Highly-multiplexed volumetric mapping with Raman dye imaging and tissue clearing

Mapping the localization of multiple proteins in their native 3D context would be useful across many areas of biomedicine, but multiplexed fluorescence imaging has limited intrinsic multiplexing capability, and most methods for increasing multiplexing can only be applied to thin samples (<100 μm). Here we harness the narrow spectrum of Raman spectroscopy and introduce Raman Dye Imaging and Tissue Clearing (RADIANT), an optical method that is capable of imaging multiple targets in thick samples in one shot. We expanded the range of suitable bioorthogonal Raman dyes and developed a tissue clearing strategy for them (rDISCO). We applied RADIANT to image up to eleven targets in millimeter thick brain slices, extending the imaging depth 10–100 fold compared to prior multiplexed protein imagining methods. We showcased the utility of RADIANT in extracting systems information including region-specific correlation networks and their topology in cerebellum development. RADIANT will facilitate the exploration of the intricate 3D protein interactions in complex systems.