Figure 5

Mechanical mapping using atomic force microscopy. A) In mechanical mapping measurements, the tip oscillates in the vertical direction while the tip scans the sample surface (xy plane), generating force versus distance curves at each pixel of the image. In the approaching trace (gray line), the tip approaches the sample until it makes contact (change in slope) reaching the force setpoint, which determines the topography of the sample. If the sample is soft, the tip deforms it as force increases. The tip then retracts back until the initial position is reached (black line). The shape of the curve during contact depends on the contact geometry between the tip and the sample and allows estimating the elasticity of the sample. The sketch represents the setup with the AFM tip deforming the bacteriorhodopsin sample. The actual size of the tip is much larger than the protein. Actually, AFM tips are a few microns high and the radius of the sharp apex of a few nm. B) Topography and elasticity maps of bacteriorhodopsin obtained from mechanical mapping using AFM, revealing the trimeric organization in 2D hexagonal arrays. C) Average images of trimer repeats from B (same colour scale). Overlapped is the top view crystal structure of the bacteriorhodopsin trimer coloured by B-factors. Data replotted from ref. [31] using a tip of ~2 nm radius.