Time-Correlated Single-Photon Counting Fluorescence Lifetime Imaging–FRET Microscopy for Protein Localization

This chapter discusses the development and characterization of a two-photon excitation time-correlated single-photon counting (TCSPC) FLIM–FRET microscopy system that uses the TCSPC imaging mode hardware. Fluorescence lifetime imaging–FRET microscopy relies on the ability to capture weak and transient fluorescent signals efficiently and rapidly from the interactions of labeled molecules in single living or fixed cells. Forster resonance energy transfer (FRET) microscopy has the advantage because the spatial distribution of FRET efficiency can be visualized throughout the image, rather than registering only an average over the entire cell or population. The advantage of using FLIM is that lifetime measurements are independent of change in concentration and excitation intensity. One of the principal and unique benefits of FRET microscopic imaging is that not only co-localization of the donor- and acceptor-labeled probes can be seen but also intimate interactions of molecules labeled with donor and acceptor can also be demonstrated. The TCSPC FRET-FLIM imaging system has the added advantage of tracking dynamic protein–protein interactions and provides more precise determinations of the distance between the donor and the acceptor molecules as compared to the intensity-based (wide-field, confocal, and multiphoton) methods.