Bridging the resolution-sectioning gap in stimulated emission depletion microscopy: theory and experiment

Abstract

AbstractMicroscopes generally exhibit superior performance in 2D compared to 3D. Fluorescence super-resolution imaging often intensifies this discrepancy, such as with the gold-standard vortex-based stimulated emission depletion (STED) microscope, which narrows the point-spread function only laterally. In this study, we developed a semi-analytical theory based on the Nijboer-Zernike expansion, conducted simulations and performed experiments to establish the merits of the alternative bivortex-based STED. We find that this mode reduces the axial-lateral resolution mismatch and effectively emulates noisier multi-beam approaches by providing access to point-spread function geometries that are strictly forbidden to the two conventional single-beam modes, 2D-STED and z-STED. Notably, theory and experiment indicate that, besides filling the gap, bivortex STED delivers a higher signal-to-background than the two modes it bridges.