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

Abstract

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). 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 (Raman 3D imaging of solvent-cleared organs (rDISCO)). 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 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.

Super-resolution vibrational imaging using expansion stimulated Raman scattering microscopy

“…Particularly, these amplification strategies will further increase the multiplexity with a practical goal of over 20 channels 7 . Second, specimens become optically transparent after expansion and thus MAGNIFIERS has promising applications for thick specimens of millimeter scales, considering successful precedents in integrating tissue clearing with label-free SRS 58 or highly-multiplexed Raman dye imaging 11 . This would open exciting applications to probe interactions between ultrastructure with high content over a long distance, such as with neuronal tracing.…”

“…Epr-SRS enables Raman detection of interesting biomarkers ranging from proteins, transcripts, and enzyme activities 6,50 . In particular, immuno-eprSRS with MARS dye-conjugated antibodies has emerged as an encouraging platform for one-shot multiplexed protein imaging 6,11 , bypassing technical troubles involved in cyclic techniques. Yet, it only works in a diffraction-limited paradigm.…”

“…In particular, by combining electronic pre-resonance spectroscopy with stimulated Raman scattering (SRS) microscopy (i.e., epr-SRS), the Raman cross sections of electronically coupled vibrational modes in light-absorbing dyes can be enhanced by 10 13 -fold 6,9 . Owing to this drastic enhancement, we achieved nanomolar sensitivity of Raman-active dyes (such as commercial far-red fluorescent dyes and specially-designed MARS probes), and demonstrated epr-SRS imaging of specific proteins 6,10,11 .…”

“…Collectively, SRS imaging circumvents the fundamental bottlenecks of fluorescence microscopy and provides unique benefits including label-free chemical imaging of endogenous biomolecules 1,12 , nonperturbative mapping of small metabolites 4 , and simultaneous profiling of a large group of specific biomarkers 6,11 . Yet, being an optical technique that uses near infrared or visible light, SRS microscopy is still constrained to diffraction-limited spatial resolution (~300 nm).…”

Understanding immunity in a tissue‐centric context: Combining novel imaging methods and mathematics to extract new insights into function and dysfunction*

“…3D-IBEX represents a major advance in the emerging field of multiplexed high-resolution 3D imaging, 87,[113][114][115][116][117][118] but requires further refinements. For example, the uniform penetration of high molecular weight antibodies to regions deeper than >300 µm, especially in dense tissues or those with a fibrous capsule, remains challenging.…”