Mass Spectrometry Imaging of Complex Microbial Communities

Abstract

In the two decades since mass spectrometry imaging (MSI) was first applied to visualize the distribution of peptides across biological tissues and cells, the technique has become increasingly effective and reliable. MSI excels at providing complementary information to existing methods for molecular analysis-such as genomics, transcriptomics, and metabolomics-and stands apart from other chemical imaging modalities through its capability to generate information that is simultaneously multiplexed and chemically specific. Today a diverse family of MSI approaches are applied throughout the scientific community to study the distribution of proteins, peptides, and small-molecule metabolites across many biological models. The inherent strengths of MSI make the technique valuable for studying microbial systems. Many microbes reside in surface-attached multicellular and multispecies communities, such as biofilms and motile colonies, where they work together to harness surrounding nutrients, fend off hostile organisms, and shield one another from adverse environmental conditions. These processes, as well as many others essential for microbial survival, are mediated through the production and utilization of a diverse assortment of chemicals. Although bacterial cells are generally only a few microns in diameter, the ecologies they influence can encompass entire ecosystems, and the chemical changes that they bring about can occur over time scales ranging from milliseconds to decades. Because of their incredible complexity, our understanding of and influence over microbial systems requires detailed scientific evaluations that yield both chemical and spatial information. MSI is well-positioned to fulfill these requirements. With small adaptations to existing methods, the technique can be applied to study a wide variety of chemical interactions, including those that occur inside single-species microbial communities, between cohabitating microbes, and between microbes and their hosts. In recognition of this potential for scientific advancement, researchers have adapted MSI methodologies for the specific needs of the microbiology research community. As a result, workflows exist for imaging microbial systems with many of the common MSI ionization methods. Despite this progress, there is substantial room for improvements in instrumentation, sample preparation, and data interpretation. This Account provides a brief overview of the state of technology in microbial MSI, illuminates selected applications that demonstrate the potential of the technique, and highlights a series of development challenges that are needed to move the field forward. In the coming years, as microbial MSI becomes easier to use and more universally applicable, the technique will evolve into a fundamental tool widely applied throughout many divisions of science, medicine, and industry.

Figure 1

General overview of MSI. A focused microprobe desorbs molecules into the gas phase, where they are ionized and electrically focused into the mass analyzer. Subsequent mass-to-charge ratio (m/z) differentiation and detection produce a mass spectrum for each point across the sample. The abundance of each ion of interest is then plotted as a function of location to produce false-color ion images of specific molecular features.

Figure 2

Examples of MALDI MSI in microbiology. (a) Alkyl quinolines produced by P. aeruginosa in the presence of S. aureus. Reprinted with permission from ref (15). Copyright 2016 Macmillan Publishers Ltd. (b) Surfactants and peptides produced by colony biofilms of B. subtilis. Adapted from ref (16). Copyright 2016 American Chemical Society. (c) Nutritionally dependent P. aeruginosa proteins from a heterogeneous biofilm grown in a drip-flow reactor. Adapted with permission from ref (17). Copyright 2016 Nature Publishing Group under a Creative Commons CC-BY license. (d) Chemical response of two strains of P. aeruginosa in the presence of the antibiotic azithromycin. Adapted with permission from ref (18). Copyright 2015 Springer. See the original references for more information on the specific bacterial strains used and the identities of all of the ions. *, Surfactin-C14; , Plipastatin-C17-Val.

Figure 3

Examples of SIMS imaging in microbiology. (a) (i) FISH and (ii–iv) NanoSIMS imaging of the filamentous cyanobacterium Anabaena sp. and Rhizobium sp. In (ii), Rhizobium sp. is labeled with fluorine using ALF968 dye, while in (iii) and (iv) Anabaena sp. metabolically incorporates ¹⁵N-dinitrogen and ¹³C-bicarbonate. Adapted with permission from ref (24). Copyright 2008 American Society for Microbiology. (b) Bi₃⁺-TOF-SIMS imaging of a B. subtilis swarming community imprinted onto a silicon wafer. (i) Microscopy image of the community prior to imprinting. (ii) Low- and (iii–v) high-resolution TOF-SIMS images of the sum of all surfactant ions. Adapted with permission from ref (26). Copyright 2008 John Wiley and Sons. (c) Quinolones and quinolines produced by static (i) P. aeruginosa microcolonies, (ii) planktonic culture, and (iii) 7 h biofilms. Mass spectra are averages of four pixels from the regions indicated by the red arrows. Reproduced with permission from ref (28). Copyright 2015 Royal Society of Chemistry.

Figure 4

Examples of DESI MSI in microbiology. (a) Imprint imaging of interacting communities of B. subtilis and S. coelicolor. Adapted from ref (32). Copyright 2010 American Chemical Society. (b) Ion images of iron-scavenging siderophores at different times during the growth of Streptomyces wadayamensis. Samples were grown on thin agar and vacuum-desiccated prior to imaging. Adapted from ref (34). Copyright 2015 American Chemical Society. (c) NanoDESI liquid microjunction probe design and ion images of living S. coelicolor colonies on agar. Adapted from ref (35). Copyright 2013 American Chemical Society.

Figure 5

C₆₀-SIMS product ion imaging to differentiate the PQS/HQNO and C₉-PQS/NQNO isomeric pairs on two adjacent regions of a P. aeruginosa biofilm. (a) Product of m/z 288 for C₉-PQS and NQNO. (b) Product of m/z 260 for PQS and HQNO. Fragments arising from PQS and C₉-PQS are shown in purple, while those arising from N-oxides are shown in red. The white arrows indicate the approximate locations of the subsequent higher-magnification images. Reproduced with permission from ref (28). Copyright 2015 Royal Society of Chemistry.