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Super-resolution Microscopy: Novel Developments and Optimisations 10.17863/CAM.43503 Abdul Rehman, Sohaib 0000000211706610 O'Holleran, Kevin 0000000310392127 Lee, Steven F. 0000000344925139 This thesis describes the design, development and optimisation of a multifunctional localisation based super-resolution microscope at Cambridge Advanced Imaging Centre. The microscope is optimised to perform single and dual-colour imaging with high localisation precision and accuracy. Moreover, three-dimensional imaging capability is included in the microscope using the double-helix point spread function and the light field imaging modality. The thesis also describes the application of the microscope to image challenging biological samples, in collaboration with the research groups at the Department of Physiology, Development and Neuroscience. This includes, studying dynamics of a DNA-binding transcription factor for the Notch signalling pathway, deep within the whole salivary glands of Drosophila. Characterisation and optimisation of the microscope and the subsequent image analysis pipeline, to extract dynamics of single molecules at such depths is also discussed. Another application of the microscope, discussed in the thesis, is the study of chromatin architecture in primary spermatocytes of Drosophila. This includes optimisation of imaging conditions and data analysis software to reconstruct features with different densities of labelling dye in the imaged nuclei. Calibration and application of dual-colour localisation microscopy, to visualise the arrangement of active transcription sites in chromatin fibres is also discussed. Finally, the thesis also presents the application of light field imaging technique to extend the depth of field of localisation microscopy to over 20 μm. Modification of the microscope for light field imaging and a method to localise point emitters with high precision in all three spatial dimensions is discussed. The effectiveness of the technique for single molecule imaging is shown by detecting emissions from single fluorophores in labelled cells. University of Cambridge 2019-10-26 Thesis Doctoral Doctor of Philosophy (PhD) en https://www.repository.cam.ac.uk/handle/1810/296453 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/c64cabed-79fa-4ed3-90c1-3ea8987cce3f/download f55aaa80746dbae209cec9edc58999b5 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/74133a90-be44-45c3-9149-e454e1744ef7/download 87eda9de84448d1f82354d60eee3eb5f https://creativecommons.org/licenses/by-nc-sa/4.0/ Fluorescence Microscopy Super-resolution Microscopy Localisation Microscopy Three-dimensional Microscopy Lightfield Microscopy Single Molecule Tracking Clustering Notch Pathway Chromatin Architecture Optics