Shaping the illumination beams for STED imaging through highly scattering media

Abstract

Stimulated emission depletion (STED) microscopy has been widely applied in single-cell and single-molecular studies benefiting from its ability of super-resolution imaging. However, it is challenging to perform STED imaging in highly scattering media, such as biological tissues, due to high scattering, which destroys the structured illumination beams. Here, we present a technique to produce the desired illumination beams beyond high scattering for STED microscopy. A dual-wavelength transmission matrix method is employed for shaping the excitation beam and the STED beam simultaneously, achieving the temporal and spatial overlap of the two beams behind a scattering medium. Experimentally, the overlapped foci were constructed by using a digital micromirror device (DMD). The extinction ratio of the STED focus reaches -23.8 dB, which compares favorably to the oft-quoted desirable value for STED applications. In particular, raster scanning of overlapped foci and the switching of the STED focus were demonstrated by adopting the fast switching ability of the DMD. Construction of the illumination beams beyond scattering will benefit STED microscopy in deep tissues.