Photobleaching-corrected FRET efficiency imaging of live cells

Fluorescent resonance energy transfer (FRET) imaging techniques can be used to visualize protein-protein interactions in real-time with subcellular resolution. Imaging of sensitized fluorescence of the acceptor, elicited during excitation of the donor, is becoming the most popular method for live FRET (3-cube imaging) because it is fast, nondestructive, and applicable to existing widefield or confocal microscopes. Most sensitized emission-based FRET indices respond nonlinearly to changes in the degree of molecular interaction and depend on the optical parameters of the imaging system. This makes it difficult to evaluate and compare FRET imaging data between laboratories. Furthermore, photobleaching poses a problem for FRET imaging in timelapse experiments and three-dimensional reconstructions. We present a 3-cube FRET imaging method, E-FRET, which overcomes both of these obstacles. E-FRET bridges the gap between the donor recovery after acceptor photobleaching technique (which allows absolute measurements of FRET efficiency, E, but is not suitable for living cells), and the sensitized-emission FRET indices (which reflect FRET in living cells but lack the quantitation and clarity of E). With E-FRET, we visualize FRET in terms of true FRET efficiency images (E), which correlate linearly with the degree of donor interaction. We have defined procedures to incorporate photobleaching correction into E-FRET imaging. We demonstrate the benefits of E-FRET with photobleaching correction for timelapse and three-dimensional imaging of protein-protein interactions in the immunological synapse in living T-cells.