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Title: Scanning near-field optical microscopy studies of cell membrane proteins labelled with fluorescent quantum dots
Author: Walker, Kelly-Ann D.
Awarding Body: Swansea University
Current Institution: Swansea University
Date of Award: 2010
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Scanning near-field optical microscopy (SNOM) has been employed to simultaneously acquire high-resolution fluorescence images along with shear-force atomic force microscopy of cell membranes. Implementing such a technique overcomes the limits of optical diffraction found in standard fluorescence microscopy and also yields vital topographic information. However, one of the biggest challenges of imaging fluorescent biological specimens with SNOM, is the photostability and low yield of fluorescent labelling agents. Semiconductor quantum dots are a recently developed class of fluorophores which exhibit superior optical properties. They are significantly brighter and more resistant to photo-degradation than organic fluorophores. In this study, SNOM has been utilised in conjunction with quantum dot labelling to interrogate the biomolecular composition of cell membranes. The technique has been applied to investigate cell-cell adhesion in human epithelial cells. This has been realised through immunofluorescence labelling of the cell-cell adhesion protein E-cadherin. Moreover, a dual labelling protocol has been optimised to facilitate a comparative study of the adhesion mechanisms, and the effect of aberrant adhesion protein expression, in both healthy and cancerous epithelial cells. This study reports clear differences in the morphology and phenotype of healthy and cancerous cells. In healthy prostate epithelial cells (PNT2 cells), Ecadherin was predominantly located along the cell periphery and within filopodial protrusion. The presence of E-cadherin appeared to be enhanced when cell-cell contact was established. Furthermore this study has revealed the interactions of filopodia and their functional relationship in establishing adherens junctions in PNT2 cells. In contrast, examination of metastatic prostate cancer cells (PC-3 cells) revealed E-cadherin to be predominantly localised around the nuclear region of the cell, with no E-cadherin labelling around the periphery of the cells. This lack of functional E-cadherin in PC-3 cells coincided with a markedly different morphology and PC-3 cells were not observed to form tight cell-cell associations with their neighbours. Facilitated by the high-resolution imaging afforded by the SNOM technique, this research further highlights the important role th at E-cadherin plays in the development of invasive, metastatic cancers.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available