Imaging Viruses in Physiological Conditions Figure 6. Stiffness as a source for dissipation. (a) and (d) FM-AFM topographies showing, each of them, two virus MVM particles containing genetic material. In both images the particles have different spring constant since they are sitting on different fold-symmetries [10,11]. (b) and (e) Drive amplitude maps taken simultaneously with a and d. Since the adhesion is negligible, the compositional map is only plotting the elastic modulus. As expected for the different fold-symmetries, the virus showing the 5 fold-symmetry has lower stiffness than the other one which is showing the 3 fold-symmetry. Furthermore the viruses are softer than the substrate. (c) Simulation of the drive signal versus elastic modulus and viscosity. If the adhesion is close to zero, the drive signal is basically the elastic modulus of the sample, with the exception of samples that could have an extremely high viscosity orders of magnitude higher that the expected for virus particles. Fig c despites the corresponding profiles across the two virus particles observed in fig. 6a. Cantilever oscillation amplitude 1.0 nm and frequency shift 32 Hz, scan speed 350 nm/s, Resonance frequency 21 KHz, calibrated spring constant of 0.6 N/m. doi:10.1371/journal.pone.0030204.g006 The implication is that drive amplitude images in liquids are not related to tip-sample dissipation, as in air or vacuum conditions, but maps of the short range conservative mechanical properties (local elasticity, conservative van der Waals forces, conservative electrostatic forces, etc.) of the surface with resolution at the nanometer scale. One reason for the difference in the FM-AFM drive signal images in liquid compared to vacuum environments can be traced to recent findings in AM-AFM in liquids [28,29]. In the absence of tip-sample interactions, the probe oscillates with a pure tone at the drive frequency. However, when the probe encounters nonlinear interactions from the sample, the oscillation waveform is populated with higher frequency content (higher harmonic distortions and even contributions from higher vibrational eigenmodes of the probe). The energy that is supplied to the probe from the drive is partially dissipated into the liquid medium by the higher frequency components of the oscillation, even when the cantilever is driven at small oscillation amplitude (,1 nm). Although this higher frequency content is always present in dynamic AFM to some extent, it is only when operating in liquids that the energy dissipated by higher frequency oscillations can be much larger than the energy dissipated by nonconservative tip-sample interactions [29]. This is because the lower quality factors in liquid enhance the energy transfer between eigenmodes. Unlike the tip-sample dissipation, the energy dissipated in the higher frequency oscillations can be result from purely conservative interactions with the sample [29]. The result is that the drive signal image acquired with FM-AFM can show contrast from purely conservative interactions. This is precisely the result found in both the experiments and simulations shown in Fig. 3, where the drive signal shows variations in conservative component of the interaction such as the stiffness of the virus or the conservative van der Waals force. As shown above FM-AFM has a number of advantages over other more standard AFM modes. We are sure that it will soon become the standard AFM technique used to image weakly attached soft samples immersed in buffer solution with high resolution. Materials and Methods AFM The data were taken using a Nanotec Electronics system. The data acquisition and processing was carried out with WSxM [30]. The cantilevers used for the fig. 2c, f and 3a and d are Olympus OMCL-RC800PSA with a nominal force constant of 0.75 N/m. The topographic image of figs. 2e, 3b, c, e and f were acquired with Nanosensors PPP-FMR with a nominal force constant of PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e30204