Interferometric ultra-high resolution 3D imaging through brain sections

Single-molecule super-resolution microscopy allows pinpointing 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 the 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 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 within complex tissue specimens. We demonstrated 4Pi-BRAINSPOT's capacities in revealing the molecular arrangements in various sub-cellular organelles and resolved the membrane morphology of individual dendritic spines through 50-µm 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.