Atomic force microscopy (AFM)

About this technique

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Atomic force microscopy (AFM) is the technique of choice to provide nanoscale structural and mechanical information. It can be applied to virtually any sample and experiments can be carried out in a variety of environments. In addition to topographical information about a sample, interaction forces between the substrate and a probe can be measured, potentially elucidating adhesion, magnetic and electronic forces. It can also used to measure intermolecular forces, including the forces that govern lubrication, cell adhesion and colloidal interactions such as electrostatic repulsion and van der Waals adhesion. Experiments in liquid enable biological samples such as cells and proteins to be imaged giving quantitative topological data and potentially insights into interaction sites.

The AFM operates by scanning a sharp tip across a sample surface. The tip is typically a pyramid or conical in shape and is four to five microns in height with a diameter at the apex of 10 to 20 nm. It is positioned at the end of a cantilever, which is typically 100 to 200 mm long. This probe is usually made from silicon or silicon nitride with the cantilevers' spring constants ranging from 0.05 to 50 N/m depending on the AFM mode of operation being employed. The tip or surface is raster scanned using a piezoelectric control mechanism that allows the AFM to acquire an image in three dimensions. AFM can be operated in the imaging mode, which allows an image of the substrate to be collected or you can operate in the force spectroscopic mode where the deflection of the cantilever is monitored as it is moved towards and away from the surface in question. Spectroscopic mode measures what are known as force distance curves and provides information about sample material properties. This can be used to measure adhesive forces, determine bond strengths and map local elastic properties. There are numerous imaging modes for the AFM but the two main modes are contact mode and tapping mode.

Contact-mode AFM operates by scanning the tip across the sample surface while monitoring the change in cantilever deflection. By maintaining a constant cantilever deflection, the force between the tip and the sample remains constant. Spring constants usually range from 0.01 to 1.0 N/m, resulting in forces applied to the surface ranging from nN to μN in an ambient atmosphere. It is these forces that are used to collect the image and hence it is important that the right force is used for a particular sample. For example, a high force used on a soft sample will lead to damage. Operation can take place in ambient and liquid environments.

Tapping mode AFM is used for soft samples such as lipid bilayers and polymers where contact mode imaging would damage the sample. Similar information is measured but via a different imaging mechanism. Tapping mode operates by scanning a tip attached to the end of an oscillating cantilever across the sample surface. The cantilever is oscillated at or near its resonance frequency with an amplitude ranging from 20 nm to 100 nm. The frequency of oscillation can be at or on either side of the resonant frequency of the cantilever. The tip lightly 'taps' on the sample surface during scanning, contacting the surface at the bottom of each oscillation. The feedback loop maintains a constant oscillation amplitude by maintaining a constant amplitude of the oscillation signal acquired by the split photodiode detector. By maintaining a constant oscillation amplitude, a constant tip-sample interaction is maintained during imaging. Like contact mode, tapping mode can be operated in ambient and liquid environments. Tapping mode has a number of advantages compared with contact mode, including a reduction in lateral and normal forces being applied to the sample surface.

Specimen choice and preparation can be a limiting factor with any type of AFM. The AFM scanners are usually limited to a maximum vertical movement of approximately five microns with a maximum x-y range of 100 x 100 microns. Therefore any surface with more than five microns of roughness will be extremely difficult to image with most AFMs. Sample size is also restricted to a maximum area of 1 x 1 cm and with a maximum sample thickness of approximately 3 mm.