Understanding complex biological systems requires simultaneous characterization of a large number of interacting components in their native 3D environment. However, fluorescence methods are usually hindered by the fundamental color barrier. Here we will introduce electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy, an emerging method of ultrasensitive vibrational imaging with high potential of multiplexing. To generalize epr-SRS to large-scale volumetric imaging, we further integrated it with tissue clearing technology and achieved simultaneous visualization of >10 protein targets over millimeter thickness of brain tissues. Overall, super-multiplexed vibrational imaging is a promising tool to provide a complete picture of tissue biology.
Understanding metabolism is indispensable in unraveling the mechanistic basis of biological processes. However, in situ metabolic imaging tools are still lacking. Here we introduce a framework for mid-infrared (MIR) metabolic imaging by coupling the emerging high-information-throughput MIR microscopy with specifically designed IR-active vibrational probes. We present three categories of small vibrational tags including azide bond, 13C-edited carbonyl bond and deuterium-labeled probes to interrogate various metabolic activities in cells, small organisms and mice. Our technique is uniquely suited to metabolic imaging with high information throughput. In particular, we performed single-cell metabolic profiling and large-area metabolic imaging at tissue or organ level.
Mapping the localization of multiple proteins in their native 3D context would be useful for biomedicine. We harness the narrow spectrum of Raman spectroscopy and introduce Raman Dye Imaging and Tissue Clearing (RADIANT), an optical method for imaging multiple targets in thick samples in one shot. We expanded the range of bioorthogonal Raman dyes and developed a tissue clearing strategy for them. RADIANT allowed us to image >10 targets in millimeter thick brain slices, extending the imaging depth 10-100 fold compared to prior methods. RADIANT will facilitate the exploration of the intricate 3D protein interactions in complex systems.
Understanding metabolism is of great significance to decipher various physiological and pathogenic processes. While great progress has been made to profile gene expression, how to capture organ-, tissue-, and cell-type-specific metabolic profile (i.e. metabolic tissue atlas) in complex mammalian systems is lagging behind, largely owing to the lack of metabolic imaging tools with high resolution and high throughput. Here, we applied mid-infrared imaging coupled with heavy water (D2O) metabolic labeling to a scope of mouse organs and tissues. Our premise is that, as D2O participates in the biosynthesis of various macromolecules, the resulting broad C-D vibrational spectrum should interrogate a wide range of metabolic pathways. Applying multivariate analysis to the C-D spectrum, we successfully identified both inter-organ and intra-tissue metabolic signatures of mice. A large-scale metabolic atlas map between different organs from the same mice was thus generated. Moreover, leveraging the power of unsupervised clustering methods, spatially-resolved metabolic signatures of brain tissues were discovered, revealing tissue and cell-type specific metabolic profile in situ. As a demonstration of this technique, we characterized metabolic changes during brain development and captured intratumoral metabolic heterogeneity of glioblastoma. Altogether, the integrated platform paves a way to map the metabolic tissue atlas for complex mammalian systems.
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