceptor molecules within the local environment of the donor that permit energy transfer will influence the fluorescence lifetime of the donor. By measuring the donor lifetime in the presence and the absence of acceptor one can accurately calculate the FRET efficiency and the distance between donor- and acceptor-labeled proteins. Moreover, the FRET-FLIM technique allows monitoring more than one pair of protein interactions in a single living cell.