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Robust approaches to quantitative ratiometric FRET imaging of CFP/YFP fluorophores under confocal microscopy

Ratiometric quantification of CFP/YFP FRET enables live‐cell time‐series detection of molecular interactions, without the need for acceptor photobleaching or specialized equipment for determining fluorescence lifetime. Although popular in widefield applications, its implementation on a confocal microscope, which would enable sub‐cellular resolution, has met with limited success. Here, we characterize sources of optical variability (unique to the confocal context) that diminish the accuracy and reproducibility of ratiometric FRET determination and devise practical remedies. Remarkably, we find that the most popular configuration, which pairs an oil objective with a small pinhole aperture, results in intractable variability that could not be adequately corrected through any calibration procedure. By quantitatively comparing several imaging configurations and calibration procedures, we find that significant improvements can be achieved by combining a water objective and increased pinhole aperture with a uniform‐dye calibration procedure. The combination of these methods permitted remarkably consistent quantification of sub‐cellular FRET in live cells. Notably, this methodology can be readily implemented on a standard confocal instrument, and the dye calibration procedure yields a time savings over traditional live‐cell calibration methods. In all, identification of key technical challenges and practical compensating solutions promise robust sub‐cellular ratiometric FRET imaging under confocal microscopy.