Methods for optical microscopy beyond conventional limits

Optical microscopy seeks to produce accurate and precise enlarged images of an object. It is highly desirable, particularly in live-cell biological microscopy, that the observation process does not perturb the structure, nor interfere with the dynamics, of the sample. These requirements are at odds with the fact that increased resolution necessarily entails a larger light dose. This thesis proposes methods for increasing the resolution of fluorescence microscopy beyond that of the widely employed confocal microscopy while reducing photodamage and phototoxicity. Measurements of the structures of fluorescent shells with 10 nm precision, derived from conventional wide-field micrographs and a quantitative image analysis routine, are presented and used to study bacterial spore coats. A design for a light sheet microscope capable of isotropic super-resolution is proposed and shown to be capable, in simulations, of 120 nm lateral and 190 nm axial resolution while maintaining the benefits of low intensity illumination and conventional sample mounting. An investigation into the possibility of further doubling the axial resolution of three-dimensional structured illumination microscopy (SIM) via a simple modification to the illumination system, and no need for interferometric detection, is described. Work on modifying a total internal reflection SIM instrument into one capable of optical sectioning SIM is presented, with the suitability of the converted system for investigations into HSV-1 viral particle assembly detailed. Finally, a modified instrumental design to further improve optical sectioning and axial resolution is proposed.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2018

Author dc:creator

• Manton, James

Advisor dc:contributor.advisor

• Rees, Eric

• microscopy
• optics
• fluorescence
• imaging
• light sheet microscopy
• structured illumination microscopy
• localisation microscopy

Licence

• https://www.repository.cam.ac.uk/bitstreams/46b9a36a-060f-4522-8a06-8a02fd5726b0/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

Author Identifier

0000-0001-9260-3156

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/306920