![]() ![]() We showcased the utility of RADIANT in extracting systems information, including region-specific correlation networks and their topology in cerebellum development. We applied RADIANT to image up to 11 targets in millimeter-thick brain slices, extending the imaging depth 10- to 100-fold compared to prior multiplexed protein imaging methods. We expanded the range of suitable bioorthogonal Raman dyes and developed a tissue-clearing strategy for them (Raman 3D imaging of solvent-cleared organs (rDISCO)). ![]() 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. Mapping the localization of multiple proteins in their native three-dimensional (3D) context would be useful across many areas of biomedicine, but multiplexed fluorescence imaging has limited intrinsic multiplexing capability, and most methods for increasing multiplexity can only be applied to thin samples (<100 µm).
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