Super-resolution Microscopy: Novel Developments and Optimisations

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.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2019

Author dc:creator

• Abdul Rehman, Sohaib

Advisors dc:contributor.advisor

• O'Holleran, Kevin
• Lee, Steven F.

• Fluorescence Microscopy
• Super-resolution Microscopy
• Localisation Microscopy
• Three-dimensional Microscopy
• Lightfield Microscopy
• Single Molecule Tracking
• Clustering
• Notch Pathway
• Chromatin Architecture
• Optics

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/74133a90-be44-45c3-9149-e454e1744ef7/download
• https://creativecommons.org/licenses/by-nc-sa/4.0/

Language dc:language

en

Author Identifier

0000-0002-1170-6610
0000-0003-1039-2127
0000-0003-4492-5139

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/296453