Sensitive and specific visualisation of biomolecules in living models is highly challenging because of the complexity of cellular systems. Raman spectroscopy provides chemical contrast based upon molecular vibrations within a sample. It is therefore a powerful approach in biomedicine for disease diagnosis, owing to its potential to provide a spectroscopic fingerprint of biological species. However, Raman scattering is a weak process, and therefore novel approaches are required in order to improve the detection sensitivity for biomolecular imaging in living systems. Here, novel approaches for biomolecular visualisation based upon stimulated Raman scattering (SRS) microscopy are reported. Spectroscopically bioorthogonal functional groups, those which produce spectrally isolated Raman peaks distinct from endogenous cellular Raman peaks, are utilised as a general strategy for small-molecule visualisation. A key issue in the pharmaceutical industry is the lack of available techniques which can probe drug uptake and retention in living cells. Spontaneous Raman spectroscopy and SRS imaging are used for the selective intracellular visualisation of small-molecule inhibitors using a spectroscopically bioorthogonal approach in Chapter 2. Ponatinib and erlotinib are tyrosine kinase inhibitors used in clinical cancer treatment. The spectroscopically bioorthogonal alkyne group present within each drug is used as a specific marker to visualise the uptake and distribution of these two compounds in relevant cellular models. However, only a limited number (~2%) of regulatory approved drugs contain spectroscopically bioorthogonal Raman functional groups. Hence, a general strategy for the design of novel bioorthogonal Raman labels would allow SRS microscopy to be fully exploited in the drug discovery process. Therefore, density functional theory (DFT) is utilised as an in silico screening technique for the design of novel Raman labels, which is presented in Chapter 3. A library of bioorthogonal Raman-labelled analogues of the natural product, anisomycin, have been synthesised in order to validate the DFT screening approach. The effects of labelling upon the biological activity of anisomycin is also assessed. Spectroscopically bioorthogonal Raman imaging of several biomolecules was performed using SRS imaging and the results of which are reported in Chapter 4. The intracellular detection of de novo DNA synthesis was investigated using SRS imaging through metabolic incorporation of alkyne-containing nucleosides. Secondly, the intracellular detection of the Raman-labelled anisomycin analogues is reported using both spontaneous Raman spectroscopy and SRS imaging. This approach has enabled the rate of drug uptake to be assessed in real-time. Furthermore, combining multi-colour SRS imaging and fluorescence imaging in a dual-modality approach, enabled the analysis of drug uptake to be visualised across intracellular structures, and to be correlated with markers of cell-cycle status. These studies represent novel approaches for the direct intracellular visualisation of the uptake and retention of small-molecule inhibitors in live cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:739071 |
| Date | January 2017 |
| Creators | Tipping, William James |
| Contributors | Brunton, Valerie ; Hulme, Alison |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/29512 |
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