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

A label-free approach for relative spatial quantitation of c-di-GMP in microbial biofilms

“…For positive mode imaging, a 1:1 mixture of α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) matrices were applied using a sieve method for bacterial colonies grown on agar media. 21 We tested two other MALDI matrices that are known to facilitate ionization of nucleotides and nucleosides, 2′,4′,6′-trihydroxyacetophenone (THAP) and 3-hydroxypicolinic acid (HPA), 22,23 in addition to CHCA and DHB in negative ionization mode and determined that the 1:1 CHCA:DHB matrix provided the best limit of detection for c-di-GMP detection (Methods, Fig. S1).…”

“…At present, microbial MSI requires optimization for different microbial cultures and analyte classes. 21…”

Reducing Mass Confusion over the Microbiome

“…There are limited examples of this due to the complexities and consent required to collect these samples. , (D) Microbial cultures, whether monoculture or complex community, can be analyzed spatially to gain metabolomic information about heterogeneity throughout the culture. These samples can also be extracted and analyzed for presence/absence of metabolites without spatial information. ,, (E) Some samples are only compatible with bulk analysis of metabolites due to their complexity. For example, human microbial communities can often only be interrogated via surface sampling using a swab, then cultured and extracted.…”

Imaging mass spectrometry reveals complex lipid distributions across Staphylococcus aureus biofilm layers

“…Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) [22] is a technology that is effective for mapping hundreds-to-thousands of molecules, including small metabolites [23] , [24] , glycans [25] , [26] , [27] , lipids [28] , [29] , peptides [30] , [31] and intact proteins [32] , [33] in tissue at biologically-relevant spatial resolutions [22] , [34] . The ability to interrogate molecular distributions within bacterial communities by MALDI IMS has been previously demonstrated for a wide range of microbial molecules including phenol-soluble modulins [35] , [36] , [37] , metabolites [35] , [38] , [39] , [40] , [41] , [42] , [43] , [44] , [45] , oligosaccharides [46] and some lipids [40] , [41] , [46] , [47] , [48] . However, most of this work has been focused on analytes other than lipids, or has been performed with bacteria other than S. aureus or a co-culture of S. aureus with other microbes.…”

“…One challenge posed by microbial imaging is the small size of an individual bacterium. Most MALDI IMS studies of bacterial communities have been performed at spatial resolutions greater than 50 µm, with rare exceptions [47] , [45] . Because these S. aureus biofilm layers are approximately 100 µm thick, higher spatial resolutions are necessary to effectively investigate inter-layer variations.…”