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bioRxiv · 10.64898/2026.06.20.733419

High-speed volumetric single-molecule imaging using dual-wavelength light sheets and PSF-engineered enhanced biplane detection

Joshi, P.·Saliba, N.·Cheng, S.·Nakatani, Y.·Xiao, D.·Orange-Kedem, R.·Shechtman, Y.·Gustavsson, A.-K.
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Abstract

Single-molecule localization microscopy (SMLM) enables nanoscale imaging but remains limited in three-dimensional (3D), high-speed, and high-density applications due to background fluorescence, photon inefficiency, and large point-spread function (PSF) footprints. Here, we present single-objective light-sheet microscopy with PSF-engineering enhanced biplane detection (SoLiD-3D), a versatile imaging platform that integrates dual-wavelength light-sheet illumination with dual-color, multi-configuration biplane imaging for parallel acquisition with PSF engineered detection for high-speed volumetric SMLM. Parallelized single-objective light-sheet excitation combined with PSF engineering overcomes key limitations of conventional wide-field and biplane approaches. Independent control of two excitation wavelengths for optical sectioning enables simultaneous dual-target imaging and single-target dual-color imaging with improved contrast and temporal resolution utilizing dynamically displaced light sheets for volumetric coverage. Using SoLiD-3D, we demonstrate high-speed single- and dual-target dual-color imaging that doubles localization density without sacrificing photon efficiency and continuous volumetric imaging via PSF-engineering enhanced biplane detection for whole-cell 3D imaging with improved axial localization performance over extended depth ranges. We further demonstrate improved speed by utilizing the Hummus PSF, a compact engineered PSF that enables high-precision 3D localization with a substantially reduced spatial footprint, for the first time for super-resolution imaging applications. Taken together, SoLiD-3D mitigates the trade-off between axial range and localization precision and offers improved speed compared to conventional 3D SMLM approaches.

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Joshi, P., Saliba, N., Cheng, S., Nakatani, Y., Xiao, D., Orange-Kedem, R., Shechtman, Y., Gustavsson, A.-K.. 2026-06-25. High-speed volumetric single-molecule imaging using dual-wavelength light sheets and PSF-engineered enhanced biplane detection. https://doi.org/10.64898/2026.06.20.733419

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