The objective of this thesis is to present the development of Raman microscopy for biochemical imaging of living cells. The main aim was to construct a Raman micro-spectrometer with the ability to perform time-course spectral measurements for the non-invasive study of biochemical processes in individual cells. The work can be divided into two parts: first, the development and characterization of the instrument; and second, completion of two experiments that demonstrate the suitability of Raman technique for studies of live cells. Instrumental development includes the design of optics and software for automated measurement. The experiments involve data collection and development of mathematical methods for analysis of the data. Chapter One provides an overview of techniques used in cell biology, with a special focus on Raman spectroscopy. It also highlights the importance of experiments on living cells, especially at the single cell level. Chapter Two explains the theoretical background of Raman spectroscopy. Furthermore, it presents the Raman spectroscopy techniques suitable for cell and biological studies. Chapter Three details the instrumentation and software development. The main parts of the confocal Raman micro-spectrometer, as designed for studying living cells, are: inverted microscope, 785 nm laser and high quality optics, environmental enclosure for maintaining physiological conditions during measurements of cells, and fluorescence wide-field microscopy facility for validation and confirmation of biochemical findings by Raman studies. Chapter Four focuses on the evaluation of the performance of the Raman setup and explains calibration and analysis methods applied to the data. Chapter Five and Six describe experiments performed on living cells. Chapter Five focuses on studies of the immunological synapse formed between primary dendritic and T cells indicating the polarisation of actin. Chapter Six describes time-course experiment performed on cancerous cells in the early phases of the apoptosis process, which enabled detection of the DNA condensation and accumulation of unsaturated lipids. Chapter Seven summarizes the work and gives concluding remarks.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:539199 |
| Date | January 2011 |
| Creators | Zoladek, Alina |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/13338/ |
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