Accurate Background Subtraction in STED Nanoscopy by Polarization Switching

STED nanoscopy helped usher in resolution revolution of optical microscopy and has been widely used to study subcellular structures and dynamics. To achieve optical resolution beyond the diffraction limit, STED nanoscopy employs 10⁴-10⁵ times more intense light compared to conventional imaging methods, giving rise to additional background that can be detrimental to imaging and quantification of dense samples. Here, we report a simple and easy-to-implement method to suppress background noise by polarization switching (psSTED). STED imaging requires a well-defined polarization state of a STED laser (typically left-circular polarization). By switching to right-circular polarization, the spatial profile of the STED laser changes to fill up the center of the donut-shaped beam, enabling an accurate background recording. Through a straightforward subtraction of the recorded background from the regular STED image, we achieved background-subtracted STED imaging. With simulation and experiment, we demonstrate that psSTED outperforms other background subtraction methods.