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Correlated imaging with C60-SIMS and confocal Raman microscopy: Visualization of cell-scale molecular distributions in bacterial biofilms.

Lanni EJ, Masyuko RN, Driscoll CM, Dunham SJ, Shrout JD, Bohn PW, Sweedler JV - Anal. Chem. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.

ABSTRACT
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.

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C60-SIMS and MS/MS of biofilm. (a) C60-SIMSdirect analysis of an untreated biofilm surface yielded a profilethat included multiple quinolones detected as MH+ pseudomolecularions. (b) In situ MS/MS of putative quinolones supportsthe mass assignments, e.g., for HHQ at m/z 244.17 shown here, yielding characteristic fragments at m/z 159.07 and 172.08.
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fig3: C60-SIMS and MS/MS of biofilm. (a) C60-SIMSdirect analysis of an untreated biofilm surface yielded a profilethat included multiple quinolones detected as MH+ pseudomolecularions. (b) In situ MS/MS of putative quinolones supportsthe mass assignments, e.g., for HHQ at m/z 244.17 shown here, yielding characteristic fragments at m/z 159.07 and 172.08.

Mentions: To complement the CRM images, C60-SIMS was conductedin a semitargeted manner, i.e., the acquisition parameters were optimizedfor detection of quinolones and other small metabolites in the <300AMU range. A detail of the P. aeruginosa biofilmMS profile is shown in Figure 3a; quinoloneswere consistently detected as MH+ ions, in agreement withprevious reports,28,30 as well as with our own recentobservations using MSI with other probe types (metal-assisted LDIand Au-SIMS, validated by CE–ESI MS/MS). Assignments were initiallymade by mass match with previous reports, and in situ MS/MS was also performed to confirm identities when signals wereadequate, as shown for HHQ in Figure 3b andfor other analytes in Table S1. A totalof nine quinolones were detected and confirmed with MS/MS, includingtwo isobaric quinolone pairs—PQS/HQNO and C9:1-PQS/C9:1-NQNO—yielding unique characteristic fragments. Amass list summary of these results is presented in Figure S2.


Correlated imaging with C60-SIMS and confocal Raman microscopy: Visualization of cell-scale molecular distributions in bacterial biofilms.

Lanni EJ, Masyuko RN, Driscoll CM, Dunham SJ, Shrout JD, Bohn PW, Sweedler JV - Anal. Chem. (2014)

C60-SIMS and MS/MS of biofilm. (a) C60-SIMSdirect analysis of an untreated biofilm surface yielded a profilethat included multiple quinolones detected as MH+ pseudomolecularions. (b) In situ MS/MS of putative quinolones supportsthe mass assignments, e.g., for HHQ at m/z 244.17 shown here, yielding characteristic fragments at m/z 159.07 and 172.08.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221875&req=5

fig3: C60-SIMS and MS/MS of biofilm. (a) C60-SIMSdirect analysis of an untreated biofilm surface yielded a profilethat included multiple quinolones detected as MH+ pseudomolecularions. (b) In situ MS/MS of putative quinolones supportsthe mass assignments, e.g., for HHQ at m/z 244.17 shown here, yielding characteristic fragments at m/z 159.07 and 172.08.
Mentions: To complement the CRM images, C60-SIMS was conductedin a semitargeted manner, i.e., the acquisition parameters were optimizedfor detection of quinolones and other small metabolites in the <300AMU range. A detail of the P. aeruginosa biofilmMS profile is shown in Figure 3a; quinoloneswere consistently detected as MH+ ions, in agreement withprevious reports,28,30 as well as with our own recentobservations using MSI with other probe types (metal-assisted LDIand Au-SIMS, validated by CE–ESI MS/MS). Assignments were initiallymade by mass match with previous reports, and in situ MS/MS was also performed to confirm identities when signals wereadequate, as shown for HHQ in Figure 3b andfor other analytes in Table S1. A totalof nine quinolones were detected and confirmed with MS/MS, includingtwo isobaric quinolone pairs—PQS/HQNO and C9:1-PQS/C9:1-NQNO—yielding unique characteristic fragments. Amass list summary of these results is presented in Figure S2.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.

ABSTRACT
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.

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