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bioRxiv · 10.1101/2025.02.03.636258

Interferometric Ultra-High Resolution 3D Imaging through Brain Sections

Gao, H.-C.·Xu, F.·Cheng, X.·Bi, C.·Zheng, Y.·Li, Y.·Chen, T.·Chubykin, A. A.·Huang, F.
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Abstract

Single-molecule super-resolution microscopy allows pin-pointing individual molecular positions in cells with nanometer precision. However, achieving molecular resolution through tissues is often difficult because of optical scattering and aberrations. We introduced 4Pi single-molecule nanoscopy for brain with in-situ point spread function retrieval through opaque tissue (4Pi-BRAINSPOT), integrating 4Pi single-molecule switching nanoscopy with dynamic in-situ coherent PSF modeling, single-molecule compatible tissue clearing, light-sheet illumination, and a novel quantitative analysis pipeline utilizing the highly accurate 3D molecular coordinates. This approach enables the quantification of protein distribution with sub-15-nm resolution in all three dimensions in complex tissue specimens. We demonstrated 4Pi-BRAINSPOTs capacities in revealing the molecular arrangements in various sub-cellular organelles and resolved the membrane morphology of individual dendritic spines through 50-{micro}m transgenic mouse brain slices. This ultra-high-resolution approach allows us to decipher nanoscale organelle architecture and molecular distribution in both isolated cells and native tissue environments with precision down to a few nanometers.

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Gao, H.-C., Xu, F., Cheng, X., Bi, C., Zheng, Y., Li, Y., Chen, T., Chubykin, A. A., Huang, F.. 2025-02-06. Interferometric Ultra-High Resolution 3D Imaging through Brain Sections. https://doi.org/10.1101/2025.02.03.636258

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