153 Dylan M. Owen (ed.), Methods in Membrane Lipids, Methods in Molecular Biology, vol. 1232, DOI 10.1007/978-1-4939-1752-5_13, © Springer Science+Business Media New York 2015 Chapter 13 Measuring Cytoskeleton and Cellular Membrane Mechanical Properties by Atomic Force Microscopy Charles Roduit, Giovanni Longo, Giovanni Dietler, and Sandor Kasas Abstract Atomic force microscope is an invaluable device to explore living specimens at a nanometric scale. It permits to image the topography of the sample in 3D, to measure its mechanical properties and to detect the pres- ence of specific molecules bound on its surface. Here we describe the procedure to gather such a data set on living macrophages. Key words AFM, Macrophages, CD40 ligand, Stiffness tomography, Chemical mapping 1 Introduction Almost all cellular physiological functions depend on the mechani- cal properties of diverse cellular components [1, 2]. The filamen- tous 3D networks of the cytoskeleton play an important role in the mechanical properties of cells. It permits cellular motion, intracel- lular displacement of organelles and plays a cornerstone role in cell division. The function of this highly organized network mostly depends on its mechanical properties that are unfortunately still very poorly known today. Similarly, the mechanical properties of lipid membranes that delimit cells and organelles play an important role in numerous cellular processes. Beside physiology, recent stud- ies have pointed out that cellular mechanical properties are strongly modified in numerous pathological conditions such as cancer [3, 4] or muscular dystrophy [5] opening novel avenues to innovative diagnostic tools or complementary investigation techniques. The exploration of the cytoskeleton and lipid membrane structure is possible nowadays by means of various optical or electronic micros- copy techniques. While these instruments can reveal very precisely the 3D structure and the organization of the cytoskeletal filaments and cellular membranes, they have no access to their mechanical properties and often are usable only in non-physiological condi- tions. This type of investigation domain is therefore addressed by