Improved resolution in fluorescence microscopy with the FRET pairs by time gating

We investigate the temporal information of nonlinear donor fluorescence in the FRET frustration microscopy. The arrival time of the donor fluorescence varies with the distribution of the excitation laser. The differences of the arrival times between different positions in the excitation spot can be further enhanced in the case of adding a depletion beam upon the FRET probes. The spatial information is encoded in the temporal dynamics of the fluorescent photons from donor molecules and time-gating in detection can be used to increase the spatial resolution.