In the two decades since mass spectrometry imaging (MSI) was first applied to visualize the distribution of peptides across biological tissues and cells, the technique has become increasingly effective and reliable. MSI excels at providing complementary information to existing methods for molecular analysis-such as genomics, transcriptomics, and metabolomics-and stands apart from other chemical imaging modalities through its capability to generate information that is simultaneously multiplexed and chemically specific. Today a diverse family of MSI approaches are applied throughout the scientific community to study the distribution of proteins, peptides, and small-molecule metabolites across many biological models. The inherent strengths of MSI make the technique valuable for studying microbial systems. Many microbes reside in surface-attached multicellular and multispecies communities, such as biofilms and motile colonies, where they work together to harness surrounding nutrients, fend off hostile organisms, and shield one another from adverse environmental conditions. These processes, as well as many others essential for microbial survival, are mediated through the production and utilization of a diverse assortment of chemicals. Although bacterial cells are generally only a few microns in diameter, the ecologies they influence can encompass entire ecosystems, and the chemical changes that they bring about can occur over time scales ranging from milliseconds to decades. Because of their incredible complexity, our understanding of and influence over microbial systems requires detailed scientific evaluations that yield both chemical and spatial information. MSI is well-positioned to fulfill these requirements. With small adaptations to existing methods, the technique can be applied to study a wide variety of chemical interactions, including those that occur inside single-species microbial communities, between cohabitating microbes, and between microbes and their hosts. In recognition of this potential for scientific advancement, researchers have adapted MSI methodologies for the specific needs of the microbiology research community. As a result, workflows exist for imaging microbial systems with many of the common MSI ionization methods. Despite this progress, there is substantial room for improvements in instrumentation, sample preparation, and data interpretation. This Account provides a brief overview of the state of technology in microbial MSI, illuminates selected applications that demonstrate the potential of the technique, and highlights a series of development challenges that are needed to move the field forward. In the coming years, as microbial MSI becomes easier to use and more universally applicable, the technique will evolve into a fundamental tool widely applied throughout many divisions of science, medicine, and industry.
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