Abstract
Förster resonance energy transfer (FRET) is a powerful tool for the visualization of molecular signaling events such as protein activities and interactions in cells. In its different implementations, FRET microscopy has been mainly used for monitoring single events. Recently, there has been a trend of extending FRET imaging towards the simultaneous detection of multiple events and interactions. The concomitant increase in experimental complexity requires a deeper understanding of the biophysical background of FRET. The presence of multiple acceptors for one donor affects the well-known formalism for FRET between two molecules, increasing distance sensitivity through mechanisms that have become known as the 'antenna' and 'surplus' effect. We will discuss the nature of these effects and present the imaging methods that have been used to unravel the combined transfer rates in the multi-protein interactions of multiplexed FRET experiments. Multiplexing strategies are becoming invaluable analytical tools for the elucidation of biological complexes and for the visualization of decision points in cellular signaling networks in physiological and pathological conditions.