Imaging unfixed cell membranes by atomic force microscopy

Due to their softness, cell membranes are difficult to imagine by AFM without chemical fixation or drying, Using the simple erythrocyte we present a procedure of tensing the erythrocyte membrane by spreading and immobilizing cells on a poly-L-lysine layer. This spreading tenses the upper membrane and generates a spherical cap, facilitating AFM imaging of the unfixed erythrocytes. Images were obtained in tapping mode at low scan rates avoiding damage of the membrane and proving highly reproducible. On both red cells and ghost cells indentation could be made deep enough to reach the steric core of the two membranes (upper and lower) giving a thickness of 42 +/- 15 nm (126 cells) under a maximum compression force of 2 nN. Detailed images of the erythrocyte membrane show stable corrugated structures about 7 nm in height, suggesting indentation of the membrane lipid bilayer down to the underlying cytoskeletal network.