Optimization of FRET Microscopy for Live-Cell Imaging of Multiple Protein-Protein Interactions

Abstract

Recent advances in fluorescence resonance energy transfer (FRET) microscopy have made it possible to measure simultaneously, multiple protein-protein interactions inside living cells. To further these methods, we integrated an improved multi-spectral imaging analysis method (N-way FRET) with optimized fluorophores for live-cell FRET microscopy. For 3-way FRET we determined the best cyan, yellow, and red (C-Y-R) FRET-trio by comparing the acceptor photobleaching FRET efficiencies of structurally similar linked fluorescent protein constructs. The pool of molecules examined was constrained to the available C, Y, R molecules with the best photophysical properties (CyPet/TFP1, mCitrine/YPet and TagRFP/mCherry, respectively). From this set, we determined that TFP, YPet or mCitrine and mCherry were the optimal FRET trio. For increasingly complex pathways, more fluorescent probes may be required. To extend this method to the analysis of 4-way FRET we examined fluorophore pairs with highly overlapping excitation and emission spectra (e.g. TFP and GFP or Citrine and GFP). These improvements should enhance the spatiotemporal resolution, the number of possible interacting probes and the reliability of FRET experiments leading to improved monitoring of multiple protein-protein interactions in complex pathways.