Optical metabolic imaging of the tricarboxylic acid cycle

Abstract The tricarboxylic acid (TCA) cycle lies at the core of cellular metabolism, integrating energy production, biosynthesis and redox homeostasis, yet direct quantitative imaging of its activity in living systems remains challenging. Here we introduce MATRIX-SRS (Metabolic Activity TRacing of the trIcarboXylic acid cycle by Stimulated Raman Scattering microscopy), a platform enabling spatially resolved quantification of TCA-linked metabolism in live cells. Using emerging deuterium-labeled probes, MATRIX-SRS visualizes subcellular TCA-associated carbon-deuterium bonds in live cancer cells and neurons. We then integrate density functional theory, reaction network mapping, and hyperspectral MATRIX-SRS to construct a robust in situ metabolic quantification pipeline. Integrating MATRIX-SRS with isotope-tracing mass spectrometry, we reveal a global attenuation of TCA activity during epithelial-to-mesenchymal transition, providing deep molecular insights. Applying this framework, we further quantify changes in deuterium-labeled biomass in absolute concentrations for the first time, under native and drug-treated conditions, establishing a generalizable foundation for live quantitative spatial metabolomics.