Joseph Ellis oral presentation (PB3-Thu1-2-2)
Evaluation and Mitigation of Ion Suppression in Biomolecular Secondary Ion Mass Spectrometry Imaging
1 Department of Chemistry and Beckman Institute for Advanced Science and Technology, 405 N. Mathews St., IL 61801 Urbana, United States
2 Department of Chemistry and Biochemistry and Department of Chemical and Biomolecular Engineering, Nieuwland Science Hall, IN 46556 Notre Dame, United States
3 Department of Civil and Environmental Engineering and Earth Sciences and Department of Biological Sciences, Fitzpatrick Hall, IN 46556 Notre Dame, United States
Secondary ion mass spectrometry (SIMS) imaging is a valuable tool for chemical imaging in both biology and materials science. Although the acquisition of qualitative small molecule information has become somewhat routine, obtaining quantitative data is difficult, partially due to (1) molecule-to-molecule variability in ionization efficiency and (2) matrix effects arising from heterogeneous microenvironments. Here we investigate matrix effects present in agar-bound Pseudomonas aeruginosa biofilms, and develop a strategy to partially alleviate them. P. aeruginosa is a ubiquitous bacterium found in a large variety of environments, from soil to nosocomial infections. Formation and dissolution of P. aeruginosa biofilms is partially regulated via the pqsA-E operon, which utilizes alkyl quinolones (AQs) as signaling molecules. Perhaps more broadly interesting to the SIMS community as a whole, biofilms exhibit significant architectural and chemical heterogeneity, and therefore present an excellent model for investigating matrix effects caused by both attributes.
Differences in ionization efficiency were first addressed through simple linear regression calibration to an external standard curve, which provided relative intrapixel quantitation. We evaluated the extent of matrix effects by depositing a uniform array containing equimolar quantities of several AQ standards and a bioorthogonal compound, 9-aminoacridine (9-AA), across the surface of neat agar and several P. aeruginosa biofilms. C60-SIMS imaging and imaging principal component analysis revealed substantial matrix effects within the biofilm sphere of influence, including in pqsA- mutant biofilm, which is a significantly simplified strain of P. aeruginosa deficient in the production of AQs or their downstream biosynthetic products. In pqsA- a region of substantially diminished ionization was found to correlate with several endogenous ions found specifically at the biofilm boundary (Fig 1). Electron microscopy investigations into the physical environment revealed the suppression region to be morphologically similar to the surrounding areas. The 50% methanol standard solution produced 17 (± 2.9) % larger spots in the suppression zone than in adjacent areas, suggesting that this zone is more hydrophobic. Ongoing work focuses on computationally mitigating the observed matrix effects by calculating the amount of suppression at each standard spot, and extrapolating to the pixels in the intervening regions. We expect this strategy to enable pixel-to-pixel quantitation for biomolecular SIMS imaging.