Developing Raman microspectroscopy as a technique to analyse single Caco-2 Cell

Smith, Rachael and Ashton, Lorna and Wright, Karen (2020) Developing Raman microspectroscopy as a technique to analyse single Caco-2 Cell. PhD thesis, Lancaster University.

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Abstract

Raman spectroscopy is rapidly advancing as a cell imaging technique due to its advantages over existing techniques: it requires little sample preparation, is label-free and can be carried out in aqueous environments to image both fixed and live cells. However, Raman spectroscopy is not commonly used in clinical practice due to perceived long acquisition times and complex data analysis. The aim of this work is to develop Raman spectroscopy as a technique to image cells, specifically Caco-2 cells, and to use Raman spectroscopy to measure the response of these cells to abiotic perturbations. We have developed methodologies for mapping both fixed and live Caco-2 cells, as well as robust shading parameters to allow for direct comparisons. Using a metal rhenium complex to identify the mitochondria of these cells, we have demonstrated the difficulties in shading Raman maps to specific peaks of interest, and how the shading range needs to be carefully considered to avoid over or under interpretation of the data. Raman spectroscopy was also used to evaluate the effect of the cannabinoids cannabidiol (CBD) and anandamide (AEA) on Caco-2 cells. CBD affects Caco-2 cells differently at different concentrations; at low concentrations it may induce proliferation, but at high concentrations it causes cell death characterised by DNA breakdown. Investigating the mechanism of CBD induced cell death in Caco-2 cells suggested that it is not apoptosis or necrosis, but is mediated by caspases 3/7 with the broken down DNA being exported from the cell, which is more consistent with autophagy-dependent cell death or lysosomal-dependent cell death. At low concentrations, AEA may also induce proliferation, but at high concentrations it affects Caco-2 cells differently and does not cause cell death; instead, we hypothesise that the drug has an anti-proliferative effect on these cells. Overall, we have demonstrated how Raman spectroscopy can be used to gain valuable visual and biochemical information about cells.