Development of Probes for Cellular Functions Using Fluorescent Proteins and Fluorescence Resonance Energy Transfer

Many genetically encoded probes that employ fluorescent proteins and fluorescence resonance energy transfer (FRET) have been developed to better understand the spatiotemporal regulation of various cellular processes. The different types of FRET and measurement techniques necessitate characterization of their specific features. Here I provide theoretical and practical comparisons of bimolecular and unimolecular FRET constructs, intensity-based and lifetime-based FRET measurements, FRET imaging using live- and fixed-cell samples, green fluorescent protein–based and chemical fluorophore-based FRET, and FRET efficiency and indices. The potential benefits and limitations of a variety of features in the technologies using fluorescent proteins and FRET are discussed.