Ultrafast, temporally stochastic STED nanoscopy of millisecond dynamics

Temporally stochastic STED nanoscopy with electro-optical deflector–based scanning allows ultrafast super-resolution imaging. The method was used to monitor vesicles and viruses moving at ~2 μm/s with no motion blur. Electro-optical scanning (>1,000 frames/s) with pixel dwell times on the order of the lifetime of the fluorescent molecular state renders stimulated emission depletion (STED) nanoscopy temporally stochastic. Photon detection from a molecule occurs stochastically in one of several scanning frames, and the spatial origin of the photon is known with subdiffraction precision. Images are built up by binning consecutive frames, making the time resolution freely adjustable. We demonstrated nanoscopy of vesicle motions in living Drosophila larvae and the cellular uptake of viral particles with 5- to 10-ms temporal resolution.