Force volume and stiffness tomography investigation on the dynamics of stiff material under bacterial membranes { Giovanni Longo a *, Laura Marques Rio b , Charles Roduit a , Andrej Trampuz b , Alain Bizzini c,d† , Giovanni Dietler a and Sandor Kasas a† The determination of the characteristics of micro-organisms in clinical specimens is essential for the rapid diagnosis and treatment of infections. A thorough investigation of the nanoscale properties of bacteria can prove to be a fundamental tool. Indeed, in the latest years, the importance of high resolution analysis of the properties of microbial cell surfaces has been increasingly recognized. Among the techniques available to observe at high resolution specific properties of microscopic samples, the Atomic Force Microscope (AFM) is the most widely used instrument capable to perform morphological and mechanical characterizations of living biological systems. Indeed, AFM can routinely study single cells in physiological conditions and can determine their mechanical properties with a nanometric resolution. Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work, we exploit the capabilities and peculiarities of AFM to analyze the mechanical properties of Escherichia coli in order to evidence with a high spatial resolution the mechanical properties of its structure. In particular, we will show that the bacterial membrane is not mechanically uniform, but contains stiffer areas. The force volume investi- gations presented in this work evidence for the first time the presence and dynamics of such structures. Such information is also coupled with a novel stiffness tomography technique, suggesting the presence of stiffer structures present underneath the membrane layer that could be associated with bacterial nucleoids. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: AFM; stiffness; mechanical properties; stiffness tomography; bacteria; E. coli; nucleoids INTRODUCTION Bacterial infections constitute one of the most common public health problems causing increasing mortality and morbidity worldwide. In this framework, one of the most fascinating challenges for microbiology is to improve the knowledge of bacterial physiology with a high resolution. This will require the transition from an analysis of an entire population to a character- ization at the single-cell level. Indeed, up to now, the most common techniques used to identify and characterize the properties of bacteria (first of all their resistance to antibiotics) are based on the assumption that the tested organisms repre- sent a homogenous population in relation to their physiological and physical properties. Moreover, most techniques require long incubation times (even days) to obtain a detectable cellular density. Such incubations can last for days to reach a detectable cellular density (Turnidge and Paterson, 2007). More modern techniques, such as mass spectroscopy (matrix-assisted laser desorption ionization-time of flight) (Lay, 2001) or PCR-based detection systems (Poppert et al., 2005), deliver a faster identifi- cation of the strain and analysis of the bacterial characteristics and can be employed to perform bacterial genetic typing and to identify mutations of an entire population. On the other hand, an increasing attention is devoted to the determination of the properties of single bacteria through high resolution analysis. For example, several recent works have focused on the determination of the mechanical properties of * Correspondence to: Giovanni Longo, Laboratory of Physics of Living Matter, EPFL-IPSB-LPMV, BSP/Cubotron 414, CH-1015 Lausanne, Switzerland. E-mail: giovanni.longo@epfl.ch † These two authors contributed equally to the manuscript { 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 G. Longo, C. Roduit, G. Dietler, S. Kasas Laboratory of Physics of Living Matter, EPFL, Lausanne, Switzerland b L. M. Rio, A. Trampuz Infectious Diseases Service, Department of Medicine, University Hospital Lausanne (CHUV), Lausanne, Switzerland c A. Bizzini Institute of Microbiology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland d A. Bizzini University Hospital of Lausanne (CHUV), Lausanne, Switzerland Present address: C. Roduit, S. Kasas, Department of Cellular Biology and Morphology, University of Lausanne, Lausanne, Switzerland Abbreviations: PDMS, polydimethylsiloxane; PBS, phosphate buffered saline; LB, Lysogeny broth; APTES, 3-aminopropyltriethoxysilane; FD, force distance; FV, force volume. Research Article Received: 31 October 2011, Revised: 6 January 2012, Accepted: 10 January 2012, Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jmr.2171 J. Mol. Recognit. 2012; 25: 278–284 Copyright © 2012 John Wiley & Sons, Ltd. 278