In vitro AFM is a powerful technique that provides images of biological specimens with a resolution far beyond optical microscopy. AFM can achieve molecular or even atomic resolution [14] in the classical contact mode when applied to hard samples where the friction tip-sample force is not very relevant. The so called purple membrane is a good example of a bio-sample that can be scanned with molecular resolution using contact mode [15]. On the contrary, small virus particles (25– 50 nm), which are particular but very relevant type of weakly attached (to a substrate) samples, cannot be scanned in contact mode. In fact, amplitude modulation (AM-AFM) (also known as Tapping ModeTM) imaging of small particles turns out to be a very demanding task that in many cases requires special types of cantilever [16]. AM-AFM is the most widespread dynamic AFM mode that is present in almost every commercial AFM microscope [17]. Jumping mode, [18] an AFM technique that is midway between contact and AM-AFM, is less restrictive, but again the cantilever stiffness has to be low, otherwise the residual lateral forces [19] can still move the virus particles. Larger size viruses, as is the case of the Giant Mimivirus [20], can be scanned using AM- AFM with good resolution. Nevertheless, the lateral forces in a 750 nm virus particle (well above the tip size) are not very relevant and scanning this type of sample is similar to scanning the above described membranes. The restrictions observed in both jumping mode and AM-AFM methods, as we shall see, can be overcome using FM-AFM. Figure 1 outlines the basic features of FM-AFM. A cantilever is oscillated at its resonance frequency, which changes as the tip approaches the surface due to the tip-sample interaction. This magnitude, usually referred to as the frequency shift, is the main control parameter in FM-AFM. For small oscillation amplitudes (linear regime), the frequency shift is proportional to the force gradient (stiffness) of the tip-sample interaction. The topography of the sample is obtained by adjusting the tip sample distance (by means of the z piezoelectric actuator) to track a certain resonance frequency. Therefore a topography image in FM-AFM, in the linear regime, is a map of constant force gradient (dashed line in fig. 1). More generally, for larger oscillation amplitudes (nonlinear regime), the FM-AFM topography image is a more complex mapping of the conservative interaction potential [21]. An additional feedback loop is used to keep the cantilever oscillation amplitude constant at the drive frequency by adjusting the drive amplitude. The record of the drive amplitude over the course of a scan provides a second image where variations in the drive amplitude are proportional to the energy dissipated by nonconser- Figure 1. In liquid FM-AFM basis. Artistic draw of a virus particle immersed in a buffer solution and adsorbed on a surface. The colored environment represents the total gradient of short and long-range interactions between the cantilever tip and the sample. FM-AFM follows the constant gradient lines to get the sample topography. doi:10.1371/journal.pone.0030204.g001 Imaging Viruses in Physiological Conditions vative tip-sample interactions (viscoelasticity, friction, hysteretic tip-sample forces). The drive signal image is known as a compositional map, because, unlike the frequency shift, the drive signal is free to vary during the course of a scan according to the composition of the sample. Please observe that the macroscopic radius of the tip is not directly related with the final resolution of the probe but with the decay length of the interaction. In AFM there are both long and short range interactions, both of them can be used to obtain the topography but only short ones provide atomic resolution. FM-AFM provides a way to access these short range interactions. Results Figure 2a shows an atomically resolved image of a mica surface in a buffer solution [22] and serves as a benchmark of the performance of our system when working in FM-AFM. We take this result as the upper bound for the resolution of FM-AFM that is not reached by the other AFM working modes. Figure 2b is a view of the parvovirus minute virus of mice (MVM), oriented with a 5- fold symmetry axis on top as obtained from a molecular surface model derived from X-ray data [23]. According to the data provided by this technique, the parvovirus capsid can be inscribed into a 25 nm diameter sphere and it is formed by 60 structurally equivalent subunits arranged in a simple (T = 1) icosahedral symmetry. Figure 2c is a FM-AFM topographic image of a MVM particle adsorbed on a functionalized glass surface. Most of the structural features in fig. 2b (including the pentameric arrange- ment of the structural capsomeric units) can be readily observed in fig. 2c where features as small as 1 nm can be discerned in the image. Figure 2d shows a three-dimensional volume reconstruc- tion of the w29 viral particle derived from a large set of images taken by Cryo-EM [24]. It has a prolate head around 55 nm long, with an equatorial diameter of about 45 nm, which presents a tail structure in a singular vertex. It is important to note that in this case, as in the case of X-ray data, the features observed in the specimen are the result of a reconstruction process that hide singular features of individual viruses, as these methods are based on heavy averaging to increase the periodic common features of these specimens. In order to make a more objective estimation of the degree of similarity with AFM images, the cryo-TEM derived volume was subjected to a dilation using a tip of 0.2 nm [25] that takes into account the finite dimensions of the tip apex. Figures 2e and 2f are FM-AFM images of two different w29 virus. In these images, the three main domains of the virus particle are clearly defined: the prolate head, the collar region and the tail. Besides, fine structural features, as the corrugation of the capsid due to the capsomeric subunit arrangement and, the periodic aspect of the collar due to the presence of the collar appendages can be also clearly seen. Furthermore, a number of individual details are captured in the images, for instance the virus in fig. 2e has the tail bent, probably due to distortions derived from the adsorption process, while the fig. 2f shows the presence of two particular protrusions on the surface of the capsid. As they are located at the interface of the equatorial region with the icosahedral caps, it is tempting to suggest that they might be derived from some kind of aggregate of the virus fibers, but we cannot exclude that they could be caused by small particles floating in the buffer solution that were finally adsorbed onto the virus surface. Also, it is important to note that in the distal part of the cylindrical tail extension there is a cone-shaped knob that has been previously described in some cryo-EM reconstructions of w29 viral particles prepared under specific conditions, that might be related to the release of the DNA from the capsid [26]. PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e30204