As we move into the heart of the 21st century we are finally beginning to understand the extent of microbial influence over everyday life. Truth be told, we humans are but guests on a planet ruled by a largely unseen, and often undiscovered, microbial population. In order to understand how the microbial dimension influences our own we must first understand the microbes themselves. Far from existing as a collection of single cells floating about aimlessly, bacteria largely reside in highly cooperative multicellular communities where they work in concert to colonize and mold their surrounding environments, harvest nutrients, and wage warfare. How can these seemingly simple life forms facilitate such complicated behaviors? The answer largely resides in chemistry, for the backbone of microbial influence is built with the chemicals that they produce, secrete, sense, and consume. With clever implementation of the proper analytical tools this chemical information is ripe for our discovery and exploitation. This dissertation primarily focuses on the adaptation and application of an existing chemical imaging technique, secondary ion mass spectrometry (SIMS) imaging, to study the chemistry underlying surface-bound microbial communities. The first chapter provides a general overview of the dissertation. The second chapter provides an introduction to mass spectrometry imaging – which encompasses a broad family of techniques including SIMS imaging – with a focus on its application to microbiology. The remaining six chapters, detailed below, describe both method development for SIMS imaging and application of the technique to explore several questions in microbiology.SIMS is applied in conjunction with a complimentary analytical technique, confocal Raman microscopy (CRM), to study early stage biofilms formed by the gram-negative bacterium Pseudomonas aeruginosa, which is an opportunistic pathogen for both plants and humans. In addition to methodological advancements, this work revealed that P. aeruginosa accumulates highly concentrated clusters of alkyl-quinolines – including 2-heptyl-4-quinoline-N-oxide (HQNO) and 2-nonyl-4-quinoline-N-oxide (NQNO) – during the early stages of biofilm formation. HQNO and NQNO are known to disrupt the formation of healthy communities of gram-positive bacteria, and their high abundance during biofilm development suggests that P. aeruginosa utilizes these molecules for a competitive advantage for establishing new colonies. In a purely methodological study, we examined the effects of applying a thin (~2 nm) layer of gold to the biofilm surface prior to SIMS imaging. This investigation revealed a signal enhancement for cluster-SIMS that, remarkably, only applied to analytes contained within biological samples. Examination of gold coated standards deposited on hard silicon wafers did not yield a signal enhancement, suggesting that the SIMS community needs to look beyond simple standard formulations when developing or adapting sample treatment strategies. Separately, a simple nitrogen desiccation procedure was developed for imaging microbial communities on semi-solid agar, which can be a challenging substrate due to the high water content. Traditionally, most SIMS studies are carried out using hard, conductive surfaces, however microbiology assays commonly require growth on semi-solid agar.Both analyte-to-analyte differences in ionization efficiency and interfering signal from compounds with the same or similar mass-to-charge ratio (m/z) have largely prevented biological SIMS imaging from becoming a quantitative technique; the distribution of an analyte can be determined however the absolute quantity usually cannot. We therefore developed a quantitative SIMS imaging strategy where (1) SIMS product ion imaging is used to increase analyte specificity, (2) analyte-analyte differences in ionization efficiency are accommodated through calibration to an external quadratic calibration curve, and (3) competing ion signal is algebraically removed from each image pixel. This strategy is demonstrated by imaging the surface density of two different alkyl quinolone/quinoline isomeric pairs across several agar-based P. aeruginosa bacterial biofilms. Another major challenge for SIMS imaging is the regiospecific differences in ionization efficiency, which impede direct comparison of ion intensity to analyte abundance. Heterogeneous ionization efficiency arises from a myriad of factors, including changes in the chemical microenvironment, local morphology, and conductivity of the sample. A microspot array methodology for evaluating regiospecific differences in ionization efficiency is presented, and its utility is demonstrated by evaluating several different strains of P. aeruginosa cultivated on semi-solid agar.In a highly collaborative study that required the expertise of three separate research groups, the spatiochemical response of P. aeruginosa to antibiotic exposure was examined with SIMS imaging, CRM, and a number of traditional microbiology techniques. This study showed that P. aeruginosa swarms migrate away from the antibiotic source and increase their production of several alkyl quinolones in a dose-dependent manner. Interestingly, the quorum sensing molecule known as Pseudomonas quinolone signal (PQS) was found to be more abundant in regions closest to the antibiotic. These results suggest that PQS is regulated independent of the other alkyl quinolones, and acts as a transient, short-range signal.In an effort with relevance to microbial induced corrosion, a method is presented for cultivating, preparing, and examining drip-flow biofilms on metallic surfaces. Two species of bacteria, Pseudomonas putida and Shewanella oneidensis, were cultivated on both stainless and low carbon steel and examined with SIMS imaging, matrix-assisted laser desorption/ionization mass spectrometry imaging, scanning electron microscopy, and energy-dispersive x-ray spectroscopy. This study partially identifies and maps the distribution of 25 lipids and polysaccharides on P. putida drip-flow biofilms, examines the spatiochemical interactions between P. putida and S. oneidensis grown adjacent to one another, and examines the chemical and morphological environment of the two bacteria on corroding low-carbon steel. Taken together, the research described in this dissertation enhances our fundamental knowledge of the chemistry underlying microbial communities. The developed analytical methodologies can be applied by other researchers to further advance our collective understanding of the microbial world.
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Secondary ion mass spectrometry imaging of surface-bound microbial communities
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