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Title: Overcoming resolution limits in fluorescence microscopy with adaptive optics and structured illumination
Author: Shaw, Michael
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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This thesis presents an investigation of two dynamic optical techniques for restoring and extending the spatial frequency response of fluorescence microscopes. In the first method, adaptive optics (AO), imaging performance is improved through the measurement and compensation of wavefront aberrations. The use of fluorescent guide stars contained within the sample is explored to allow direct wavefront sensing in a microscope. This guide star method is tested using an artificial phantom object and is applied to measure the wavefront aberrations induced by a commonly studied biological organism. It is shown that such a scheme can be used in combination with a confocal pinhole to reject out of focus light and allow effective wavefront sensing in thick biological samples. The direct wavefront sensing technique is then used in a closed loop AO system incorporated in a combined widefield and confocal fluorescence microscope. The design and validation of the microscope system are presented and the device is used for aberration corrected imaging of synthetic samples and a biological organism. Whilst AO makes possible the restoration of diffraction limited imaging performance, structured illumination microscopy (SIM) seeks to increase effective spatial resolution through frequency mixing between the sample and a spatially modulated excitation field. A high speed SIM system is presented in which the excitation patterns are generated using a liquid crystal on silicon spatial light modulator configured as a binary phase grating. The optical system and image reconstruction methods are described and the effect of light polarisation state on pattern formation is investigated using vectorial ray tracing and experimental measurements. The ability of the system to generate superresolution and optically sectioned images is tested using fluorescent microspheres and a range of biological samples.
Supervisor: Paterson, Carl; Hall, Simon Sponsor: National Physical Laboratory
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available