Mass spectrometry imaging (MALDI-MSI) has revolutionized the study of host–microbe interactions by allowing direct and simultaneous mapping of diverse metabolites within biological tissues. However, the precise localization of microbes and the assignment of metabolites to either the host or members of the microbiome present challenges in this dynamic environment. To address this, we introduce a correlative imaging approach, metaFISH, which seamlessly integrates MALDI-MSI with fluorescence in situ hybridization (FISH) on the same tissue section for the identification and localization of microbial cells.
MetaFISH is a robust and user-friendly method that enables the spatial distribution of metabolites to be assigned to specific microbiome members, achieving single-cell resolution through the use of nucleic acid probes. Through metaFISH, we demonstrate the ability to map hundreds of metabolites and multiple microbial species at the micrometer scale on a single tissue section. Notably, intra- and extracellular bacteria, host cells, and their associated metabolites are precisely localized in animal tissues, providing insights into complex metabolic interactions.