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Bacteria growing as surface attached biofilms differ significantly from planktonic cells in several important traits that are reflected in the spatiotemporal organization of the cells and the extracellular polymeric substances they secrete. The structural and chemical features that define these biofilms are explored here using a combination of matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and confocal Raman microspectroscopies (CRM) to characterize and compare the composition and distribution of biomolecules found in biofilms and planktonic cells of the bacterium Pseudomonas aeruginosa. Three-day old P. aeruginosa biofilms show dramatic differences in molecular composition compared to planktonic cultures. CRM reveals that wild-type planktonic cell Raman spectra are characterized by bands linked to cellular constituents and are dominated by contributions from DNA- and RNA-related bands. In contrast, biofilm spectra are dominated by bands characteristic of glycolipids - rhamnolipids - polysaccharides and by secreted proteins. LDI MS was applied in turn to identify the rhamnolipids present in the biofilm. Experiments were also conducted using an acyl homoserine lactone quorum sensing-deficient mutant (ΔlasIΔrhlI), which is incapable of producing rhamnolipids. CRM and LDI MS analyses revealed that while molecular composition of the planktonic quorum sensing-deficient cells is similar to that of the wild-type planktonic cells, several compositional differences are observed in the mutant after biofilm growth, including complete absence of detectable rhamnolipids. CRM vibrational spectra of the mutant cells are very similar for planktonic and biofilm growth conditions, indicating that biofilm formation is greatly hindered in the absence of functioning quorum sensing machinery.

Secondary ion mass spectrometry (SIMS) and confocal Raman microscopy (CRM) are combined to analyze the chemical composition of cultured Pseudomonas aeruginosa biofilms, providing complementary chemical information for multiple analytes within the sample. Precise spatial correlation between SIMS and CRM images is achieved by applying a chemical microdroplet array to the sample surface which is used to navigate the sample, relocate regions of interest, and align image data. CRM is then employed to nondestructively detect broad molecular constituent classes-including proteins, carbohydrates, and, for the first time, quinolone signaling molecules-in Pseudomonas-derived biofilms. Subsequent SIMS imaging at the same location detects quinolone distributions in excellent agreement with the CRM, discerns multiple quinolone species which differ slightly in mass, resolves subtle differences in their distributions, and resolves ambiguous compound assignments from CRM by determining specific molecular identities via in situ tandem MS.

A novel method of correlated imaging, combining confocal Raman microscopy (CRM) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) was developed in order to investigate the structural and chemical diversity inherent in three-dimensional (3D) cell cultures. These 3D spheroidal cell cultures are high throughput in vitro model systems that recapitulate some of the chemical and physiological gradients characteristic of tissues. As a result, they are ideal for testing new imaging approaches due to the native diversity of cellular phenotypes found within a single culture. Individually, confocal Raman microscopy (CRM) and mass spectrometry imaging (MSI) produce different kinds of chemical information. CRM imaging reveals differences in cellular integrity and protein secretion across a typical near-equatorial transverse slice, while MSI shows localization of small molecules to discrete regions of the spheroid section. Correlating information obtained from these disparate imaging methods begins with an external fiducial mask, added to the spheroidal samples to orient image acquisition on the two orthogonal platforms. Rather than combine the images directly, principal component analysis is used to reveal the most chemically-informative elements, which are then combined using digital image correlation. Using this approach, relationships between the principal components of each method are visualized so that they may be compared on commensurate spatial length scales.

Mass spectrometry imaging (MSI) is a versatile tool for visualizing molecular distributions in complex biological specimens, but locating microscopic chemical features of interest can be challenging in samples that lack a well-defined anatomy. To address this issue, we developed a correlated imaging approach that begins with performing matrix-assisted laser desorption/ionization (MALDI) MSI to obtain low-resolution molecular maps of a sample. The resulting maps are then used to direct subsequent microscopic secondary ion mass spectrometry (SIMS) imaging and tandem mass spectrometry (MS/MS) experiments to examine selected chemical regions of interest. By employing MALDI undersampling, the sample surface is left mostly unperturbed and available for the SIMS analysis, while also generating an ablation array that can be used for navigation in SIMS. We validated this MALDI-guided SIMS approach using cultured biofilms of the opportunistic pathogen Pseudomonas aeruginosa; bioactive secondary metabolites, including rhamnolipids and quinolones, were detected and visualized on both macro- and microscopic size scales. MSI mass assignments were confirmed with in situ MALDI MS/MS and capillary electrophoresis-electrospray ionization MS/MS analysis of biofilm extracts. Two strains of P. aeruginosa were compared, wild type and a quorum sensing mutant, and differences in metabolite abundance and distribution were observed.

Applying complementary experiments, like laser desorption-ionization mass spectrometry (LDI-MS) and confocal surface-enhanced Raman microscopy, to the same physical sample location has the potential to elucidate the behavior of complex chemical and biochemical systems in ways that are not available to either method applied in isolation. In these experiments surface-enhanced Raman scattering (SERS) and LDI-MS are applied to the same sample spot using a common structure, deposited Ag colloids, both as ionization matrix and simultaneously as enhancing media for surface-enhanced Raman scattering of small organic molecules, dyes and lipids, and the behavior is compared. Three compounds-p-aminothiophenol (ATP), rhodamine 6G and cholesterol-which exhibit different strengths of interaction with Ag are examined in detail by correlated SERS and LDI-MS. The related mechanisms of nanoparticle-assisted desorption-ionization and Raman enhancement are explored by correlating mass and Raman spectra. The correlated spectra highlight the manner in which the different test compounds interact with plasmonic metal nanostructures. These coupled studies yield new insight into the transition of analyte from the metal-solution interface to gaseous ions, including, in the case of organothiols, a rich set of mixed clusters that provide chemical insight into the ion formation process.