AFM imaging artifacts due to bacterial cell height and AFM tip geometry

Atomic force microscopy (AFM) has become an important tool for investigating various biological materials, and it is now being applied more routinely for imaging bacteria. By imaging bacteria in water, AFM can provide in-situ images of viable cells and be used to measure interaction forces between the AFM tip (or a colloid probe) and the cell surface. However, the relatively large height and compliance of the bacterium can also lead to imaging artifacts. AFM images of Escherichia coli K12 were consistently found to contain image shadows that were oriented in parallel lines 27degrees from the direction of the cantilever tilt, regardless of the scan direction. Similar image shadows were also observed for 1 mum diameter polystyrene latex microspheres. Using a simple geometric model for the interaction of the tip and the bacterium, it is demonstrated here that these lines observed for bacteria are image artifacts produced by the pyramidal shape of the tip, the 10degrees tilt of the cantilever, and the height of the bacterium relative to the size of the tip. Such image artifacts disappear when we image dehydrated bacteria that are lower in height, or bacteria that become damaged and deflated during imaging in water. The interaction of the edge of the tip with the bacterium is also shown to result in inconsistent shapes of force curves unless the force curve is centered on the crest of the rounded bacterial surface.