Seeing between the lines: matrix-assisted laser desorption/ionization mass spectrometry imaging advances for microbial interactions.

This review delves into the technical advancements of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) applications from the last 15 years, highlighting its importance as an analytical tool for exploring the complex interactions within microbial communities. Recognizing the limitations of standard laboratory cultivation practices, we examine how recent research has worked toward bridging the gap between controlled laboratory experiments and in situ measurements. We discuss the shift from traditional agar-based co-culture to newer methods, such as 3D MALDI-MSI and artificial environments, ultimately progressing toward in situ analysis. We highlight the innovative aspects of these methodologies, emphasizing the technical challenges of sample preparation and subsequent metabolite identification. While MALDI-MSI has proven to be an invaluable tool for mapping chemical communication within complex microbial communities, we acknowledge the significant bottlenecks that remain. We call on the researchers to use establish reporting standards and support the expansion of metabolomic databases for future discoveries.