Postdoctoral Researcher University of California San Diego, United States
Introduction: : Cells undergo dynamic changes in metabolic pathways in response to physiological or pathological cues. This metabolic rewiring is especially critical in systems, such as neurons, cancer, stem cells, and immune response, where metabolic needs and biosynthetic demands rapidly shift. Existing metabolic imaging techniques report synthetic and catabolic activity of single metabolite at each time by isotope labeling, yet do not trace the functional utilization of multiple metabolites derived from anabolic products and their turnover rate. Here we report a subcellular multiplexed metabolic isotope tracing (SuMMIT) microscopy technique to trace individual macromolecules derived from various metabolites.
Materials and
Methods: : Here we report a subcellular multiplexed metabolic isotope tracing (SuMMIT) microscopy technique to trace individual macromolecules derived from various metabolites. Based on stimulated Raman-scattering imaging, SuMMIT simultaneously visualizes multiple metabolic dynamics of newly synthesized macromolecules, such as DNA, protein and lipids, via the enrichment and distinct spectra of carbon–deuterium bonds transferred from the deuterated amino acid, lipid and monosaccharide precursors.
Results, Conclusions, and Discussions:: We developed SuMMIT (Subcellular Multiplexed Metabolic Isotope Tracing), a platform that integrates stimulated Raman scattering (SRS) microscopy with deuterium-labeled metabolic probes and linear unmixing algorithms to visualize and quantify the synthesis of major macromolecules—including proteins, lipids, and nucleic acids—with high spatial and temporal resolution. SuMMIT enables simultaneous tracing of multiple biosynthetic pathways back to their original carbon sources in vivo, providing unprecedented insight into how distinct metabolic precursors are differentially allocated across macromolecular pools. We applied this platform in diverse biological contexts—including metabolic gene mutations, intermittent fasting, and neuronal development and aging—to uncover context-specific metabolic rewiring, particularly in lipid and protein synthesis pathways. By identifying major shifts in biosynthetic activity, SuMMIT reveals key metabolic vulnerabilities and regulatory nodes that may serve as potential targets for therapeutic intervention or offer mechanistic insights into developmental and aging processes.
