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

Area of Science:

• Biomedical Optics
• Molecular Imaging
• Systems Biology

Background:

• Multiplexed fluorescence imaging is limited in its ability to image multiple proteins in thick samples.
• Existing methods for increasing multiplexity in imaging are restricted to thin samples (<100 µm).

Purpose of the Study:

• To develop a novel optical method for imaging multiple protein localizations in native 3D context within thick samples.
• To overcome the limitations of current multiplexed imaging techniques in terms of sample thickness and multiplexing capability.

Main Methods:

• Introduction of Raman dye imaging and tissue clearing (RADIANT), an optical method utilizing Raman spectroscopy.
• Expansion of bioorthogonal Raman dye range and development of a tissue-clearing strategy (rDISCO).
• Application of RADIANT to image up to 11 targets in millimeter-thick brain slices.

Main Results:

• RADIANT enables imaging of multiple targets in thick samples in a single acquisition.
• Imaging depth was extended 10- to 100-fold compared to previous multiplexed protein imaging methods.
• Demonstrated utility in extracting systems information, including cerebellum development correlation networks.

Conclusions:

• RADIANT significantly advances the capability to explore 3D protein interactions in complex biological systems.
• The method overcomes previous depth limitations in multiplexed protein imaging.
• Facilitates deeper understanding of intricate protein interactions in native tissue environments.