Single molecule multi-color multi-pair FRET microscopy

Single molecule Förster resonance energy transfer (smFRET) microscopy is an important tool to understand function of biomolecules by measuring their structural dynamics at nanometer length scale. However, to monitor more than one distances at once, simultaneous multi-pair FRET measurement is challenging due to the spectral bandwidth required for the donor and acceptor fluorophores. We have recently developed Continuously Controlled Spectral resolution (CoCoS) microscopy by integrating a pair of coaxially rotating amici prisms in front of the camera in a standard epi-fluorescence microscope. We show that CoCoS microscopy allows us to perform smFRET experiments on DNA molecules to simultaneously register multiple distinguishable FRET signals, and with a five-fold larger field of view compared to traditional dual-channel TIRF optics. These multicolor experiments are possible owing to the unique feature of CoCoS to simultaneously register several emission channels without compromising throughput. Using the herein described multi-color multi-pair smFRET, we would be able to simultaneously monitor multiple distances in large bio-molecular assemblies in order to decipher their structural dynamics.