Single cell dielectrophoretic trapping for the analysis of cellular membrane dynamics
Author(s)
Gielen, Fabrice Matthieu
Type
Thesis
Abstract
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.
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.
Date Issued
2011-11
Date Awarded
2012-03
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
de Mello, Andrew
Cass, Tony
Edel, Joshua
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)