Measuring the dynamics of suspended lipid bilayers and their reaction to membrane proteins using label free microscopy

Turley, Freya 2024. Measuring the dynamics of suspended lipid bilayers and their reaction to membrane proteins using label free microscopy. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Cell membranes play crucial roles in biological processes, acting as a barrier and controlling exchange of molecules. This includes intercellular signalling via receptor mediated processes and budding of vesicles from the membrane. For these functions membrane dynamics are fundamental. The sorting of components on and within the membrane leads to a large portion of the complex organisation that exists at a cell surface. There is a reciprocal effect between these components and the membrane, exerting their effects predominantly by altering dynamics. Membranes are described using a fluid mosaic model with areas of differing fluidity, which have different properties due to the lipids they are made up of. Lipids can be in different phases such as gel, a solid state that confers stability, or liquid which can be ordered or disordered. Through this action they can influence the movement of structures such as proteins to sort them into areas with certain properties. This is a reciprocal effect with membrane components affecting the phase behaviour of the lipids and modulating membrane function. This interplay at a crucial structure makes the dynamics an interesting and important area of study. In this work we aimed to measure the dynamics of a membrane via thickness and height changes. We demonstrate methods to probe these dynamics, generating suspended model membranes and measured with various techniques. Epifluorescence measurements allowed the visualisation of phase separation across the surface of a bilayer, to assess it’s interaction with a pore forming toxin. We also show fitting and simulation methods developed using the label free method, quantitative Differential Interference Contrast microscopy (qDIC) to characterise vesicles and their internal volumes. We introduce our novel label free technique interferometric Gated Off-axis Reflectometry (iGOR) to measure the thickness and axial position of the suspended membrane to sub nanometer resolution. This revealed the undampened dynamics occurring in these membranes at 327Hz in a widefield conformation. These measurement conditions allowed fast acquisition of every pixel in the field of view simultaneously providing sufficient temporal resolution. Due to it’s sensitivity and high resolution, iGOR is a step change in this field of imaging for these dynamics. We also employed iGOR to track nanoparticles in a 3D volume to ascertain their hydrodynamic radii and scattering cross-section. Combined these features of the iGOR technique make it a powerful tool for the investigation of model membrane dynamics and their interaction with proteins. In this work we show how we made model membranes and how each measurement technique used is a step change in measuring membrane properties. We show measurement of lamellarity and internal refractive index of vesicles using qDIC and thickness and height changes with high temporal resolution using iGOR. Whilst we showed adequate sensitivity there is still outstanding optimisation to acquisition and analysis for full quantitative analysis.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Physics and Astronomy
Subjects:	Q Science > QC Physics
Uncontrolled Keywords:	Membranes, Microscopy, Label-free, Dynamics, Giant unilamellar vesicles, Interferometric off-axis reflectometry
Funders:	EPSRC
Date of First Compliant Deposit:	17 June 2025
Last Modified:	17 Jun 2025 14:29
URI:	https://orca.cardiff.ac.uk/id/eprint/179109

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