In Vivo Interaction Studies by Measuring Förster Resonance Energy Transfer Through Fluorescence Lifetime Imaging...

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.