Biomedical imaging is a key tool for the study of host-pathogen interactions. New techniques are enhancing the quality and flexibility of imaging systems, particularly as a result of developments in laser technologies. This work applies the combination of two advanced laser imaging methods to study the interactions between a virus and the host cells it infects. The first part of this work describes the theory and experimental implementation of coherent anti-Stokes Raman scattering microscopy. This technique-first demonstrated in its current form in 1999-permits the imaging of microscopic samples without the need for fluorescent labelling. Chemical contrast in images arises from the excitation of specific vibrations in the sample molecules themselves. A laser scanning microscope system was set up, based on an excitation source consisting of two titanium-sapphire lasers synchronized with a commercial phase-locked loop system. A custom-built microscope was constructed to provide optimal imaging performance, high detection sensitivity and straightforward adaptation to the specific requirements of biomedical experiments. The system was fully characterized to determine its performance. The second part of this work demonstrates the application of this microscope platform in virology. The microscope was configured to combine two nonlinear imaging modalities: coherent anti-Stokes Raman scattering and two-photon excitation. Mouse fibroblast cells were infected with a genetically modified cytomegalovirus. The modification causes the host cell to express the green fluorescent protein upon infection. The host cell morphology and lipid droplet distribution were recorded by imaging with coherent anti-Stokes Raman scattering, whilst the infection was monitored by imaging the viral protein expression with two-photon excitation. The cytopathic effects typical of cytomegalovirus infection were observed, including expansion of the nucleus, rounding of the cell shape, and the appearance of intracellular viral inclusions. In some cases these effects were accompanied by dense accumulations of lipid droplets at the nuclear periphery. Imaging was performed both with fixed cells and living. It was demonstrated that the lipid droplets in a single live cell could be imaged over a period of 7 hours without causing noticeable laser-induced damage. The system is shown to be a flexible and powerful tool for the investigation of virus replication and its effects on the host cell.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764174 |
| Date | January 2010 |
| Creators | Robinson, Iain Thomas |
| Contributors | Hossack, Will ; Crain, Jason |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/33314 |
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