Imaging Viruses in Physiological Conditions Figure 5. Adhesion force as a source of dissipation. (a) Bare HOPG topography taken in FM-AFM in the same buffer solution used for the MVM viruses. (b) Dissipation map simultaneously taken with a. (d) and (e) are 2 force distance curves taken in the bright and dark regions of the dissipation respectively. As can be seen from the curves there is a clear correspondence between adhesion and dissipation. Cantilever oscillation amplitude 1.0 nm and frequency shift 35 Hz, scan speed 350 nm/s, Resonance frequency 21 KHz, calibrated spring constant of 0.6 N/m. (c) Simulation of the drive signal versus elastic modulus and adhesion. For a given sample with a homogeneous elastic modulus, the drive signal increases as the adhesion gets higher. doi:10.1371/journal.pone.0030204.g005 buffer solution used for imaging the MVM virus. The drive amplitude image shows regions with different contrast. In traditional FM-AFM, this contrast in drive amplitude would indicate that the different sample regions had differing amounts of tip-sample dissipation (non-conservative tip-sample interactions). In the present case we do not expect that there should be any difference in dissipation between the regions. However, by acquiring force vs. distance static curves (figs. 5d and e) it can be easily shown that these regions exhibit very different conservative adhesion force (probably due to different van der Waals forces arising from the adsorption of molecules from the buffer solution) but the stiffness, given by the slope of the curve near the contact region, is the same. In particular, the darkest regions show almost zero adhesion force. Therefore, we conclude in this case that the drive amplitude is correlated to the conservative van der Waals forces. Figure 5c shows a computer simulated surface map of the drive signal as a function of the sample stiffness and the adhesion force that confirm the variation of the drive signal for regions with the same stiffness but different adhesion force. Before discussing the reason for this curious result, we provide a second example. Figure 6a and d displays FM-AFM topographies, each of them with two MVM virus particles, adsorbed on a graphite substrate with negligible adhesion along the 5 fold symmetry (upper particle) and 3 fold symmetry (lower particle). It is know that full MVM virus particles (containing genetic material) present anisotropic stiffness. By performing individual indentation curves on the particles it has been found stiffness of 0.6 N/m and 0.8 N/m for the 5 and 3 fold symmetry respectively [10,11]. Figures 6b and e are the corresponding drive amplitudes maps simultaneously acquired with the topography images. There is an obvious striking correspondence between the variations of this magnitude and what it is expected for the virus stiffness. Figure 6f despites the corresponding profiles across the two virus particles observed in fig. 6a. As can be seen the topographical height of both viruses is basically the same indicating the absence of cross talking between the topography and the compositional map images. This is also the case for fig. 6d. In order understand this correspondence we have simulated the variation of the driving signal as a function of the sample stiffness (elastic modulus) and its viscosity (assuming a sample with low adhesion). The drive signal is actually invariant with respect to viscosity except in the combination of extremely low elasticity and high viscosity. For moderate to low viscosities, as expected for virus particles, the driving signal is monotonically increasing with respect to the elastic modulus. PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e30204