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Multiplex Coherent Anti-Stokes Raman Scattering Correlation Spectroscopy and Imaging
This dissertation focuses on the utilization of multiplex coherent anti-Stokes Raman scattering (CARS) for correlation spectroscopy and chemical imaging.
Multiplex CARS imaging via supercontinuum excitation has the spectral and spatial resolution to probe changes in biomolecules on the cell surface with chemical treatment. By ascertaining the nonresonant and resonant contributions of the nonlinear CARS intensity, the peak ratio of methylene to methyl functional groups can distinguish between molecular markers, such as lipids and proteins. Our results indicate that OvCa429 and SKOV3ip epithelial ovarian cancer cells undergo similar morphological and chemical responses to treatment with lysophosphatidic acid (LPA). The microvilli in multicellular aggregates are removed by treatment of LPA. The CARS analysis shows a distinct decrease in protein and increase in lipid composition on the surface of LPA-treated cells. Importantly, the CARS signals from cellular sheddings from OvCa429 and SKOV3ip cells with LPA treatment are consistent with cleavage of microvilli and proteins originally present.
Multiplex CARS correlation spectroscopy is shown as a chemically specific approach for monitoring the molecular mobility of particles on the millisecond time scale. The CARS signal provides a quantitative measurement for the number of particles in the focal volume, whereas the autocorrelation of spectral data elucidates dynamic events, such as diffusion. The measured diffusion coefficients for polymer beads ranging from 100 nm to 1.1 µm in diameter are in good agreement with predicted Stokes-Einstein values. Multivariate curve resolution analysis of distinct spectral features in multiplex CARS measurement distinguishes different composition lipid vesicles in a mixture diffusing through the focal volume. The observed diffusion is consistent with results obtained from single particle tracking experiments.
Single particle tracking (SPT) measurements are used to monitor the interactions between ligand functionalized nanoparticles and membrane receptors in real-time. Dark-field SPT detects scattering from peptide-conjugated gold nanoparticles interacting with integrin receptors immobilized on glass surfaces and in intact cells to characterize ligand-receptor binding. Our results indicate cRGD and CisoDGRC peptide sequences are recognized differently by the αvβ3 receptor. We observed a preferential binding and higher binding affinity for cRGD-receptor complexes compared to CisoDGRC-receptor complexes on integrin-immobilized glass and in live colon cancer cells.
History
Date Created
2016-10-24Date Modified
2022-03-17Defense Date
2016-09-30Research Director(s)
Zachary D. SchultzDegree
- Doctor of Philosophy
Degree Level
- Doctoral Dissertation
Alternate Identifier
961213789Library Record
4471790OCLC Number
961213789Additional Groups
- Chemistry and Biochemistry
Program Name
- Chemistry and Biochemistry