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Biochemical phenotyping of live single cells using confocal Raman spectroscopy

Gala de Pablo, Julia (2019) Biochemical phenotyping of live single cells using confocal Raman spectroscopy. PhD thesis, University of Leeds.

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Abstract

Colorectal cancer is the 3rd most common cancer worldwide with the second highest mortality rates. Around 20% of the tumours of diagnosed patients have metastasis. Understanding cancer development and progression represents a major challenge. As a proof of concept, hundreds of live single-cells derived from different stages of colorectal adenocarcinoma – from primary to metastasis (SW480, HT29, SW620) – were compared to a cell line from colorectal carcinoma (HCT116) and leukaemia (HL60) using Raman confocal spectroscopy, obtaining >92% correct rate classification using principal component analysis and linear discriminant analysis. Photodynamic therapy is a treatment that uses photosensitisers that selectively accumulate in cancerous cells. Light activates the photosensitiser, killing the cancer cells while sparing the surrounding tissue, with promising results on colorectal cancer. Experiments proved if Raman spectroscopy could detect the presence of these agents and their effects in live single cells. Experiments were done to detect Protoporphyrin IX accumulation in live cells. Moreover, the novel photosensitizer DC473 (LightOX) accumulation was imaged within fixed SW480 cells using Raman and fluorescence. Raman spectroscopy detected the accumulation of DC473 in live single-cells, with higher accumulation in HT29 and SW620 than in SW480 and causing a drop in the main lipid’s bands. The activation of the photosensitiser was tracked on live single-cells using Raman, observing the chemical reaction of the photosensitiser and a drop of the cells' viability over time. The bands of the fraction of DC473 that did not react were observed to broaden and shift to higher wavenumbers, indicating a change in the environment of the photosensitiser after activation. To test if Raman spectroscopy could be pushed to higher throughput using microfluidics, Raman acquisition was made in Polydimethylsiloxane microfluidic devices on the same cell before and following incubation with DC473. Results showed that Raman spectroscopy has great potential for single-cell time-dependent drug treatment studies.

Item Type: Thesis (PhD)
Keywords: Raman, live, single-cell, colorectal, photodynamic, cancer, phenotyping
Academic Units: The University of Leeds > Faculty of Maths and Physical Sciences (Leeds) > School of Physics and Astronomy (Leeds)
Identification Number/EthosID: uk.bl.ethos.786537
Depositing User: Dr. Julia Gala de Pablo
Date Deposited: 01 Oct 2019 10:20
Last Modified: 18 Feb 2020 12:50
URI: http://etheses.whiterose.ac.uk/id/eprint/24935

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