The objective of the work described in this thesis was to develop novel experimental and analytical methods to study samples of anisotropically oriented fluorophores. The primary development was the fluorescence detection method for linear dichroism spectroscopy (FDLD). The experimental configuration of the measurement has the detector placed facing the propagation direction of the exciting incident light. Long-pass edge filters, selected so that their cut-off wavelength lies between the excitation and emission maxima of the sample’s fluorophore, were placed between the sample and detector to block transmitted incident light, whilst allowing Stoke’s shifted fluorescence emissions to pass freely. The experiment was designed to be conducted using a commercial circular dichroism (CD) spectropolarimeter, which has been adapted to measure linear dichroism (LD). A theoretical framework to calculate FDLD spectra using the output of such an instrument is presented, and used to generate the FDLD spectra of small molecules oriented on stretched oxidised polyethylene films and biological samples oriented in Couette flow. It was found that much of the information that can be obtained from LD may also be derived from FDLD spectra, however, FDLD possessed two clear advantages: i) fluorescence detection is highly sensitive, which significantly lowered the sample volume requirement of a measurement, and ii) fluorescence detection is more selective than absorption methods, as only chromophores that are also fluorophores are detected. A method for the cloning, expression and purification of the Escherichia coli actin homologue MreB is also presented. The composition of secondary structure elements within the protein obtained was analysed using CD and found to be in good agreement with literature values, taken from the crystal structure of Caulobacter crescentus MreB. Fluorescence spectra of the protein were recorded, which indicated that it may be possible to study its in vitro polymerisation kinetics using FDLD. Unfortunately, we were unable to obtain our Escherichia coli MreB in a sufficient yield to develop this assay. Finally, a novel method for detecting the wall shear stress (WSS) exerted on a specific point of a surface by a fluid flowing over it is reported. This information was derived from the response to a shear flow of a fluorescently labelled particle of M13 bacteriophage, which was tethered to the surface. The focus of this thesis was primarily on the analysis of the fluorescence signals. Using a custom made algorithm, microscopy images of the tethered phage particle were segmented to define the region of each image occupied by the particle, and to calculate its orientation and length at all time points. These data were used to calculate the WSS at the point of the particle’s attachment, and show that it varied when the surface was not uniform — highlighting a potential problem with commonly used methods for determining WSS that average over surface dynamics, such as particle image velocimetry and particle tracking velocimetry.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:714960 |
| Date | January 2016 |
| Creators | Wemyss, Alan M. |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/89284/ |
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