Utilizing imaging mass spectrometry to analyze microbial biofilm chemical responses to exogenous compounds.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is an appealing label-free method for imaging biological samples which focuses on the spatial distribution of chemical signals. This approach has been used to study the chemical ecology of microbes and can be applied to study the chemical responses of microbes to treatment with exogenous compounds. Specific conjugated cholic acids such as taurocholic acid (TCA), have been shown to inhibit biofilm formation in the enteric pathogen Vibrio cholerae and MALDI-IMS can be used to directly observe the chemical responses of V. cholerae biofilm colonies to treatment with TCA. A major challenge of MALDI-IMS is optimizing the sample preparation and drying for a particular growth condition and microbial strain. Here we demonstrate how V. cholerae is cultured and prepared for MALDI-IMS analysis and highlight critical steps to ensure proper sample adherence to a MALDI target plate and maintain spatial distributions when applying this technique to any microbial strain. We additionally show how to use both manual interrogation and statistical analyses of MALDI-IMS data to establish the adequacy of the sample preparation protocol. This protocol can serve as a guideline for the development of sample preparation techniques and the acquisition of high quality MALDI-IMS data.