Composition-Dependent Alterations in Thickness and Physical Properties of Lipid Bilayer FILM Revealed by Frequency Modulation Atomic Force Microscopy

Abstract

Assembly of microdomains and membrane viscosity due to these physical properties of membrane have been highlighted as important factors supporting proper functions of membrane proteins. Although advanced imaging technologies including scanning probe microscopy and super-resolution optical microscopy have revealed dynamics of microdomains and visco-elastic properties of cell membranes, it is still difficult to link modifications of nanoscale membrane structure and specific diseases. In this study, we aimed to obtain high-resolution structural information of supported planner membrane with different fatty acid lengths using liquid dynamic-mode AFM and FM-AFM (Frequency Modulation Atomic Force Microscope). In FM-AFM, the cantilever is moved in a non-contact state, maintaining the cantilever frequency shift (Δf) is constant. This enables a highly sensitive force detection, 20 times better S/N than with existing methods, thereby markedly improving the image resolution. We used supported phosphatidylcholine membranes with fatty acids ranged from PC 12:0 to PC 22:6. For saturated PCs, membrane thickness increased along with the number of carbon from 4.32 nm (PC12:0) to 6.37nm (PC18:0), but diarachidoylphosphatidylcholine (PC20:0) showed reduced membrane thickness: PC20:0 thickness had 6.09 nm, that is ≈0.3 nm thinner than PC18:0. Unsaturation of fatty acid further reduced membrane thickness to 4.12 nm for PC20:4, suggesting that structural arrangement of PCs with fatty acid chain length and unsaturation level occurred. These data demonstrated that our imaging modality can detect membrane thickness with minimal membrane compression by scanning probe while maintaining high detection sensitivity. We will also present modulations of mechanical properties of membrane in the functions of carbon chain length and the level of unsaturation.