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

Hirokazu Ishii; Kohei Otomo; Jui-Hung Hung; Motosuke Tsutsumi; Hiroyuki Yokoyama; Tomomi Nemoto

doi:10.1364/BOE.10.003104

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

Biomed Opt Express. 2019 Jun 3;10(7):3104-3113. doi: 10.1364/BOE.10.003104. eCollection 2019 Jul 1.

Authors

Hirokazu Ishii¹, Kohei Otomo^{1

2}, Jui-Hung Hung³, Motosuke Tsutsumi¹, Hiroyuki Yokoyama^{3

4}, Tomomi Nemoto^{1

2}

Affiliations

¹ Research Institute for Electronic Science, Hokkaido University, Kita 20 Nishi 10, Kita-ku, Sapporo 001-0020, Japan.
² Graduate School of Information Science and Technology, Hokkaido University, Kita 14 Nishi 9, Kita-ku, Sapporo 060-0814, Japan.
³ New Industry Creation Hatchery Center (NICHe), Tohoku University, 6-6-10, Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan.
⁴ Graduate School of Engineering, Tohoku University, 6-6-05, Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan.

Abstract

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.