Figure 1
From: QuanTI-FRET: a framework for quantitative FRET measurements in living cells
The QuanTI-FRET approach. (A) A schematic of a widefield epifluorescence setup used for the validation of the framework is shown. Three images are acquired in two snapshots by automatically alternating the laser excitation and splitting the camera in two detection channels corresponding the donor and acceptor channels. (B) Framework for quantitative FRET analysis. The analysis requires three images combining the detection in the donor and the acceptor channels with the excitation of the donor and the acceptor. A calibration step allows the determination of four factors correcting for the crosstalks and the relative excitation and detection efficiencies of the donor and acceptor fluorophores. As a result, instrument-independent FRET probabilities and stoichiometries are calculated. Scale bar: 20 μm.
