In vivo interaction studies by measuring förster resonance energy transfer through fluorescence lifetime imaging microscopy (FRET/FLIM)

Combinations of multiple fluorescent fusion proteins are commonly generated and used for colocalization studies in live cell imaging but also biochemical analysis of protein-protein interactions by co-immunoprecipitation in vitro. Advanced microscopy techniques like Förster resonance energy transfer through fluorescence lifetime imaging microscopy (FRET/FLIM) nowadays enable the combination of both approaches. This opens up the possibility to perform a location-specific protein-protein interaction analysis in vivo. To this end, the nonradiant energy transfer from a donor to an acceptor fluorophore (FRET) is harnessed to test for close proximity as an indicator for interaction, while the spectromicroscopical measurement of the fluorescence lifetime by FLIM serves as a readout. Here, we describe FRET/FLIM measurements performed with a Leica TCS SP8/PicoHarp 300 combination to demonstrate the interaction between a RFP-tagged GFP-nanobody and its epitope, GFP, in the cytoplasm of tobacco mesophyll protoplasts.