Principle of Operation

AFM (Fig. 1) utilizes an ultrasharp microfabricated tip (Fig. 2A) mounted on the end of a flexible cantilever 100-250 ^m long. This is scanned across the sur-

Fig. 1. Schematic diagram of an atomic force microscope. The volume of a fluid-filled cell depends on the thickness of the O-ring and is approx 30-70 |L.
Fig. 2. (A) SEM image of the oxide sharpend Si3N4 AFM probe. (B) SEM image of a nanotube tip (arrow) attached to an Si cantelever. (C) TEM image of CVD grown nan-otube tip (arrow) on an Si cantelever. (B and C, courtesy of A. Noy, Lawrence Livermore Laboratory.)

face of a sample in a systematic raster manner, with the lateral and vertical movements of either the tip or the sample under piezoelectric positional control. Because of interactions between atoms on the surface of the sample and those on the tip, deflection of the cantilever takes place. The deflection of the cantilever is monitored with an optical beam deflection system, whereby a laser beam focused on the top of the cantilever is reflected onto a split photodiode. Cantilever deflections as small as 1-2 A can be readily measured and utilized to correct the tip-sample distance in order to maintain a constant imaging force.

Subsequently, the height corrections are used to construct a topographic image of the sample.

AFM can operate in either contact or tapping mode. In the former mode, the AFM probe remains in contact with the sample during the scanning, whereas in the latter mode the tip oscillates a few angstroms from the sample surface, tapping it only during a short interval in its oscillation cycle. In both tapping and contact modes, the feedback mechanism adjusts, through the piezoelectric positioner, the vertical height of the sample (or AFM probe) in order to maintain a constant amplitude of the oscillating probe (tapping mode), or deflection of the cantilever (contact mode).

In contact mode, lateral forces caused by probe-surface interactions typically complicate the imaging of soft biological samples. This often results in poor imaging or even the physical displacement of virions adsorbed on the substrate. In tapping mode, virtually no lateral forces are applied to the sample during imaging, which makes it the preferred approach to probe structures of virions and their disassembly at high resolution.

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