Applications and technical challenges of MALDI mass spectrometry imaging in microbial ecosystems and metabolic network research.

Microbial communities are highly organized systems in which metabolic activities, functional states, and interspecies interactions are distributed across distinct spatial microenvironments. Conventional microbiological and omics approaches rely largely on bulk measurements or sample homogenization and therefore provide limited insight into spatial heterogeneity and localized metabolic processes. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has emerged as a label-free analytical technique that enables in situ, spatially resolved visualization of metabolites, lipids, peptides, and proteins within intact biological samples, offering a powerful approach for investigating complex microbial systems. This review summarizes the technological development of MALDI-MSI and its applications in spatial microbial metabolism and ecosystem research, including the analysis of metabolic heterogeneity and functional compartmentalization in biofilms, molecular profiling of microbial individuals and populations, and stratified metabolic organization in complex environmental samples. Applications of MALDI-MSI in studies of microbe–host–environment interactions and antimicrobial research are also discussed, with emphasis on spatial mapping of chemical communication, host responses, and antimicrobial distribution within infected microenvironments. Finally, current technical challenges and future perspectives are outlined, highlighting the importance of multimodal integration and advanced data analysis strategies. Overall, MALDI-MSI represents a versatile platform for spatially resolved molecular analysis in microbiology, linking microbial metabolic function with ecological and pathological contexts.