Development of a Raman microscope for applications in radiobiology




Matthews, Quinn

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Raman microscopy (RM) is a vibrational spectroscopic technique capable of obtaining sensitive measurements of molecular composition, structure, and dynamics from a very small sample volume (~1 µm). In this work, a RM system was developed for future applications in cellular radiobiology, the study of the effects of ionizing radiation on cells and tissues, with particular emphasis on the capability to investigate the internal molecular composition of single cells (10-50 µm in diameter). The performance of the RM system was evaluated by imaging 5 µm diameter polystyrene microbeads dispersed on a silicon substrate. This analysis has shown that RM of single cells is optimized for this system when using a 100x microscope objective and a 50 µm confocal collection aperture. Quantitative measurements of the spatial, confocal, and spectral resolution of the RM system have been obtained using metal nanostructures deposited on a flat silicon substrate. Furthermore, a spectral investigation of several substrate materials was successful in identifying low-fluorescence quartz as a suitable substrate for RM analysis of single cells. Protocols have been developed for culturing and preparing two human tumor cell lines, A549 (lung) and DU145 (prostate), for RM analysis, and a spectroscopic study of these two cell lines was performed. Spectra obtained from within cell nuclei yielded detectable Raman signatures from all four types of biomolecules found in a human cell: proteins, lipids, carbohydrates, and nucleic acids. Furthermore, Raman profiles and 2D maps of protein and DNA distributions within single cells have been obtained with micron-scale spatial resolution. It was also found that the intensity of Raman scattering is highly dependent on the concentration of dense nuclear material at the point of Raman collection. RM shows promise for studying the interactions of ionizing radiation with single cells, and this work has been successful in providing a foundation for the development of future radiobiological RM experiments.



Medical Physics, Radiobiology, Raman spectroscopy, Raman microscope