Characterising nanoscale organisation of repair foci during repair of double strand breaks using expansion microscopy.

Faulkner, Emma (2021). Characterising nanoscale organisation of repair foci during repair of double strand breaks using expansion microscopy. University of Birmingham. Ph.D.

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Abstract

Double strand breaks are highly deleterious lesions, and their inefficient repair can lead to mutations and chromosomal translocations. These events cause severe phenotypes such as cancer, immunodeficiencies and neurodegenerative disease. Double strand breaks can be repaired by several mechanisms and the decision of repair pathway is crucial to faithful repair and preservation of genomic integrity.
Repair pathway choice is regulated by numerous factors including cell cycle phase, kinetics of repair and resection of DNA at the break site. Recent studies applying fluorescence microscopy have provided compelling evidence that repair factors are assembled at the break site in a highly spatially and temporally organised manner, and that the spatial dimension of repair is critical to faithful damage resolution. The aim of this thesis is to characterise the spatial organisation of double strand break repair factors to corroborate a spatial model of repair progression.
In this work, I applied novel fluorescence microscopy technique, Expansion Microscopy. This technique enables three-dimensional nanoscale imaging of specimens on a conventional microscope. This is achieved by a specialised chemical preparation in which cells are embedded into a polyelectrolyte gel which can subsequently be swollen by the addition of water. The net effect of this is a de-crowding of molecular information in the specimens and lateral and axial resolution of 70 nm and 200 nm, respectively. Super resolution images can be acquired rapidly, with minimal constraints on fluorophore choice, and without need for sophisticated hardware or software. Specimens are optically cleared during the preparation, which removes the effects of light scattering through the sample. Increased imaging depths and volumes are achievable as compared to other super resolution imaging modalities. In this work, I demonstrate an ability to visualise thousands of repair structures using expansion microscopy which are analysed by a spot detection-based analysis and provide a novel quantitative insight into spatial organisation of repair foci during double strand break repair.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):