Development and characterisation of synthetic model lipid membranes under linear and non-linear microscopy

• Main Author: McPhee, Craig
• Published: Cardiff University 2016
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.742793

Lipid domains provide a framework for localised functionality of the cellular membrane through transient coordination of certain lipids and membrane proteins into structurally distinct, stabilised heterogeneous membrane regions. Present experimental studies fall short of conclusively proving lipid domain existence within the plasma membrane due to the lack of label-free, chemically sensitive nanoscale detection. Herein, I present my progress towards developing novel, label-free optical microscopy techniques to over- come these limitations. Giant unilamellar vesicles (GUVs) represent a simple model of cellular membranes and are well suited for the study of lipid domains. In this thesis, I discuss the demonstration of a novel, label free method to directly assess GUV lamellarity: Quantitative differential interference contrast microscopy (qDIC). Under qDIC, a contrast image is produced which encodes the difference in optical phase (hence optical path length) after propagation through two adjacent points of the sample. I show that, with appropriate data analysis applied to qDIC contrast images, we are able to measure membrane lamellarity directly with sub-nm precision. I then demonstrate the application of this method to static synthetic membranes exhibiting lipid domains: Planar Lipid Bilayer Patches (PLBPs). Sub-nm thickness differences (∼9Å) attributable to coexisting lipid domains are resolved and quantified. Overall, these results demonstrate that label free qDIC is a rapid, non-perturbing, sensitive and accurate method, providing an alternative to fluorescence microscopy, for quantitative studies of lipid domains in model membranes. Furthermore, I discuss correlative qDIC and Coherent Anti-Stokes Ra- man scattering microscopy (CARS) of PLBPs with lipid domains. CARS microscopy has emerged in the last decade as a powerful, chemically specific multi-photon imaging method which overcomes the sensitivity and speed limitations of spontaneous Raman scattering, and enables rapid quantitative analysis of lipids label-free. I demonstrate application of broadband hyper-spectral CARS imaging over the CH 2,3 stretching vibrational resonances, combined with in-house developed phase-corrected Kramers Krönig (PCKK) analysis, which allowed us to resolve and quantify the chemical components of lipid domains at the single bilayer level. Stimulated Raman loss (SRL) microscopy is an alternative, chemically specific, non-linear imaging modality recently implemented within our research group. In contrast to CARS microscopy, SRL rejects non-resonant background providing high contrast imaging of single lipid bilayers comparable to fluorescence imaging. I demonstrate early application of SRL at the single bilayer level across the CH 2,3 stretch region. During this project a number of notable achievements have been made. A novel qDIC method has been developed and utilised. CARS microscopy has been applied to determine lipid liquid phase at both single frequency and hyper-spectral imaging modalities. SRL microscopy has then been applied, demonstrating superior contrast to that seen under CARS. These studies form the foundation for further chemically specific investigation.