Resolving Structure and Mechanical Properties at the Nanoscale of Viruses with Frequency Modulation Atomic Force Microscopy David Martinez-Martin1., Carolina Carrasco1,2., Mercedes Hernando-Perez1, Pedro J. de Pablo1, Julio Gomez-Herrero1*, Rebeca Perez3, Mauricio G. Mateu3, Jose L. Carrascosa2, Daniel Kiracofe4, John Melcher4, Arvind Raman4 1 Departamento F´ısica de la Materia Condensada, Universidad Auto´ noma de Madrid, Madrid, Spain, 2 Centro Nacional de Biotecnolog´ıa, CSIC, Madrid, Spain, 3 Centro de Biolog´ıa Molecular ‘‘Severo Ochoa’’ (CSIC), Madrid, Spain, 4 School of Mechanical Engineering and Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana, United States of America Abstract Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25–50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called ‘‘dissipation channel’’ in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used. Citation: Martinez-Martin D, Carrasco C, Hernando-Perez M, de Pablo PJ, Gomez-Herrero J, et al. (2012) Resolving Structure and Mechanical Properties at the Nanoscale of Viruses with Frequency Modulation Atomic Force Microscopy. PLoS ONE 7(1): e30204. doi:10.1371/journal.pone.0030204 Editor: Markus J. Buehler, Massachusetts Institute of Technology, United States of America Received January 18, 2011; Accepted December 12, 2011; Published January 25, 2012 Copyright: 2012 Martinez-Martin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors acknowledge the financial support from projects S2009/MAT-1467 (CAM), MAT2010-20843-C02. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Julio.gomez@uam.es . These authors contributed equally to this work. Introduction Millions of years of evolution have converted viruses into nanomachines optimized to carry out complex functions with a minimalistic structure [1]. Viruses are masterpieces of nanoengi- neering with a basic common architecture that consists of the capsid – a protein shell made up of repeating protein subunits- which packs within it the viral genome. Beyond this basic architecture, viruses can have further elaborations such as protein collars, tails, connectors, lipid coats, surface receptors, enzymes, and molecular motors. With this simple structure, viruses are able to withstand tremendous internal pressure [2], osmotic shocks [3,4] and chemical agents while maintaining their viability. To the materials engineer or nanotechnologist, viruses are perfectly defined organic nanoparticles which are commonly used as scaffolds or nano-vectors [5]. Because their structure can be directed by tailored evolution and their production can be commercially viable, viruses are becoming the basis of whole new approaches to the manufacture of nanomaterials far beyond applications in biology and medicine. There has been a significant increase in the use of viruses for the template assembly of nanomaterials with applications from vaccines to electronics [6,7,8]. The extreme resistance of virus particles to the external media conditions has triggered the attention over the mechanical properties of viruses [9] and their relationship with their structure [10,11]. These studies have used in vitro AFM as the main tool to characterize the topography and the stiffness of the virus particles, a current hot topic in physical virology [12]. While AFM characterization of mechanical properties is usually carried out by nanoindentation [13] in this work we present a new method that allows obtaining, in addition to the topography, spatially resolved maps of flexibility. PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e30204