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Title: Single cell dielectrophoretic trapping for the analysis of cellular membrane dynamics
Author: Gielen, Fabrice Matthieu
ISNI:       0000 0004 2718 3396
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Cellular membrane dynamics has been subject to an ever-growing research interest since the introduction of the fluid mosaic model in the early seventies. The recognition that individual components of a cell membrane are able to diffuse in a two-dimensional matrix led to the crucial questioning of the structure-function relationship. The stunning diversity of lipid or proteins making up the plasma membrane of mammalian cells prevents theoretical treatment to apprehend membrane organization and dynamics. For this reason, membrane dynamics has remained up to now predominantly an experimental field of study. The presence of membrane micro-domains including lipid rafts and the co-existence of several phases has for instance been recently confirmed using single-molecule fluorescence detection methods. These domains as well as overall membrane fluidity are thought to be essential in many key cellular processes such as signal transduction, pathogen entry or trafficking. This thesis focuses on the development, characterization and applications of novel microfluidic tools for probing cellular plasma membrane structure and dynamics. We successfully demonstrated dielectrophoretic trapping of single mammalian cells (typically 10μm in diameter) as a means to facilitate time-resolved studies on living cell membranes for timescales of minutes. Firstly, microfluidic devices embedding micro-electrodes have been fabricated. These dielectrophoretic (DEP) traps were characterized to assess their potential as a tool for performing in-vitro membrane bio-assays. DEP traps have been subsequently used to trap single-cells near a defined surface and reagents were introduced via microfluidic channels. Incorporation of a Förster Resonance Energy transfer (FRET) acceptor dye within a donor labelled cellular membrane allowed for time-resolved observation of colocalization events using a scanning confocal microscope and fluorescence lifetime imaging. The presence of cholesterol was shown to influence probes localization. Such microfluidic devices coupled with high-resolution imaging of single cells can potentially be used to study the organization dynamics of individual molecules on the membrane of live cells.
Supervisor: de Mello, Andrew ; Cass, Tony ; Edel, Joshua Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral