Accurate Background Reduction in Adaptive Optical Three-Dimensional Stimulated Emission Depletion Nanoscopy by Dynamic Phase Switching
Shijie Tu, Xin Liu, Difu Yuan, Wenli Tao, Yubing Han, Yan Shi, Yanghui Li, Cuifang Kuang, Xu Liu, Yufeng Yao, Yesheng Xu, Xiang Hao
STED nanoscopy, background suppression, adaptive optics, 3D-STED
Stimulated emission depletion (STED) fluorescence nanoscopy allows the three-dimensional (3D) visualization of nanoscale subcellular structures, providing unique insights into their spatial organization. However, 3D-STED imaging and quantification of dense features are obstructed by the low signal-to-background ratio (SBR) resulting from optical aberrations and out-of-focus background. Here, combining adaptive optics elements, we present an easy-to-implement, flexible, and effective method to improve the SBR by dynamic phase switching. By switching to a counterclockwise vortex phase mask and a top-hat one with an incorrect inner radius, the depletion pattern features a nonzero-intensity center, enabling accurate background recordings. When the recorded background is subtracted from the aberration-corrected 3D-STED image, the SBR in dense sample areas can be improved by a factor of 3–7 times. We demonstrate our method on various dense subcellular structures, showing more advantages than the software-based background subtraction algorithms.