published_or_final_version / Botany / Doctoral / Doctor of Philosophy

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This study used Raman spectral analysis to characterise the structural order of carbon in three carbonaceous chondrites and twelve achondrites. The achondrites analysed were a group of carbon-rich meteorites, known as ureilites. These meteorites are composed primarily of olivine and pyroxene and have igneous textures but contain noble gases and primitive oxygen isotopes which appear to contradict their high temperature origin, which has led to the group being described as “enigmatic” by some authors. This study used Raman spectral analysis to show that ureilite carbon is heterogeneous, even at the micrometer scale, and is derived from more than one source. In order to better understand the processes involved in ureilite formation, terrestrial analogues containing carbonaceous material with similar spectral characteristics to the meteorites were identified. Analysis of terrestrial samples showed that the sedimentary carbon can be incorporated into igneous rocks with little structural change, suggesting that the same may be true for carbonaceous material in ureilites. Although the terrestrial carbon is biogenic in origin, it is structurally similar to pre-biotic organic matter found in meteorites. Carbon can be used as an effective tracer for geological events, such as melting and heating, which appear to be ubiquitous in planetary evolution. This study concluded that carbon is a primary component of melts on the ureilite parent body (UPB) and that impact processes have increased the heterogeneity of ureilite carbonaceous material. Carbon is likely to have been remobilised by later impact events, explaining the lack of correlation between carbon content and isotopic values with other geochemical parameters. Spectral analysis suggested that most of the carbon in ureilites is derived from primitive material.

One in two people born after 1960 in the United Kingdom will be diagnosed with some form of cancer during their lifetime. Analysis of cancer at the bulk level means that individual attributes may be averaged, and single cell detection and interrogation techniques are therefore of particular interest. In recent years, significant progress has been made into the label-free detection and discrimination of individual cancer cells using Laser Tweezers Raman Spectroscopy (LTRS). However, methods have invariably involved a high degree of manual intervention, and before this technique can be translated into a clinical setting a greater degree of automation is required.\\Initial work has centred on the construction of a LTRS system for the analysis of individual prostate cancer cells and lymphocytes. A novel method of acquiring cell spectra using a microfluidic flow cell has been developed, and the optimum operating conditions for such a system are elucidated in this thesis. Using the system developed, the discrimination of epithelial prostate cells and lymphocytes has been achieved with a high degree of accuracy while requiring a significant reduction in operator input. Further developments to the system have made it possible to obtain Raman spectra for multiple cell lines in a completely automated manner, with no input required during the acquisition of spectra. \\A motivating factor behind the integration of LTRS with microfluidics is the possibility of a label-free equivalent of the Fluorescent Activated Cell Sorter (FACS), which remains the gold standard for single cell analysis. A number of significant steps toward the development of the Raman equivalent (RACS) are presented in this thesis, including the demonstration of an automated system which is capable of multivariate classification and cell translation in real time. Due to limitations relating to the microfluidic flow cells, it has not been possible to actively sort one cell line from a mixed population. However, the system presented in this thesis represents a considerable level of progress towards this objective.\\In addition to the construction of a LTRS arrangement, a 2D Raman mapping system has been developed for the analysis of adherent prostate cell line. This has allowed a direct comparison between the more common technique of Raman mapping and LTRS, and provides new insights into the level of information which can be obtained using LTRS. This thesis presents results obtained with both systems which enable malignant and normal prostate cell lines to be distinguished based on cytochrome-c levels. While cytochrome-c content has been linked with malignancy previously, this is the first demonstration of this relationship using a LTRS system which could be applied in a high throughput setting.

Sensitive and specific visualisation of biomolecules in living models is highly challenging because of the complexity of cellular systems. Raman spectroscopy provides chemical contrast based upon molecular vibrations within a sample. It is therefore a powerful approach in biomedicine for disease diagnosis, owing to its potential to provide a spectroscopic fingerprint of biological species. However, Raman scattering is a weak process, and therefore novel approaches are required in order to improve the detection sensitivity for biomolecular imaging in living systems. Here, novel approaches for biomolecular visualisation based upon stimulated Raman scattering (SRS) microscopy are reported. Spectroscopically bioorthogonal functional groups, those which produce spectrally isolated Raman peaks distinct from endogenous cellular Raman peaks, are utilised as a general strategy for small-molecule visualisation. A key issue in the pharmaceutical industry is the lack of available techniques which can probe drug uptake and retention in living cells. Spontaneous Raman spectroscopy and SRS imaging are used for the selective intracellular visualisation of small-molecule inhibitors using a spectroscopically bioorthogonal approach in Chapter 2. Ponatinib and erlotinib are tyrosine kinase inhibitors used in clinical cancer treatment. The spectroscopically bioorthogonal alkyne group present within each drug is used as a specific marker to visualise the uptake and distribution of these two compounds in relevant cellular models. However, only a limited number (~2%) of regulatory approved drugs contain spectroscopically bioorthogonal Raman functional groups. Hence, a general strategy for the design of novel bioorthogonal Raman labels would allow SRS microscopy to be fully exploited in the drug discovery process. Therefore, density functional theory (DFT) is utilised as an in silico screening technique for the design of novel Raman labels, which is presented in Chapter 3. A library of bioorthogonal Raman-labelled analogues of the natural product, anisomycin, have been synthesised in order to validate the DFT screening approach. The effects of labelling upon the biological activity of anisomycin is also assessed. Spectroscopically bioorthogonal Raman imaging of several biomolecules was performed using SRS imaging and the results of which are reported in Chapter 4. The intracellular detection of de novo DNA synthesis was investigated using SRS imaging through metabolic incorporation of alkyne-containing nucleosides. Secondly, the intracellular detection of the Raman-labelled anisomycin analogues is reported using both spontaneous Raman spectroscopy and SRS imaging. This approach has enabled the rate of drug uptake to be assessed in real-time. Furthermore, combining multi-colour SRS imaging and fluorescence imaging in a dual-modality approach, enabled the analysis of drug uptake to be visualised across intracellular structures, and to be correlated with markers of cell-cycle status. These studies represent novel approaches for the direct intracellular visualisation of the uptake and retention of small-molecule inhibitors in live cells.

