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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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Correlated SIMS/CRM imagingprovides additional information aboutsignaling molecules in P. aeruginosa biofilm. (a)Superimposed CRM “composite quinolone” image (1350–1400cm–1, quinolone ring stretch) and SIMS 2-heptyl-2-quinoloneion image (HHQ, MH+ at m/z 244.17) shows a similar molecular distribution in the selected ROI.The same images are shown individually for (b) SIMS and (c) CRM, wherehigh spatial resolution enables visualization of micron-scale featureswithin the ROI. (d) Another quinolone, 4-hydroxy-2-nonylquinolone-N-oxide(C9–NQNO, MH+ at m/z 288.20) appears to be colocalized, but the distributionis distinct from that of HHQ within the composite quinolone area.(e) Optical and (f) SIMS ion images (m/z 250.81) with a larger field of view show how the microspot arrayis visualized around the ROI, allowing precise navigation and imagealignment. Red boxes specify the ROI of the CRM/SIMS detail. Scalebars = 100 μm in a–d and 200 μm in e and f.
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fig4: Correlated SIMS/CRM imagingprovides additional information aboutsignaling molecules in P. aeruginosa biofilm. (a)Superimposed CRM “composite quinolone” image (1350–1400cm–1, quinolone ring stretch) and SIMS 2-heptyl-2-quinoloneion image (HHQ, MH+ at m/z 244.17) shows a similar molecular distribution in the selected ROI.The same images are shown individually for (b) SIMS and (c) CRM, wherehigh spatial resolution enables visualization of micron-scale featureswithin the ROI. (d) Another quinolone, 4-hydroxy-2-nonylquinolone-N-oxide(C9–NQNO, MH+ at m/z 288.20) appears to be colocalized, but the distributionis distinct from that of HHQ within the composite quinolone area.(e) Optical and (f) SIMS ion images (m/z 250.81) with a larger field of view show how the microspot arrayis visualized around the ROI, allowing precise navigation and imagealignment. Red boxes specify the ROI of the CRM/SIMS detail. Scalebars = 100 μm in a–d and 200 μm in e and f.

Mentions: Results from a correlated imagingexperiment are shown in Figure 4. CRM is performedfirst as it is nondestructive,thus providing a good way to survey the sample. A region with intensemicrometer-scale quinolone features was found and imaged by CRM; thesample was then physically transferred to the C60-SIMSinstrument where the ROI was relocated using the microspot array.MSI was performed over the entire grid cell, which included the CRM-imagedROI. Figure 4a shows the CRM “compositequinolone” image aligned and superimposed with the SIMS ionimage of quinolone HHQ (MH+ at m/z 244.17). The two images are in excellent agreement inspatial distribution and relative signal intensity within the feature,cross-validating the data and indicating that the observed ion andRaman scattering distributions are accurate, not artifactual (e.g.,arising from ion suppression effects or similar vibrational modesin other biofilm constituents). The individual SIMS and CRM imagesare also shown separately in Figure 4b andc, respectively. The microspots around the ROI are visible in theoptical image, Figure 4e, and are also detectedas intense spots in several ion images, including m/z 250.81, Figure 4f, andin the total ion count, Figure S3. Thesemarker ions were not identified, but they are likely adduct or inorganiccluster ions formed or enhanced by the presence of the Ag nanoparticlesand/or the citrate buffer. Notably, ions associated with the microspotsdid not include silver clusters such as Ag+ or Ag2+, perhaps indicating that the silver was extensivelyassociated with other compounds, or that the nanoparticles were notsufficiently fragmented by the C60+ primaryion beam. The spots are well-defined against the biofilm background,indicating that the nanoparticle solution dried into discrete 132± 9 μm (n = 6) diameter regions withoutdiffusing into the adjacent sample.


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)

Correlated SIMS/CRM imagingprovides additional information aboutsignaling molecules in P. aeruginosa biofilm. (a)Superimposed CRM “composite quinolone” image (1350–1400cm–1, quinolone ring stretch) and SIMS 2-heptyl-2-quinoloneion image (HHQ, MH+ at m/z 244.17) shows a similar molecular distribution in the selected ROI.The same images are shown individually for (b) SIMS and (c) CRM, wherehigh spatial resolution enables visualization of micron-scale featureswithin the ROI. (d) Another quinolone, 4-hydroxy-2-nonylquinolone-N-oxide(C9–NQNO, MH+ at m/z 288.20) appears to be colocalized, but the distributionis distinct from that of HHQ within the composite quinolone area.(e) Optical and (f) SIMS ion images (m/z 250.81) with a larger field of view show how the microspot arrayis visualized around the ROI, allowing precise navigation and imagealignment. Red boxes specify the ROI of the CRM/SIMS detail. Scalebars = 100 μm in a–d and 200 μm in e and f.
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Related In: Results  -  Collection

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fig4: Correlated SIMS/CRM imagingprovides additional information aboutsignaling molecules in P. aeruginosa biofilm. (a)Superimposed CRM “composite quinolone” image (1350–1400cm–1, quinolone ring stretch) and SIMS 2-heptyl-2-quinoloneion image (HHQ, MH+ at m/z 244.17) shows a similar molecular distribution in the selected ROI.The same images are shown individually for (b) SIMS and (c) CRM, wherehigh spatial resolution enables visualization of micron-scale featureswithin the ROI. (d) Another quinolone, 4-hydroxy-2-nonylquinolone-N-oxide(C9–NQNO, MH+ at m/z 288.20) appears to be colocalized, but the distributionis distinct from that of HHQ within the composite quinolone area.(e) Optical and (f) SIMS ion images (m/z 250.81) with a larger field of view show how the microspot arrayis visualized around the ROI, allowing precise navigation and imagealignment. Red boxes specify the ROI of the CRM/SIMS detail. Scalebars = 100 μm in a–d and 200 μm in e and f.
Mentions: Results from a correlated imagingexperiment are shown in Figure 4. CRM is performedfirst as it is nondestructive,thus providing a good way to survey the sample. A region with intensemicrometer-scale quinolone features was found and imaged by CRM; thesample was then physically transferred to the C60-SIMSinstrument where the ROI was relocated using the microspot array.MSI was performed over the entire grid cell, which included the CRM-imagedROI. Figure 4a shows the CRM “compositequinolone” image aligned and superimposed with the SIMS ionimage of quinolone HHQ (MH+ at m/z 244.17). The two images are in excellent agreement inspatial distribution and relative signal intensity within the feature,cross-validating the data and indicating that the observed ion andRaman scattering distributions are accurate, not artifactual (e.g.,arising from ion suppression effects or similar vibrational modesin other biofilm constituents). The individual SIMS and CRM imagesare also shown separately in Figure 4b andc, respectively. The microspots around the ROI are visible in theoptical image, Figure 4e, and are also detectedas intense spots in several ion images, including m/z 250.81, Figure 4f, andin the total ion count, Figure S3. Thesemarker ions were not identified, but they are likely adduct or inorganiccluster ions formed or enhanced by the presence of the Ag nanoparticlesand/or the citrate buffer. Notably, ions associated with the microspotsdid not include silver clusters such as Ag+ or Ag2+, perhaps indicating that the silver was extensivelyassociated with other compounds, or that the nanoparticles were notsufficiently fragmented by the C60+ primaryion beam. The spots are well-defined against the biofilm background,indicating that the nanoparticle solution dried into discrete 132± 9 μm (n = 6) diameter regions withoutdiffusing into the adjacent sample.

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.

Show MeSH
Related in: MedlinePlus