Comparison of one- and two-photon fluorescence resonance energy transfer microscopy

The physics and chemistry of fluorescent resonance energy transfer (FRET) have been well studied theoretically and experimentally for many years, but only with recent technical advances has it become feasible to apply FRET in biomedical research. FRET microscopy is a better method for studying the structure and localization of proteins under physiological conditions than are X-ray diffraction, nuclear magnetic resonance, or electron microscopy. In this study, we used four different light microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (CIEBP alpha) in living pituitary cells. In wide-field, confocal, and two-photon microscopy the FRET image provides 2-D spatial distribution of steady-state protein-protein interactions. The two-photon imaging technique provides a better FRET signal (less bleed through and photo bleaching) compared to the other two techniques. This information, although valuable, falls short of revealing transient interactions of proteins in real time. We will discuss the advantage of fluorescence lifetime methods to measure FRET signals at the moment of the protein-protein interactions at a resolution on the order of subnanoseconds, providing high temporal, as well as spatial resolution.