Life on Earth is shaped by a vast network of interacting microbes that co-operate or compete as dynamic communities or microbiomes 1, 2, 3, 4••. Interactions in microbiomes are mediated by small metabolites that play a crucial role in both inter- and intra-species communication 2, 4••. Likewise, the relationship between microbes and their host or environmental niche is often mediated by the production of small metabolites. For instance, Hezaveh et al. demonstrated that indole-related metabolites derived from tryptophan metabolism in Lactobacillus spp. within the gut microbiome can activate the aryl hydrocarbon receptor in tumor-associated macrophages, suppressing antitumor immunity and promoting pancreatic ductal carcinoma in the host organism [5]. However, one major limitation of the traditional microbial culture-based studies is that many micro-organisms cannot be cultivated in the lab, leaving an incomplete picture when it comes to reconstructing and studying the microbiome 4••, 5•. Therefore, scientists have sought to develop multidisciplinary methodologies to holistically examine polymicrobial interactions within their native environments [1]. Each method has specific strengths and limitations, but we posit that mass spectrometry imaging (MSI) has provided a unique advantage as an untargeted approach to discover key microbial metabolites in both simple and complex settings.

Measuring specific forms of chemical communication for how microbes interact and alter their local microenvironments in situ remains a significant challenge. Rigorously designing experiments to control and test community-level interactions versus collections of individual species represents another barrier [3]. Advancements in instrumental technology and multi-omics approaches have revolutionized our ability to explore microbial composition and function in situ 1, 2, 3. Among these, mass spectrometry (MS) provides depth, specificity, and coverage 2, 4••. Moreover, the application of MSI in microbiology has become an attractive approach because of its unique ability to keep spatial orientations of the small molecules intact while offering excellent mass resolution of the signals for subsequent identification.

Over the past two decades, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has proven to be a key tool for mapping chemical communication in complex microbial communities 3, 6. Specifically, in the last 15 years, microbial interactions have often been studied by co-culturing two microorganisms, usually bacteria–bacteria 7, 8, 9, 10, 11, 12, 13, 14•, 15, 16, 17, 18, bacteria–fungi 10, 19, 20, 21, 22, or fungi–fungi 23, 24, 25 on solid/semi-solid media in Petri dishes, with subsequent transfer of the interaction to stainless steel MALDI plates directly [8], indium tin oxide (ITO)/glass slides 24, 25, or porous membranes (Figure 1a) [16]. The co-cultures can be grown for one to several days, after which inhibition zones or other distinct phenotypes emerge, such as pigmentation changes or antagonism in the interaction interface. These inhibition and interaction zones can be analyzed using MALDI-MSI to map the distribution of ions across the sample surface (Figure 1b). Metabolites are detected based on their mass to charge ratio (m/z), spatial distribution, ion intensity, and the ecological context in which they are produced (Figure 1c). Preserving the spatial arrangement of the co-cultured organisms has proven critical, as it provides an essential biological context for metabolite production [20]. Finally, as a complementary tool, tandem MS (MS/MS) or ion mobility spectrometry (IMS) can be used to confirm the identity of the ion and assist in the annotation of unknown ions of interest 7, 12, 22, 23, 25.

While agar-based co-culture studies have advanced our understanding of the molecular mechanisms underlying multispecies interactions, it is crucial to recognize that the microenvironment dictates the availability of essential resources for microbial growth, which, in turn, influences metabolite production [4]. Since these laboratory conditions differ significantly from natural environments, lab-based studies may not represent the full complexity of native microbial interactions, and therefore, the emergent properties inherent to microbial communities remain poorly understood 26, 27. In this review, we explore the evolution of MALDI-MSI as a primary analytical tool for studying microbial interactions, from controlled laboratory setups to in situ analyses. Specifically, we call attention to key technical details that have enabled measurements in unique samples. There is no universally accepted sample preparation or handling that can be applied to every microbe; thus, attention and detail to sample preparation and handling is critical for adapting MSI to new samples. Additionally, we discuss future perspectives on its applications in the chemical microbiology and microbiome fields.