Advanced Methods for MALDI-MS Imaging of the Chemical Communication in Microbial Communities.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is increasingly used to visualize the chemical communication between microorganisms. However, to fully exploit the potential of this label-free technique, crucial methodological advances are still needed. In particular, with current microbial MALDI-MSI methods chemical coverage is strongly limited to well ionizing compounds and a safe MSI-compatible inactivation of microbial viability and quenching of metabolism is not possible. Here, we introduce a membrane-based culturing workflow that enables a rapid MSI-compatible steam inactivation of pathogens and generation of a flat surface. We equipped precision mass spectrometers with laser-postionization (MALDI-2) modules to increase the analytical sensitivity by up to several orders of magnitude. In this way, for example 39 different 2-alkylquinolones with differential expression patterns and a similar number of glycerophospholipids were simultaneously visualized from single cultures of at about 50 μm resolution. To visualize the metabolic exchange between competing microorganisms, we challenged commensal MG1655 and virulence factor-depleted C600 strains with enteropathogenic Shiga-toxin negative O26:H11, and with antagonistic . Insight into the three-dimensional organization of a biofilm of the probiotic Nissle 1917 at 15 μm pixel size was obtained after developing an embedding/cryosectioning protocol. Our advanced protocols could help to substantially increase the application range of microbial MS imaging.