An inertia-free beam scanning device for single-wavelength 2PE-STED nanoscopy

Abstract

Two-photon excitation stimulated emission depletion nanoscopy (2PE-STED) is a fluorescence imaging technique ideal for significantly improving the spatial resolution when observing scattering tissue in fixed, in vitro, and in vivo specimens. Both 2PE and STED are beam scanning techniques. The image acquisition is commonly realized by raster scanning, and a pair of galvanometric mirrors are the most used approach. In 2PE fluorescence microscopy, acousto-optical deflectors (AODs) are also used because they allow random-access scanning. However, since the AOD working principle is diffraction of light, these devices are of limited use when it is necessary to deflect multiple beams of different wavelengths and polarization, e.g. in STED nanoscopy. Here, we present the first implementation of single wavelength 2PE-STED that enables a smart beam scanning system based on AODs technology.