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Analysis of single cell physiology using nano-scale electrochemical methods
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Cremin, Kelsey Lauren (2022) Analysis of single cell physiology using nano-scale electrochemical methods. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3877425
Abstract
Scanning probe microscopy (SPM) can offer high-resolution, multi-functional analysis of live biological samples. This thesis focuses on two of these methods; scanning ion conductance microscopy (SICM) and scanning electrochemical microscopy (SECM), investigating both bacterial and mammalian cells. Primarily this work concentrates on the growing field of single cell analysis, as opposed to bulk analysis which can homogenise a cell population and potentially miss individualistic properties of single cells. Each study in this work aims to either non-invasively measure properties of live cells, such as the topography, local charge environment or respiration, or to induce localised environment changes to a cell population and study the cellular response. The experimental work within this thesis moves towards multifaceted cell analysis, through the combination of SPM with complementary methods, particularly finite element method (FEM) analysis and laser scanning confocal microscopy (LSCM). FEM simulations are vital in explaining and utilising electrochemical data collected through SICM/SECM, allowing a visualisation of phenomena occurring within the experiment, such as concentration gradients or material flow, and offering a theoretical playground to explore experimental conditions. As shown throughout this work, simulations are useful for examining an experiment at the local interface level to more thoroughly determine the impact of the nanopipette at the cell surface. Here, simple FEM models are expanded to better represent the investigated biological systems, and fully utilise the richness of electrochemical data achieved through SPM. The findings across this thesis include the believed first application of SICM charge mapping on live single-cell bacteria, comparing the Gram-positive and - XVII negative cell wall structures and featuring a biologically-tailored FEM model for exploring the charge values (Chapter 3). FEM is also used to investigate the requirements for successful measurement of single-cell respiration at mammalian cells via SECM (Chapter 4). Finally, this thesis also includes a method for creating localised gradient conditions using an adapted SICM delivery protocol combined with confocal microscopy. The nanopipette probe was used for the controlled delivery of acid to a plane of cells, with the intracellular and local pH of each individual each mammalian cell under a different pH condition could be measured simultaneously (Chapter 5). Each series of experiments is discussed in detail, followed by an examination on how this work may advance the application of SPM for more complex and comprehensive biological studies. The findings across this thesis include the believed first application of SICM charge mapping on live single-cell bacteria, comparing the Gram-positive and - negative cell wall structures and featuring a biologically-tailored FEM model for exploring the charge values (Chapter 3). FEM is also used to investigate the requirements for successful measurement of single-cell respiration at mammalian cells via SECM (Chapter 4). Finally, this thesis also includes a method for creating localised gradient conditions using an adapted SICM delivery protocol combined with confocal microscopy. The nanopipette probe was used for the controlled delivery of acid to a plane of cells, with the intracellular and local pH of each individual each mammalian cell under a different pH condition could be measured simultaneously (Chapter 5). Each series of experiments is discussed in detail, followed by an examination on how this work may advance the application of SPM for more complex and comprehensive biological studies.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QH Natural history Q Science > QR Microbiology T Technology > TA Engineering (General). Civil engineering (General) |
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Library of Congress Subject Headings (LCSH): | Scanning probe microscopy, Scanning electrochemical microscopy, Cells -- Analysis, Finite element method, Bacterial cell walls, Ion channels | ||||
Official Date: | May 2022 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Molecular Analytical Science Centre for Doctoral Training | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Sponsors: | University of Warwick. Molecular Analytical Science Centre for Doctoral Training ; Engineering and Physical Sciences Research Council | ||||
Format of File: | |||||
Extent: | xviii, 330 pages : illustrations (colour), charts (colour) | ||||
Language: | eng |
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