Dynamic Imaging of Lipid Membranes by Means of Water

Lipid membranes are self-assembled structures whose composition determines the properties of membranes of cells and organelles. The molecular level understanding of lipid membranes is based on spectroscopy and MD simulations of lipid monolayer systems. As spectroscopy rely on spatial and temporal averaging and are necessarily linked to mean field models, information about the molecular interactions and their spatiotemporal evolution in real membranes is currently unavailable. In this thesis, we use high throughput wide-field second harmonic (SH) microscopy to image water-membrane interactions at sub-second time scale to follow the spatiotemporal evolution of membranes in freestanding membranes.

We improve the throughput of nonlinear SH microscope by 2-3 orders and integrate the apparatus of freestanding membranes for simultaneous electrical and optical characterization of membranes. We show that the non-resonant response of water can be SH imaged on sub-second time scales and use this response as a contrast mechanism to image membrane dynamics.

We investigate how oil redistributes within bilayers after formation. Using SH imaging, there is less oil present within bilayers prepared with hexadecane versus squalene. Diffusion of excess hexadecane droplets within bilayer follow directed diffusion whereas squalene show no directed motion. Hexadecane can diffuse within a single leaflet whereas squalene span both leaflets and more branched, couples to both leaflets and moves slower.

We probe the diffusion of charged lipid domains at sub-second time scale and construct electrostatic potential maps of asymmetric membranes. The average membrane potential is quadratic to an applied external bias, modeled by nonlinear optical theory. We observe fluctuations in the membrane potential of ~100 mV implying that membranes are dynamic in structure and in their potential landscape.

We probe the interactions of divalent cations with water and negatively charged membranes and show that Ca2+, Ba2+ and Mg2+ induce short-lived ( Mg2+ > Ba2+, for all quantities. We quantify KD and observe domain values that deviate up to 4 orders of magnitude from average KD. The transient domains exhibit potential fluctuations of up to -386 mV (dG = 28.6 kT), induce strain in the membrane resulting to fluctuations in membrane curvature.

We SH image the opening and closing of voltage-gated alamethicin ion channels in freestanding membranes. The SH intensity is due to changes in the orientational distribution of water molecules induced by electric field gradients. Only 0.01 % of the transported ions arrives at the membrane interface, leading to interfacial electrostatic changes on the time scale of a second. We quantify the distribution of ion channels and obs