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

Abstract

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.