Stimulated Raman Imaging for Spatial Metabolomics: From Metabolite Mapping to Cellular Function

Metabolism encompasses the full network of chemical reactions sustaining life- the creation, utilization, and storage of biologically active small molecules known as metabolites. Understanding how these molecules are organized in space and real time, at the subcellular level, has long been a fundamental bottleneck in advancing our knowledge of cellular metabolism. In recent years, stimulated Raman scattering (SRS) microscopy has emerged as a powerful approach to visualize metabolism directly in living systems, offering high sensitivity, chemical selectivity, and a non-destructive imaging modality uniquely suited to longitudinal studies. Despite these attributes, metabolic imaging with SRS remains in its early stages relative to the well-established fluorescence-based and mass spectrometry imaging methods that dominate the field. Therefore, this thesis aims to push the boundaries of SRS microscopy toward quantitative, spatially resolved metabolomics to enable direct visualization of metabolic activity in living systems. By applying emerging Raman probes across diverse biological systems, this work reveals metabolic vulnerabilities that deepen insight into disease mechanisms and can inform therapeutic development. In Chapter 1, we introduce metabolic imaging using SRS microscopy and underscore its unique advantages for longitudinal imaging in living biosystems. We cover both label-free (untargeted) and labeled (targeted) approaches using SRS to probe metabolism in situ. We discuss both the advantages and current limitations of SRS microscopy and propose strategies for integrating it with complementary functional “-omics” techniques to gain deeper insight into cellular metabolism. In Chapter 2, we explore the use of untargeted SRS microscopy to non-invasively characterize the chemical landscape of engineered living materials (ELMs) in real-time. By correlating spectral data with the mechanical properties of genetically modified biofilms, we find multiscale metabolic heterogeneity within these systems. This approach enables quantitative, real-time monitoring of ELMs that would enable improved design in applications spanning biomedicine, sustainability, and responsive materials. In Chapter 3, we utilize targeted SRS microscopy to tackle critical questions in cardiovascular health with a focus on understanding the metabolic basis of diabetes. In collaboration with the Chen Lab at City of Hope Medical Center and the TeSlaa lab at UCLA, we observe glycogen accumulation in live endothelial cells under diabetic stress. By imaging glutamine and lactate metabolism using Raman probes for the first time in this system, we reveal how intracellular glycogen pools shape early metabolic adaptations in the endothelium during glucose deprivation- providing new insight into the vascular ramifications of hyperglycemia. In Chapter 4, we introduce MATRIX-SRS (Metabolic Activity TRacing of the trIcarboXylic acid cycle by Stimulated Raman Scattering microscopy), a platform for spatially resolved, quantitative imaging of TCA cycle activity in live cells. By combining deuterium-labeled metabolic probes with hyperspectral stimulated Raman scattering microscopy, we directly visualize and map TCA-associated metabolism at subcellular resolution. We next integrate density functional theory (DFT) with reaction network modeling to develop a robust in situ quantification pipeline in live cells. Using this approach, we identify a global attenuation of TCA activity during epithelial-to-mesenchymal transition (EMT) and, for the first time, achieve absolute quantification of deuterium-labeled biomass in live cells under native and drug-treated conditions, establishing a general framework for live-cell spatial metabolomics. In Chapter 5, we present MetaboRamics, highly multiplexed metabolic imaging using stimulated Raman for spatial metabolomics in live cells. Through rational probe design, isotope editing, and robust spectral unmixing, we establish a 16-color metabolic palette spanning key pathways alongside endogenous protein, lipid, and redox signals, with organelle-targeted probes enabling spatial interactomics. We apply this high-content platform to EMT to observe a global metabolic rewiring in mesenchymal cells. We further resolve subcellular adaptations under diverse metabolic stress conditions in live epithelial cells. MetaboRamics enables 16-plex, subcellular metabolomics in living systems and opens new avenues for applications in drug discovery and clinical diagnostics. Through these studies, I demonstrate the power of stimulated Raman imaging for subcellular, spatiotemporal metabolomics to visualize metabolism in living systems and uncover its roles in both health and disease.