Extending Observation Windows While Preserving Signal-to-Noise in Single-Molecule Spectroscopy

We introduce an approach to extend the observation window in single-molecule spectroscopy that is generalizable to any fluorophore without relying on chemical photostabilization. The bleaching time can be controlled through the repetition rate of a pulsed excitation source. We show that reducing the excitation repetition rate from 40 to 2.5 MHz delays photobleaching and therefore extends the observation window of a single-molecule fluorophore by more than an order of magnitude. Furthermore, we demonstrate that photon-stream analyses that depend on emitted photon flux suffer a decrease in signal-to-noise ratio with decreasing repetition rate, while analyses that depend on total photon number have a signal-to-noise ratio that is independent of excitation repetition rate.