Two-photon STED nanoscopy realizing 100-nm spatial resolution utilizing high-peak-power sub-nanosecond 655-nm pulses

We developed two-photon excitation stimulated emission depletion (STED) nanoscopy using high-peak-power sub-nanosecond 655-nm pulses. The STED pulse exhibited ideal optical properties and sufficient pulse energy to realize a 70-nm spatial resolution in the compact setup With electrically controllable components. For biological applications, we screened suitable fluorescent dyes or proteins and realized the sub-100 nm spatial resolution imaging of presynaptic protein clusters in fixed primary cultured neurons Without severe photobleaching. We expect this method to enable visualization of ultrastructures and the cluster dynamics of biomolecules representing physiological functions in living cells and tissue. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement