Single Molecule Localisation with Fourier Light Field Microscopy

This thesis explores the design, development and application of a novel 3D super-resolution technique, which aims to improve the temporal resolution of 3D Single Molecule Localisation Microscopy (SMLM). This technique combines the super-resolution capability of SMLM with the principles of parallax; multiple perspective views of photo-switchable fluorophores are captured using a lenslet array in the Fourier plane of a fluorescence microscope. The 3D localisation of each fluorophore is then computed in post-processing. An in-depth description of the principles of the proposed technique is first presented, followed by an investigation into how the design of the lenslet array, namely, the number of lenslets, affects the imaging capability of the technique. This is initially quantified through simulation using the Fourier optics-based model of image formation presented in this thesis and subsequently validated through practical characterisation experiments. These results highlight how the technique can be tuned to signal constraints and desired imaging specifications, giving rise to the formulation of a novel design workflow. The performance of this technique is compared to current 3D SMLM techniques, highlighting the unique strengths of its point spread function: better differentiation between fluorophores, which suggests a better performance at a high labelling density, and the ability to maintain efficient localisation over a larger axial range. Finally, this thesis discusses three applications which demonstrate the technique’s advantages. The technique was first applied to capture the organisation of B-cell receptors on the surface of a primary mouse B-cell over a continuous depth of 8 μm. This exceptional depth of field demonstrates that this tool can achieve faster 3D SMLM. The second application employed the technique’s high-density capability to image the 3D structure of chromatin inside the nucleus of Drosophila primary spermatocytes with an unprecedented axial and temporal resolution. Lastly, the localisation efficiency provided by this technique enabled the capture of the dynamics of a transcription factor for the Notch signalling pathway inside the whole salivary glands of Drosophila in 3D for the first time. Ultimately, this thesis aims to demonstrate the versatility of this tool and the potential it offers for gaining new insights into nano-scale biological architecture and mechanisms.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Benaissa, Sarah

Advisor dc:contributor.advisor

• O'Holleran, Kevin

• Light Field Microscopy
• Single Molecule Localisation Microscopy
• Super-resolution Microscopy

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/eef79e15-406f-4532-ad95-3b6331216b9f/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.112901

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/375096