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Title: Raman activated cell sorting and counting in continuous flow
Author: McIlvenna, David
ISNI:       0000 0004 5363 1885
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2015
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Bacteria play a key role in the natural maintenance of our ecosystem and are also used extensively in agriculture, environmental engineering and in the manufacturing of food products and medicines. However, it is estimated that up to 99% of all micro- organisms are currently unculturable. As a result, it is likely that a vast range of potentially useful phenotypes remain unknown. Current techniques to investigate unculturable cells, such as metagenomics, lack the ability to link a specific piece of genetic information to an originating cell. As heterogeneity of phenotypes exists in populations of genetically identical bacteria, single cell studies are becoming more popular to characterize individual microorganisms. In this thesis a continuous flow, Raman activated single cell sorting system has been developed for the first time. Single cell Raman spectroscopy provides the biochemical information of a cell, enabling the label-free and non-destructive characterisation of different cell types and physiological and phenotypic changes to living cells. The system was based on a novel microfluidic pressure divider platform, where the effects of pressure fluctuations upon the cell detection region are minimised, thereby allowing mechanical switching. With this integrated platform, sorting carbon fixing Synechocystis sp. PCC6803 bacteria containing 12C and 13C isotopes at over 96% accuracy was successfully achieved. Also presented in this thesis is a novel technique for Raman based cell counting in continuous flow. By characterising the likely errors resulting from weak Raman signals, the algorithm used allows accurate analysis of the proportions of known cell types in a mixture, at the fastest acquisition rates achievable on the Raman spectrometer used. The results, obtained in real-time, had an r2 correlation value of 0.996 to the independently measured input proportions. The control of cells at low flow rates offered by the pressure divider microfluidic platform would allow significant increases in Raman signal integration times. The combination of the algorithm with this microfluidic platform could allow real-time Raman based sample analysis and diagnostics to be realised.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General) ; QR Microbiology