Time-resolved single-molecule fluorescence microscopy: Pump-probe scheme employing bursts of pulses and gated photon counting

A practical methodology to conduct the time-resolved fluorescence microscopy has been developed; the technique employs bursts of excitation pulses (≤ ms duration) gated by an electro-optic modulator from a 76 MHz ultrafast oscillator and enables incremental stage positioning along with gated photon counting in a pump-probe scheme. A fluorescence trajectory is thus spread into segments of the multiple scans, which advances an efficient data recovery not only over limited photon-cycles but a frequently blinking trajectory owing to the innate heterogeneity present in a single-molecule environment. A typical application that probes ultrafast dynamics of an interfacial electron transfer was demonstrated in a dye-sensitized TiO2 nanoparticles at ambient condition.