Imaging Viruses in Physiological Conditions Figure 2. High resolution FM-AFM. Seeing individual features. (a) Image of a muscovite mica surface (001) showing true atomic resolution. The mica was immersed into a physiological buffer to ensure that under these conditions we are able to obtain true atomically resolved images. Parameters of the image: cantilever oscillation amplitude 0.5 nm and frequency shift 36 Hz, scan speed 800 nm/s, Resonance frequency 130 KHz, nominal spring constant 40 N/m. (b) View of a MVM particle oriented with a 5-fold symmetry axis on top as obtained from a molecular surface model derived from crystallographic data [36] as rendered in [11]. The capsid diameter is 25 nm. (c) FM-AFM topographic image of an individual MVM particle in the same orientation shown in (b).Virus particles were adsorbed on a glass surface immersed into a physiological buffer (PBS, 137 mM NaCl, 2.7 mM KCl, 1.5 mM NaH2PO4, 8.1 mM KH2PO4). Parameters of the image: cantilever oscillation amplitude 0.9 nm and frequency shift 30 Hz, speed 350 nm/s, Resonance frequency 19 KHz. Calibrated spring constant of 0.6 N/m. (d) Cryo-EM model of the w29 bacteriophage [24] (equatorial diameter ,45 nm) after a dilation process using a size tip of 0.2 nm. Reconstruction techniques are extremely precise but require the merging and averaging of a large set of images of individual viruses and, consequently, they tend to neglect individual particle features. (e) FM-AFM topographic image of an individual w29 particle in TMS buffer (50 mM Tris pH7.8, 10 mM Mg Cl2, 100 mM Na Cl). This virus presents a damaged tail (see white arrow). Parameters of the image: cantilever oscillation amplitude 1.0 nm and frequency shift 31 Hz, scan speed 350 nm/s, Resonance frequency 35 KHz, calibrated spring constant of 1.8 N/m. (f) Another w29 virus particle with a clear and well resolved tail but with some material bound to its head, forming extra protrusions. Parameters of the image: cantilever oscillation amplitude 1.0 nm and frequency shift 25 Hz, scan speed 350 nm/s, Resonance frequency 19 KHz calibrated spring constant of 0.6 N/m. doi:10.1371/journal.pone.0030204.g002 Image processing is an important feature for EM and crystallography. While this is also the case for AFM the image processing required is less intensive. In fig. 2 we have chosen a projection rendering of the virus images because an adequate lightning tends to enhance the virus edges. Figure 3 a,d,e depicts the virus images shown in fig. 2 c,e,f but just with a pseudocolor table that represents the topographical height. In addition we have added 2 new images for the MVM (fig. 3b and c) and one more for the w29 (fig. 3f). In order to quantify the virus height for each topography image we show the corresponding line profile. An outstanding feature of FM-AFM is its capability to image soft samples with a variety of cantilevers. We obtained similar images to these shown in fig. 2 and 3 with cantilever stiffness ranging from 0.6 N/m up to 40 N/m (see image supporting information fig. S1). On the contrary, acquiring AFM images with Jumping mode or AM-AFM requires cantilevers with very low stiffness. This feature can be very relevant for many applications. For instance, in order to achieve the best sensitivity when measuring mechanical properties, a cantilever stiffness as similar as possible to the sample stiffness should be used. Discussion A key feature of FM-AFM in vacuum and ambient environ- ments is that the response to conservative and non-conservative (dissipative) tip-sample interactions are decoupled in the frequency shift and the drive signal, respectively. Consequently the topography image is a reflection of purely conservative interactions while the drive signal measures the nonconservative. For this reason, the drive amplitude channel is usually referred to as the ‘‘dissipation’’ channel in FM-AFM. As we shall see, this decoupling is not generally the case in liquids and ‘‘drive amplitude’’ and ‘‘tip- sample dissipation’’ are not synonymous. The continuous line in fig. 4a is the experimental cantilever average deflection as a function of the tip sample distance (the magnitude that is accessible in the experiments is the scanner elongation along the z direction, that is the variation in the tip sample distance (absolute positions always require a theoretical model). At large z distance the average force is zero and as the tip approaches the sample it first growths with a smooth elbow and then linearly. This experimental curve can be nicely reproduced by assuming the presence of solvation layers [27], at forces of about 20 pN, that introduce a weak interaction before the mechanical contact between the tip and the surface. Importantly, the hard tip-surface contact occurs at forces below 100 pN, right at the end of the elbow region where we have set the origin for the z scale. The optimum image conditions are found at these forces, all the images in the manuscript are taken in the range of 40– 80 pN. The dashed line shows a simulated curve in which a simple conservative model for both the solvation layer and sample forces is incorporated (see Text S1 for full details). In fig. 4b we are representing the frequency shift (blue line, left scale) and the cantilever driving amplitude (green line, right scale). The average cantilever deflection, the frequency shift and the cantilever driving amplitude has been simultaneously acquired in a single z excursion. As in fig. 4b continuous lines are for the experimental results and dashed lines are the result of simulations. The central quantity in FM-AFM experiments is the frequency PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e30204