Mapping metabolism using stable isotope tracers in combination with mass spectrometry imaging

Stable isotope tracing combined with mass spectrometry imaging (MSI) offers a powerful framework for spatially and temporally resolved analysis of metabolic activity, enabling visualization of labeled metabolites at cellular and subcellular resolution. This approach provides insights into the spatial organization and dynamics of metabolic pathways such as glycolysis, the tricarboxylic acid cycle, and amino acid metabolism under both physiological and pathological conditions. However, its implementation remains technically challenging due to spectral complexity, limited spatial quantification methods, and the need for meticulous sample preparation and data processing. This review defines key considerations for integrating isotope tracers with MSI, including tracer selection, administration strategies across biological model systems, and current MSI platform capabilities. It also covers interpretation of isotopic labeling patterns and emerging experimental designs for mapping metabolic activity. By consolidating recent advances and identifying ongoing challenges, this review outlines the evolving role of MSI-based isotope tracing in spatial metabolomics. • Mass Spectrometry Imaging (MSI) combined with stable isotopes provides spatial and temporal analysis of metabolic activity • Choosing the right isotope tracer is pivotal for investigating spatial metabolic activity • MSI-based stable isotope tracing was demonstrated across experimental systems from mapping cell-type-specific dynamics in kidney injury to identifying cell-intrinsic metabolic phenotypes in glioblastoma. • Systemic flux estimates remain a challenge in MSI-based stable isotope analysis