Abstract
dc:description.abstractCapturing cellular morphology on a population level requires technologies enabling high speed imaging and sample throughput. This has motivated the evolution of flow cytometry, which uses microfluidics to measure the fluorescence and scattering signals of thousands of cells per second, towards imaging flow cytometry, which allows spatial information to be collected at extremely high throughput. However, most existing systems are limited to 2D imaging, which can be unable to answer certain biological questions. Light field microscopy is a computational imaging technique that captures 3D information in a single camera shot. In contrast to many other methods of 3D imaging, light field requires only a simple optical setup and no moving parts—making it ideally suited to fast volumetric imaging. This thesis presents the development and characterisation of the first light field-based 3D imaging flow cytometry platform and its application to characterising cellular heterogeneity when imaging protein trafficking in live cells. The development of an optical system suited to large depth-of-field, multi-channel 3D imaging of flowing samples is presented. Design considerations in the excitation optics and the use of a light sheet in conjunction with light field imaging optics are explored, and methods of characterising the optical performance and 3D resolution in flow are presented. It is shown that the setup presented here achieves sub-cellular resolution across a 30 µm depth-range at a throughput-equivalent of > 100 cells per second. The performance of novel and established 3D reconstruction techniques is characterised, and methods of optimising for imaging in flow are explored. Additionally, the development of microfluidic devices and experimental methods aiming to maximise throughput are presented. Finally, the instrumentation developed here was applied to live-cell imaging in flow to track protein trafficking in 3D. The spatial distribution of a type-1 membrane protein (LAMP-1) was imaged at multiple time points and analysed to reveal the heterogeneity in trafficking rate across large numbers of cells. In summary, this thesis charts the development, characterisation, optimisation, and application of a novel 3D imaging flow cytometry system, with the aim of advancing our understanding of cellular morphological heterogeneity.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2024
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Collins, Alexander
- Advisor dc:contributor.advisor
-
- Lee, Steven
Subjects
dc:subject × 9Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.112703
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/374757