Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells.

With its precise, sensitive, and nondestructive features, spectral unmixing-based fluorescence resonance energy transfer (FRET) microscopy has been widely applied to visualize intracellular biological events. In this report, we set up a spectral wide-field microscopic FRET imaging system by integrating a varispec liquid crystal tunable filter into a wide-field microscope for quantitative FRET measurement in living cells. We implemented a representative emission-spectral unmixing-based FRET measurement method on this platform to simultaneously acquire pixel-to-pixel images of both FRET efficiency (E ) and acceptor-to-donor concentration ratio (R C ) in living HepG2 cells expressing fusion proteins in the presence or absence of free donors and acceptors and obtained consistent results with other instruments and methods. This stable and low-cost spectral wide-field microscopic FRET imaging system provides a new toolbox for imaging molecular events with high spatial resolution in living cells.