Genetically encoded FRET-based biosensors for multiparameter fluorescence imaging

The phenomenon of Forster (or fluorescence) resonance energy transfer (FRET) between two fluorescent proteins of different hues provides a robust foundation for the design and construction of biosensors for the detection of intracellular events. Accordingly, FRET-based biosensors for a variety of biologically relevant ions, molecules, and specific enzymatic activities, have now been developed and used to investigate numerous questions in cell biology. An emerging trend in the use of FRET-based biosensors is to apply them in combination with a second biosensor in order to achieve simultaneous imaging of multiple biochemical parameters in a single living cell. Here we discuss the particular technological challenges facing the use of FRET-based biosensors in multiparameter live cell fluorescence imaging and highlight recent efforts to overcome these challenges. In addition, we survey recent applications and provide an outlook on the future opportunities in this area.