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Application of Femtosecond lasers in confocal and scanning tunnelling microscopy

Davies, Owain (2010)
Ph.D. thesis, University of Birmingham.

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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.

Type of Work:Ph.D. thesis.
Supervisor(s):Palmer, Richard E.
School/Faculty:Colleges (2008 onwards) > College of Engineering & Physical Sciences
Department:School of Physics and Astronomy
Subjects:QC Physics
Institution:University of Birmingham
ID Code:933
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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