Quantitative Mapping of Free-Standing Lipid Membranes on Nano-Porous Mica Substrates

The physic-chemistry of biological membranes is at the origin of fundamental cellular functions such as vesicle trafficking, cell adhesion and migration1-3. Because most of intracellular shapes and local demixing of membranes take place in the nanometer scale, AFM becomes an extremely powerful technique to assess the properties of these biological membranes. Porous substrates provide an elegant strategy to avoid the conundrum of placing soft and thin biomembranes on hard substrates for AFM studies, although the surface chemistry make the actual substrates rather challenging setups. Here, we have engineered porous systems on the most widely used substrate in AFM, mica muscovite, with tunable pore sizes from some tens to few hundreds nanometers for biological applications. We show that free-standing bilayers on nano-porous can be obtained by using well-established vesicle spreading methods and that they display equivalent nano-mechanical stability and phsyco-chemical properties to that of membranes on conventional mica supports. By reducing the pore radius < 40 nm and limiting the contribution of membrane tension to the elastic response of free-standing membranes we estimate a bending modulus of 18 kbT and 73 kbT for DOPC and DPPC bilayers, respectively. The quantitative mapping of suspended membranes shows a different mechanical response at the pore rims, which is more pronounced for DPPC and suggest a different lipid ordering. We find that the combination of membrane bending and the different lipid packing at the edges of pores shapes the curvature of free-standing membranes on pores in the range of few tens of nm.