Making and holding an artificial lipid bilayer horizontally in an aqueous solution within the microscopic working distance of similar to 100 mu m are essential for simultaneous single molecule imaging and single ion-channel electrical current recording. However, preparation of such a lipid bilayer without a solid support is technically challenging. In a typical supported lipid bilayer, the asymmetric local environments and the strong perturbation of the underneath solid or dense surface can diverge the normal behavior of membrane proteins and lipids. On the other hand, the suspended lipid bilayers can provide a native local environment for the membrane proteins and lipids by having fluids on both sides. In this technical report, we present a simple and novel methodology for making a suspended lipid bilayer that can be used for recording the single-molecule diffusion and single ion-channel electrical measurements of ion-channel proteins. Our approach has a higher validity for studying the molecular diffusions and conformational fluctuations of membrane proteins without having perturbations from supporting layers. We demonstrate the feasibility of such an approach on simultaneous single-molecule fluorescence imaging and electric current measurements of ion channel proteins
