Interferometric Ultra-High Resolution 3D Imaging through Brain Sections.

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 point spread function retrieval through opaque tissue (4Pi-BRAINSPOT), integrating 4Pi single-molecule switching nanoscopy with dynamic 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-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 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.