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Multimodal coherent anti-stokes raman scattering (CARS) microscopy for the detection of isotope-labelled molecules

Boorman, Dale 2019. Multimodal coherent anti-stokes raman scattering (CARS) microscopy for the detection of isotope-labelled molecules. PhD Thesis, Cardiff University.
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Coherent anti-Stokes Raman scattering (CARS) microscopy utilises intrinsic vibrational resonances of molecules to drive inelastic scattering of light. This eradicates the need for exogenous fluorescent labelling, which can be perturbative to the study of many small molecules, whilst providing high-resolution three-dimensional images with chemical specificity. Isotopic substitution of hydrogen atoms with deuterium presents a labelling strategy that introduces minimal change to molecular structure but can achieve chemical contrast when coupled with CARS microscopy. This is due to an induced down-shift of the CH2 peak into the cell-silent region of the Raman spectrum which does not contain contributions from other chemical species, thus giving contrast against other cellular components. A novel data analysis methodology, named Factorisation into Susceptibilities and Concentrations of Chemical Components (FSC3), has been developed for application with hyperspectral CARS data to generate concentration maps of chemical components. Within this thesis, the ability of FSC3 to observe a range of exogenously applied deuterium-labelled molecules within a cell has been assessed. Hyperspectral CARS datasets were initially acquired from lipid droplets of HeLa cells incubated with deuterium-labelled fatty acids, and FSC3 analysis was applied to identify and spatially resolve a chemical component corresponding to carbon-deuterium bond vibrations. Identification of a deuterium-associated component enabled quantitative study of the uptake, storage and turnover of deuterated fatty acids within cytosolic lipid droplets based on changes in the carbon-deuterium signal intensity. The ability of FSC3 to visualise lipid droplets containing deuterated lipid was also utilised for non-invasive tracking of cells of interest within mixed populations. FSC3 identification of non-lipid-based molecules was more challenging, with limited intracellular visualisation of deuterium-labelled biomolecules and small molecule compounds achieved. However, some evidence suggested detection of the compound, D7-HCMVi, within cells. The imaging capabilities of the custom-built multimodal CARS microscopy system at Cardiff University were also evaluated in the context of a similar system at the Beckman Institute, University of Illinois Urbana-Champaign and a commercially available system developed by Leica. The results of this indicated multimodal advantages of the Beckman system, accessibility advantages of the Leica system, but specifically for nonlinear imaging capabilities, the Cardiff system was the most technologically advanced.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Biosciences
Subjects: Q Science > Q Science (General)
Funders: BBSRC
Date of First Compliant Deposit: 11 March 2020
Last Modified: 12 Mar 2020 10:11

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