Bacterial biofilm sample preparation for spatial metabolomics.

Spatial metabolomics using mass spectrometry imaging (MSI) has become an important approach to study the surface of biological systems. MSI can probe bacterial metabolic processes through the direct analysis of bacterial colonies. In this review, we explore recent advancements made for bacterial metabolomics of primary and secondary metabolites using MSI, focusing on improvements in agar-based sample preparation and the use of membranes for improved sample preparation. The application of derivatization agents on bacterial samples enhances select metabolite signals and can aid analyte identification. Implementing dual imaging or multi-omics techniques also aids in identifying analytes and elucidating metabolic pathways active during the host-microbe interactions. Finally, we explore improvements towards robust three-dimensional protocols for whole colony MSI analysis. These advances enhance MSI analysis of bacterial samples and pose promising avenues for future studies.