A Multimodal Imaging Pipeline for the Discovery of Molecular Markers of Cellular Neighborhoods.

Insights into tissue microenvironments are constrained by profiling cellular and molecular components independently. To address this, we developed an integrated, multimodal workflow combining imaging mass spectrometry and highly multiplexed immunofluorescence on the same sample. Custom computational pipelines enable precise spatial co-registration, facilitating direct integration of cellular and molecular profiles. CODEX-derived cellular and neighborhood masks enable mining of IMS-derived lipid data to identify molecular markers of cell types and spatial neighborhoods in human kidney tissue. We discovered distinct carnitine distributions in cortical and medullary regions, indicating metabolic heterogeneity driven by microenvironment-specific oxygen levels and energy demands. Glomerular cell-type-specific analysis found ether-linked phosphatidylcholine and sphingomyelin, species associated with slit diaphragm integrity, localized to podocytes. Immune cell neighborhoods exhibited molecular signatures consistent with cell signaling and activation pathways in damaged tissue regions. This multi-omic framework links cellular organization with molecular signatures to unveil cellular and molecular relationships underlying tissue function and pathology.