Stiffness tomography exploration of living and fixed macrophages † C. Roduit a *, G. Longo b , I. Benmessaoud b , A. Volterra a , B. Saha c , G. Dietler b and S. Kasas a,b Stiffness tomography is a new atomic force microscopy imaging technique that allows highlighting structures located underneath the surface of the sample. In this imaging mode, such structures are identified by investigating their mechanical properties. We present here, for the first time, a description of the use of this technique to acquire detailed stiffness maps of fixed and living macrophages. Indeed, the mechanical properties of several macrophages were studied through stiffness tomography imaging, allowing some insight of the structures lying below the cell’s surface. Through these investigations, we were able to evidence the presence and properties of stiff column-like features located under- neath the cell membrane. To our knowledge, this is the first evidence of the presence, underneath the cell membrane, of such stiff features, which are in dimension and form compatible with phagosomes. Moreover, by exposing the cells to cytochalasin, we were able to study the induced modifications, obtaining an indication of the location and mechanical properties of the actin cytoskeleton. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: stiffness tomography; AFM; macrophage; mechanical properties; cytoskeleton INTRODUCTION The atomic force microscope (AFM; Binnig et al., 1986) has initially been developed to image non-conductive samples with an atomic resolution. Basically, the instrument consists in a very sharp tip (less than 10 nm of apical radius) fixed at the end of a soft cantilever and kept at a very small distance from the sample’ s surface (1–2nm). When, using piezoelectric actuators, such tip is moved over the sample, the surface features deflect the cantilever and such small movements are detected through a laser beam reflected off the cantilever’ s end. The laser beam ends its path on a two or four segment photodiode that converts the cantilever’ s deflection in a voltage that is proportional to the sample’ s height at the probed spot. This information is eventually collected by a computer and displayed on a screen reconstructing the sample’ s topography in 3D with a lateral resolution of some nanometers. Soon after the invention of the microscope, it appeared clear that the instrument could indifferently be operated in air, vacuum or liquid. Indeed, this made the instrument very popular among biol- ogists who wanted to exploit its high-resolution working in physio- logical buffer (Hansma et al., 1988; Kasas et al., 1993; Dufrene, 2001). Indeed, the interaction between the AFM and the field of biology has been particularly fruitful, opening the way to inter- esting and unique investigation techniques capable of studying force and adhesion, protein–protein interactions or even protein unfolding. Particularly interesting is the use of the AFM to measure with high spatial and force resolution the mechanical properties of soft samples. This type of measurement is performed by indenting (i.e. pushing) the tip into the sample and by recording the deflection of the cantilever during the process (force curve) (Tao et al., 1992). By estimating the shape of the tip (typical AFM tips have an apex whose properties can be approximated to a sphere or a cone of known radius of curva- ture and opening angle) and that the cantilever deforms as a perfect spring (i.e. that the force it exerts on the sample is directly proportional to its deformation), it becomes possible to calculate the mechanical properties of the sample. To perform such calculation, the curve that relates the tip’ s indentation to the force exerted by the cantilever is fitted with theoretical models such as those of Hertz or Sneddon (Sneddon, 1965) to obtain the Young’ s modulus of the sample. This modulus reflects the mechanical properties and indicates the extent of sample deformation under an applied load. By repeating the measure- ment all over the surface, a complete stiffness map of the sample, in which each pixel is indicated through its Young’ s modulus, can be reconstructed. This imaging mode has been widely applied to explore the mechanical properties of living cells in nearly physiological conditions (Weisenhorn et al., 1993; Radmacher, 1997; Kasas et al., 2005; Berquand et al., 2010; Mostowy et al., 2011). Recently, we developed a new method to analyze indentation curves (ICs), named stiffness tomography (Roduit et al., 2009). This technique allows identifying the pres- ence of structures located underneath the surface of the sample * Correspondence to: C. Roduit, Département de Biologie Cellulaire et de Morphologie, Université de Lausanne, CH-1015 Lausanne, Switzerland. E-mail: charles.roduit@a3.epfl.ch † This article is published as part of the AFM BioMed Conference on Life Sciences and Medicine, Paris 2011 of the Journal of Molecular Recognition, edited by Simon Scheuring, Pierre Parot and Jean-Luc Pellequer. a C. Roduit, A. Volterra, S. Kasas Département de Biologie Cellulaire et de Morphologie, Université de Lausanne, CH-1015, Lausanne, Switzerland b G. Longo, I. Benmessaoud, G. Dietler, S. Kasas Institut de Physique des Systèmes Biologiques, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015, Lausanne, Switzerland c B. Saha National Centre for Cell Science, Ganeshkhind, Pune 411007, India Research Article Received: 31 October 2011, Revised: 21 February 2012, Accepted: 22 February 2012, Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jmr.2184 J. Mol. Recognit. 2012; 25: 241–246 Copyright © 2012 John Wiley & Sons, Ltd. 241