Atomic force microscopy - a brief explanation

Atomic force microscopy is a method for investigating the surface topography of a sample. It uses a very fine tip on the end of a cantilever to probe the surface contours just like a pick-up on an old fashion record player. A laser beam is focused on the backside of the cantilever, which is reflected onto a photodiode. When the cantilever moves up and down corresponding to the surface landscape the laser beam gets deflected as well. Thus the deflection of the laser spot on the photodiode will correspond to the height displacement on the surface. Scanning the cantilever from side to side on the sample will therefore reveal what the surface looks like.

: Schematic illustration of the cantilever scanning action.

The atomic force microscope has a very high resolution, which makes it a valuable technique for studying micro- and nano-structured surfaces. The curvature of the cantilever tip is in the order of nanometers and it is this curvature that defines the resolution of the atomic force microscope. The sharper the cantilever tip the higher resolution can be achieved – the vertical resolution is usually below 0,1 nm.

Two different scanning modes can be applied when using atomic force microscopy. In contact mode the cantilever is kept in constant contact with the sample surface by applying a constant force between the cantilever and the surface. This is a common used scanning method especially suited hard samples. The second mode is called tapping mode and this mode utilizes a piezo crystal to oscillate the cantilever up and down (usually 100-200 nm) only tapping the surface while it scans across the sample. The tapping can be finely tuned by changing the voltage applied to the piezo crystal but a common tapping frequency is commonly between 50-500 MHz. The tapping mode is very gentle in contrast to contact mode while still enabling very high resolution. Even soft surfaces as supported lipid bilayes can be visualized this way without damaging them.

Not only does atomic force microscopy allow for the visualization of minute surfaces it can also be applied to measure forces down to the piconewton range, which is ten thousand times smaller than a single covalent bond. The ability of the atomic force microscope to measure such minute forces enables investigation of binding between molecules such as proteins, single strands of DNA or cell surface receptors and their corresponding ligands. In order to perform such a measurement the target molecule has to be fixated for instance on a solid support. The cantilever tip, coated with a biomolecule capable of binding to the fixated molecule, is then brought into contact with the fixated molecule, thus attaching itself to the molecule. By gently pulling the cantilever away from the support at force profile can be measured – consequently when the force drops noticeably the molecule has been detached and the force measured corresponds to the bonding force.