Tosheva, Kalina Latchezarova;
(2024)
Defining the Limits of Live-Cell Microscopy.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Phototoxicity is the damage to living matter caused by light. It is simultaneously the most prevalent and a rarely spoken of problem present in live-cell microscopy. It poses a particular issue in specialised methods, such as super-resolution microscopy, which often rely on the use of high-intensity and ultraviolet illumination. Methods for both minimising and detecting it exist but continue to be underdeveloped. This work outlines progress on both fronts. Firstly, the fluctuation-based low-illumination super-resolution microscopy method, super-resolution radial fluctuations, or SRRF, is established as a universal analytical approach for several microscopy setups commonly used for live-cell imaging. The method was utilised for short- and long-term imaging, as well as on data obtained with both laser and LED illumination, the latter being the first demonstration of live-cell super-resolution microscopy using this illumination type. Secondly, an easy and statistically powerful assay was developed for quantitative measurement of phototoxicity. It uses a straightforward and sensitive biological readout in the form of perturbation of mitosis, employing a commonly available cell system. Photodamage is detected using a common widefield microscope setup and includes a fully developed semi-automated image analysis pipeline which utilises freely available software and open-source scripts. The simplicity of the setup and the nature of the analysis additionally allow for customisation and adaptation according to a specific biological question or microscopy modality. The methods described here serve as important milestones towards improvement of live-cell-friendly imaging. SRRF is already a widely-used method that is continuously further developed, with newer installments including higher image fidelity and, with the employment of specialised optics, super-resolution in full 3D. Meanwhile, the development of the phototoxicity assay lays down the foundation for a streamlined method for widely applicable detection of phototoxicity across different light microscopy modalities. Conceivably, it could become a benchmark for future works to study phototoxicity in live cells.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Defining the Limits of Live-Cell Microscopy |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2024. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Lab for Molecular Cell Bio MRC-UCL |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10191676 |
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