Live-cell quantitative FRET imaging made simple by autocalibration in QuanTI-FRET

SIMPLIFIED PROTOCOL FOR QUANTITATIVE FRET IN LIVING CELLS WITH DIRECT QUANTI-FRET CALIBRATION FROM THE IMAGES OF INTEREST: Genetically encoded biosensors based on the fluorescence resonance energy transfer (FRET) between two fluorescent proteins have the power to measure biochemical activity in living cells with the spatio-temporal resolution given by optical microscopy. The generalization of their usage is limited by the difficulties in obtaining quantitative results independent of the instrumental system or the expression level. We recently developed quantitative three-image FRET (QuanTI-FRET), a method for calibrating the system and obtaining absolute values of the FRET probabilities. The method proved to be efficient but required additional constructs for the calibration, thereby adding experimental steps. Here, we propose taking advantage of the constant and known stoichiometry of intramolecular FRET biosensors to directly calibrate the system using the dataset of interest, e.g., biosensor experiments. We demonstrate this idea by comparing the results of both standard calibration and autocalibration obtained on live-cell images of the FAK biosensor. This autocalibration is possible because of the strong robustness of the QuanTI-FRET calibration with respect to the quality of the calibration dataset. With this work, we simplify the experimental protocol to obtain quantitative FRET by autocalibration, and we make it accessible through a publicly available Python software and a napari plug-in.