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bioRxiv · 10.1101/2022.06.25.497623

Accurate background reduction in adaptive optical 3D-STED nanoscopy by dynamic phase switching

Tu, S.·Liu, X.·Yuan, D.·Tao, W.·Han, Y.·Shi, Y.·Li, Y.·Kuang, C.·Yao, Y.·Xu, Y.·Hao, X.
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Abstract

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 with adaptive optics, we present an easy-to-implement and flexible method to improve 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 3-6 times. We demonstrate our method on various dense subcellular structures, showing more advantages than the software-based background subtraction algorithms. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/497623v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@7aacd6org.highwire.dtl.DTLVardef@1e91f14org.highwire.dtl.DTLVardef@1f774acorg.highwire.dtl.DTLVardef@10ccf5d_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Tu, S., Liu, X., Yuan, D., Tao, W., Han, Y., Shi, Y., Li, Y., Kuang, C., Yao, Y., Xu, Y., Hao, X.. 2022-06-29. Accurate background reduction in adaptive optical 3D-STED nanoscopy by dynamic phase switching. https://doi.org/10.1101/2022.06.25.497623

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