Ambarian, J. A. | Search bioRxiv

bioRxiv2026

MetaboRamics: Highly multiplexed metabolic imaging by stimulated Raman for spatial metabolomics in live cells

Cellular metabolism is highly dynamic, intertwined and spatially heterogenous, yet methods that can simultaneously visualize multiple metabolic pathways in living systems remain largely limited. Here, we present MetaboRamics: highly multiplexed metabolic imaging by stimulated Raman for spatial metabolomics in live cells. Through rational probe selection and careful optimization, isotope editing, and robust spectral unmixing, we establish a metabolic palette of 16-colors spanning glucose uptake and utilization, lipid uptake and synthesis, choline metabolism, DNA synthesis, and amino acid incorporation in addition to endogenous proteins, lipids and redox signals. Incorporation of organelle-targeted Raman probes further enables spatial interactomics and assessment of organelle activities. Applying MetaboRamics to epithelial-mesenchymal transition (EMT), we observe metabolic rewiring in mesenchymal cells, reflected by reduced glucose-derived biomass, lipid turnover, protein synthesis, and altered redox balance. Finally, we perform optical phenotyping of cellular states under metabolic stress of serum deprivation, nutrient overload (fructose, and saturated fatty acid), inflammation, and pharmacological perturbations to reveal subcellular metabolic changes. This work fully realizes the potential of stimulated Raman scattering (SRS) microscopy for super-multiplexed metabolic imaging by establishing, for the first time, a 16-plex platform for live-cell spatial metabolomics.

cell biology↗

bioRxiv2025

Single-Cell Metabolic Imaging Reveals Glycogen Driven-Adaptations in Endothelial Cells

Endothelial dysfunction (ED) is a defining feature of diabetes mellitus (DM) and a key contributor to many metabolic and cardiovascular diseases. Endothelial cells (ECs) are known to be highly glycolytic and primarily rely on glucose to meet their energy demands. However, the role of glycogen metabolism in ECs remains poorly characterized due to a lack of suitable tools. Here, we utilize stimulated Raman scattering (SRS) microscopy to investigate subcellular glycogen metabolism in live ECs under stress conditions associated with highly prevalent diabetes and diabetic complications. We demonstrate that ECs exposed to a diabetes-mimicking milieu- high glucose and tumor necrosis factor (TNF-)- divert excess glucose toward subcellular glycogen storage, and that this storage capacity is significantly enhanced by the inhibition of glycogen synthase kinase 3 (GSK3). Pulse-chase experiments uncover glycogen dynamics and reveal that glycogen is rapidly mobilized under glucose starvation, highlighting its role as an immediate energy reserve in ECs. We further extend the capabilities of SRS metabolic imaging to visualize glutamine and lactate metabolism for the first time, directly showcasing the reliance of ECs on these alternative carbon substrates during glucose deprivation. Our results indicate that ECs containing glycogen exhibit a reduced immediate metabolic demand for these gluconeogenic substrates in the absence of extracellular glucose. These findings suggest that glycogen may play a broader role beyond energy reserves in ECs by modulating stress-responsive metabolic adaptations and may offer potential therapeutic opportunities to address diabetes-induced ED and related cardiometabolic diseases.

cell biology↗