Application of a novel high resolution widefield surface plasmon microscope in cell engineering, wound healing and development of new binding assays
Surface Plasmon (SP) microscope systems are mostly built around the prism based Krctschmann configuration. In thcsc systcms 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...
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| Format: | Thesis |
| Language: | English |
| Published: |
2007
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| Subjects: | |
| Online Access: | http://eprints.uthm.edu.my/7366/1/24p%20MUHAMMAD%20MAHADI%20ABDUL%20JAMIL.pdf |
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| Summary: | Surface Plasmon (SP) microscope systems are mostly built around
the prism based Krctschmann configuration. In thcsc systcms 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 dcpendent 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 scquence of binding events between
micropattcrncd cxtracellular 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 attachmcnt as compared with cclls 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 cvents and examination of cell surface
intcrfacial intcractions at latcral scales of less than one micron without the need
for traditional immunoOuorescent labelling. These rcsults have significant
implications in the developmcnt of ncw brecd fast binding assays system and in
enabling high resolution detailed examination of the cell surface couplings and
ccll signalling proccsses involvcd in cell attachmcnt and migration. |
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