Application of a novel high resolution widefield surface plasmon microscope in cell engineering, wound healing and development of new binding assays

Abdul Jamil, Muhammad Mahadi (2007) Application of a novel high resolution widefield surface plasmon microscope in cell engineering, wound healing and development of new binding assays. PhD thesis, University of Bradford.

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Abstract

Surface Plasmon (SP) microscope systems are mostly built around the prism based Kretsehmann configuration. In these systems the generation of Surface Plasmons (SPs) is achieved by p-polarized light striking a metallised prism surfacc at a spccific angle and then monitoring thc intensity of the reOected light. Thus in these systems. an image of the material can be obtained in terms of an intensity map. in which the intcnsity of thc image is dependent on the way the light couples into the SPs. The drawback of these systems is that lateral resolution relies on the ability of plasmons to propagate along the metallised layer. The lateral resolution is thus limited to a few microns. Therefore, a new microscope systcm was developed. i.e. thc Widcficld Surface Plasmon Resonance (WSPR) microscope. that is not only capable of analysing molecular interactions at high vertical resolutions. but also enables SP imaging at much higher lateral resolution than prism based systcms. The functionality of thc novel (WSPR) microscope has been investigated by imaging a sequence of binding events between micropattcrncd extracellular matrix proteins and their specific antibodies both in air and real-time. Using the WSPR systcm a changc in contrast was observed with each protein binding cvcnts. Images produced via the WSPR system were analyzcd and comparcd qualitatively and quantitatively. The preliminary results acquired for these binding studics between antibody/antigens dcmonstrate that the WSPR systcm capablc of resolving features down to 260nm although the theoretically proven lateral resolution of the WSPR system is -500nm. Cell surface interactions undcr two diffcrent culture conditions. i.e. HaCaTs cultured on SPR substrate with Transforming Growth Factor ~3 (TGF~3) (50ng/lII/) and without TGF~3 were also invcstigated. It was found that I-IaCaTs cultured in the presence of TGF~3 showed enhanced division and motility along with decreased cell attachment as compared with cells maintained in TGF~3 free media. It is believed that cellular signalling by TGF~3 is very important for enhancing tissue development in wound rcpair. It is confirmed that the WSPR microscope described here can be used to study sequential monomolecular layer of antibody/antigen interactions binding events and examination of cell surface interfacial interactions at lateral scales of less than one micron without the need for traditional immunofuorescent labelling. These results have significant implications in the development of ncew breed fast binding assays system and in enabling high resolution detailed examination of the cell surface couplings and cell signalling processes involved in cell attachment and migration.

Item Type:Thesis (PhD)
Subjects:Q Science > QH Natural history > QH301 Biology
ID Code:1287
Deposited By:M.Iqbal Zainal A
Deposited On:20 Apr 2011 12:05
Last Modified:29 Apr 2011 14:43

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