All-Optical Multimodal Mapping of Single Cell Type-Specific Metabolic Activities via REDCAT

Abstract

Metabolism underlies cell growth, survival, and function, yet its activities vary widely across cell types and tissue environments. Spatially resolving these processes in situ at single-cell resolution is essential to advance our understanding of cellular function and tissue physiology in health and disease. However, existing approaches are limited by either destructive workflows, insufficient spatial resolution and biochemical specificity, or lack of direct linkage to cell identity. Here, we present Raman Enhanced Delineation of Cell Atlases in Tissues (REDCAT), a multimodal all-optical platform that integrates stimulated Raman scattering, autofluorescence redox imaging, second harmonic generation, and high-plex immunofluorescence to co-map metabolic activities and cell types within the same tissue section. REDCAT achieves subcellular resolution profiling of protein, lipid, redox, and nuclear acid metabolism, together with extracellular matrix composition, in both FFPE and fresh-frozen human tissues. Applied to normal lymph nodes, REDCAT delineated distinct redox and lipid remodeling programs across germinal center B-cell zones and immune subsets, highlighting cell-type-specific metabolic specialization. In lymphoma, it revealed profound metabolic reprogramming, including extensive lipid accumulation, nuclear metabolic heterogeneity, and a transitional metabolic state associated with transformation from chronic lymphocytic leukemia to diffuse large B-cell lymphoma, thereby illuminating tumor evolution in situ. In human liver, REDCAT resolved cell-type-specific lipid droplet diversity and zonation-dependent nuclear metabolic gradients, uncovering new principles of spatial metabolic organization. By directly linking cell identity with spatial metabolic states at single-cell or subcellular resolution, REDCAT establishes a broadly applicable framework for studying immune function, tumor progression, and tissue physiology, and offers a new path to deciphering the metabolic basis of health and disease.