Title Spatial distribution of lipid headgroups and water molecules at membrane / water interfaces visualized by three-dimensional scanning force microscopy

At biological interfaces, flexible surface structures and mobile water interact with each other to present non-uniform three-dimensional (3D) distributions. In spite of their impact on the biological functions, molecular-scale understanding of such phenomena has remained elusive. Here we show direct visualization of such interfacial structures with subnanometer-scale resolution by 3D scanning force microscopy (3D-SFM). We measured a 3D force distribution at an interface between a model biological membrane and buffer solution by scanning a sharp tip within the 3D interfacial space. We found that vertical cross sections of the 3D image taken along a specific lateral direction shows characteristic molecular-scale contrasts tilted at 30 ◦ †Membrane/Water Interfaces Visualized by 3D-SFM ∗To whom correspondence should be addressed ‡Bio-AFM Frontier Research Center, Kanazawa University ¶Division of Electrical and Computer Engineering, Kanazawa University §Bio-AFM Frontier Research Center, Kanazawa University 1 to the membrane surface. Detailed analysis of the 3D image reveals that the tilted contrast corresponds to the time-averaged conformation of fluctuating lipid headgroups. Based on the obtained results, we discuss the relationships among the hydration structure, headgroup fluctuation, molecular fluidity and mechanical strength of the membrane. The results demonstrate that 3D-SFM is capable of visualizing averaged 3D distribution of fluctuating surface structures as well as that of mobile water (i.e . hydration structure) at interfaces between biological system and water. At the interface between a biological system and its surrounding physiological solution, water molecules interact with biomolecules constituting the surface. Through the interaction, water molecules give significant influence on the structure and function of biomolecules and their assembly. 1–3 Therefore, understanding of the structure and function of a biological system requires investigations on the behavior of interfacial water. The surface of a biological membrane mainly consists of hydrophilic lipid headgroups. So far, the membrane/water interface has been extensively studied by various techniques. 4–11 These previous works have shown that the water molecules adjacent to a membrane strongly interact with the headgroups and give significant influence on its mechanical strength and fluidity. 12–14However, molecular-scale origin for such a grave influence has remained elusive. One of the major difficulties in such a study lies in the measurement of molecular-scale structure of a membrane/water interface. As the lipid headgroups exhibit thermal fluctuations, the surface structure of a membrane is inherently ill-defined. In addition, the fluctuating lipid headgroups interact with mobile water, through which the interfacial water presents non-uniform density distribution known as hydration structure. Thus, the understanding of the whole structure of membrane/water interface should require a method to visualize three-dimensional (3D) distribution of mobile water as well as fluctuating lipid headgroups. Atomic force microscopy (AFM) 15 has been widely used as a nanoscale surface imaging tool. In a typical AFM setup, a sharp tip is scanned in XY directions ( i.e. parallel to the surface) on a sample to produce a two-dimensional (2D) image of the surface topography. In contrast, several