Conca, Dario Valter;
(2020)
Development of novel optical techniques for the study of cell-surface proteins in living cells at the single-molecule level.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
The plasma membrane separates the cell interior from the external environment, protecting the cell from adverse conditions and pathogens. However, cells need to continually interact with their surroundings to survive and perform their metabolic functions. These tasks are performed by a great variety of macromolecules that crowd the cellular membrane, from cell-surface receptors to transport channels. The physical and dynamic properties of many of these receptors, as well as their interaction with their target ligand, are poorly understood. This is mainly due to the technological challenges posed by studying single-molecule properties and interactions on living cells, in particular the ability to reach the required spatial and temporal resolution at which these interactions take place. This thesis focuses on the design and development from scratch of an innovative, fully custom-made microscopy platform, which combines light-sheet fluorescence microscopy and optical tweezers for the study of the physical properties of receptors in the plasma membrane of living cells. The technological developments regarding fluorescence microscopy include the demonstration of single-molecule resolution using light-sheet microscopy on single cells. With this method, the tracking of individual receptors in the plasma membrane is achieved with a temporal resolution of tens of milliseconds. Only the top surface of adherent cells is imaged. This prevents artefacts that typically arise in the cell membrane in contact with hard glass substrates, and enables measurements of cell-surface receptor mobility with increased physiological relevance. Furthermore, we have developed an optical tweezers setup optimised for the application of vertical forces on living cells using a counter-propagating beam configuration. Single-molecule force spectroscopy on living cells is demonstrated by quantification of the strength of antigen-antibody bonds. The response of cells to weak and localised mechanical stimuli is likewise measured.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Development of novel optical techniques for the study of cell-surface proteins in living cells at the single-molecule level |
| Event: | UCL (University College London) |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/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. |
| Keywords: | Microscopy, Light-sheet, Optical tweezers, Single-molecule |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Physics and Astronomy |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10108789 |
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