Imaging Viruses in Physiological Conditions Figure 3. Quantifying virus particle height: (a,c) are MVM virus particles showing the 5-fold symmetry. (b) is a MVM virus particle adsorbed along the 3-fold symmetry. Fig d-f are w29 bacteriophages. For each topography image we have included a profile that informs about the height of the virus particle that coincides with the expected height as obtained by SB techniques. For the sake of comparison we are showing the images with a simple top-view rendering with a pseudocolor table that encodes the particle heights. Figures 3a, 3d and 3e are the same as figs. 2c, 2f and 2e respectively. The calibrated cantilever stiffness for images 3a and d is 0.6 N/m, oscillation amplitude 1 nm, frequency shift about 25 Hz. The calibrated cantilevers stiffness for rest of the images was 1.8 N/m, the oscillation amplitude 1 nm and the frequency shift was about 30 Hz. All the images were acquired with 350 nm/s scanning speed. doi:10.1371/journal.pone.0030204.g003 shift, as it is used to fix the tip-sample separation and generate the topography image. In the virus images reproduced in figs. 2 and 3, the frequency shift is set at about 40 Hz. Imaging at a higher frequency shift can cause the virus particles to deform under the force from the tip or even become displaced or damaged. Also, although it is still possible to acquire images at a smaller frequency shift, where applied forces are extremely weak, the resolution is degraded because the tip is essentially probing the solvation layers at this point. To achieve optimum resolution the tip must penetrate these layers and start to graze the surface atoms of the sample. Near the grazing contact with the sample, the amplitude of the oscillation will be large compared to indentation of the sample and the probe will be operating in a nonlinear regime. In order to understand the contrast within drive amplitude images (also known as dissipation images in the literature) for a surface immersed in liquid, we start by scanning a bare graphite sample. Figures 5a and b show the topography and drive amplitude images obtained in this sample immersed in the same Figure 4. In liquid FM-AFM basis. (a) Experimental (solid line) and simulated (dashed line) average deflection (mean force) of the cantilever as the tip approaches the sample surface. The horizontal axis is the piezo elongation. (b) Experimental (solid lines) and simulated (dashed lines) frequency shift and drive amplitude as the tip approaches de sample surface. The horizontal axis is the piezo elongation. All the magnitudes in (a) and (b) were acquired simultaneously. Oscillation amplitude 1.2 nm., calibrated spring constant of 0.6 N/m. doi:10.1371/journal.pone.0030204.g004 PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30204