The European Space Agency's ExoMars 2020 mission gives the astrobiology community the opportunity to scrutinise instrumentation, landing sites and proposed biosignatures in preparation for this mission. A miniaturised Raman spectrometer will be included as part of the payload instrumentation. The main focus of this work is to test the capabilities of this technique in support of this mission. The impact crater environment is one of the most valuable targets in search for life on Mars. One of the most well preserved craters on earth has evidence of microbial life in the post-impact hydrothermal deposits and the surface mineral crusts. Raman spectroscopy is able to identify more habitable forms of sulphate and detect photo-protective microbial pigments in the crusts. One of the building blocks of life is organic carbon. Raman spectroscopy will primarily be used to characterise organics, and as Mars has a limited atmosphere and lacks a global magnetic field, the surface of Mars is heavily oxidised. The effect that oxidation has on the Raman carbon signal is therefore important. Results show that hematite has an interfering band at the same frequency as the carbon D band, which could result in a misinterpretation of the carbon order, so caution must be taken. Oxidised iron could be a viable energy source of iron-reducing bacteria. One of the most common reduction morphologies in the geological record is the reduction spheroid. They are most likely formed by iron-reducing bacteria, and could be used as a biosignature on Mars. Results show that Raman spectroscopy is able to detect reduction spheroids by tracking the presence of hematite. Experimental work aiming to simulate the formation of a reduction spheroid was unsuccessful but shed light on the complexity of crystalline Fe(III) reduction. This work has highlighted the capabilities and limitations of Raman spectroscopy prior to the ExoMars mission.

This thesis presents several Raman spectroscopic studies of bovine milk-derived products (skim and whole milk powder, anhydrous milk fat, processed cheese and soy cheese made from milk protein). Raman spectroscopy, unlike infrared spectroscopy (both mid- and near-infrared), has not been widely used as an analytical tool within the dairy industry. The purpose of this project was to assess the utility of Raman spectroscopy in several dairy industry relevant problems. FT-Raman spectroscopy coupled with partial least squares (PLS) reliably predicted the concentration of fat and protein within whole milk and protein within skim milk powders as evidenced by ratios of prediction to deviation in excess of three. It was shown that sample geometry did not significantly affect the calculated predictive models, thereby allowing the simplest, quickest method of sample presentation to be utilised without harming quantification model efficacy. The fat fraction of milk was further investigated, and it was shown that Raman spectra collected from anhydrous milk fat samples were a good basis for solid fat content (SFC) determination. Such spectra were collected when the samples were held at 40 �C, pretreated by baselining and area normalisation, and analysed using PLS with modified jack-knifing (PLS1-JK). Triacylglycerol class concentrations were also well predicted by PLS1-JK models developed from Raman spectroscopic data. Resonance enhancement of β-carotene within fat was shown to have little effect on the reliability of the optimal prediction model when compared to SFC Raman quantification. Raman microscopic mapping for heterogeneity description of processed cheese and soy cheese mimics was examined. The FT-Raman milk powder constituents study had shown fat and protein Raman spectra within complex dairy mixtures could be distinguished, hence this utility was applied to mapping of cheese systems. Univariate, principal components analysis and multivariate curve resolution methods of analyses were compared; as expected the more complex samples required multivariate methods of analyses. Raman microscopic mapping was not a time efficient method in comparison to the standard method of confocal laser scanning microscopy. However, Raman spectroscopic mapping is a more universal technique which allowed the presence of an unexpected localised constituent within the soy cheese mimics to be observed. It was proposed that this constituent was a crystalline form of a short saturated fatty acid.

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The present work examines line measurements of pressure, temperature, and density in high speed, non-isobaric flows emanating from an underexpanded jet nozzle. Line images of rotational and vibrational Raman spectra are collected for a 8-mm linear laser probe, and are combined onto the same EMCCD detector. Combining the two techniques allows for a single-shot measurement of major species, pressure, and temperature in a turbulent non-isobaric environment that is chemically reacting. Temperature measurements along the laser test section are extracted from the rotational Raman spectrum, whereas major species densities are measured by examining the intensities of their respective vibrational Raman lines. Pressure can be calculated using an equation of state, in every location along the linear laser probe. The technique feasibility is examined by performing measurements of pressure, temperature and density in a non-reacting underexpanded air jet where the chemical composition is constant and known. Future work will extend the technique to chemically reacting supersonic flows with unknown chemical composition.