Photoswitching FRET - A New Approach to Imaging Energy Transfer

Abstract

Forster Resonance Energy Transfer (FRET) is a powerful approach to study the interactions of fluorescent molecules. As it has become more commonplace in fluorescence microscopy, numerous techniques have been developed to measure FRET. Amongst them is fluorescence lifetime imaging (FLIM) which is often considered a rigorous standard for determining the level of energy transfer from a donor to acceptor molecule. However, FLIM requires specialized equipment and expertise unavailable to many microscopists. Here, we present a new approach which can be performed on a conventional widefield or confocal microscope to image FRET between a donor photoswitchable fluorescent protein, Dronpa, and an acceptor based on a conventional fluorescent protein. It is based on Dronpa's photoswitching properties in the presence and absence of an acceptor. The technique which we call photoswitching FRET (psFRET) is similar to an established but underutilized technique called photobleaching FRET (pbFRET) with the major difference being the molecules are switched “off” rather than photobleached. Therefore, less illumination light is required to switch “off” the molecule and it can be photoswitched “on” to repeat the cycle. We will present data from experiments testing the validity of this new approach and demonstrate its utility in monitoring intracellular protein-protein interactions.