Davies, Owain (2010)
Ph.D. thesis, University of Birmingham.
This thesis reports the use of a Ti:sapphire ultrafast laser with a confocal microscope to precisely induce DNA damage in the nuclei of live cells by multi-photon absorption, the development and comparison of foci counting algorithms for the quantitative assessment of radiation damage and work towards the development of an ultrafast Scanning Tunnelling Microscopy (STM) technique, employing a Ti:sapphire pulsed laser, called Shaken Pulse Pair eXcitation (SPPX) STM. Measurements of the laser intensity, pulse duration and point spread function are used to estimate the peak intensity at the focus of the confocal microscope. A UV absorption in DNA is excited by the simultaneous absorption of three IR photons (3P). This process leads to the formation of cyclobutane pyrimidine dimmers (CPDs) in the DNA chain. Proliferating Cell Nuclear Antigen (PCNA), involved in the repair of these lesions is tagged with Green Fluorescent Protein (GFP) to visualise the repair process. Damage is detected at peak intensities as low as 23±3 GW/cm2 which is lower than previous studies. PCNA localises at the DNA damage sites with an exponential localisation. Three foci counting algorithms were implemented: a simple intensity threshold algorithm; a Compact Hough transform and Radial Mapping (CHARM) algorithm and a watershed algorithm. The watershed algorithm was particularly effective for the assessment of foci in 3D datasets, providing counts and other properties relating to the foci. It is applied to a study of y-H2AX foci in radiation dosed cells to assess various properties of y-H2AX foci as a function of radiation. Work on the SPPX-STM apparatus led to the development of a novel high frequency translation stage, allowing a retro-reflector to be periodically oscillated without coupling the vibration into the optical table.
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