Atomic Force Microscopy(AFM)

Theory

The atomic force microscope (AFM) is a member of a new class of microscopes termed the scanning probe microscopes (SPMs). Unlike optical and electron microscopes which 'look' at the sample, the probe microscopes image by 'feeling' the structure of its surfaces. The first of the probe instruments to be developed was the scanning tunnelling microscope (STM) which earned its inventors, Gerd Binnig and Heinrich Rohrer the Nobel Prize in physics in 1986 (Binnig et al., 1982). The principle at the heart of the STM results from one of the most intriguing aspects of modern physics - quantum mechanics. Imagine a simple electrical circuit such as the light switch in a room. When the electrical contacts in the switch are closed, a current flows and the light comes on. Now imagine a similar situation where the contacts are very close, but not quite touching. Surprisingly, quantum theory suggests that a small current can still flow if the separation between the contacts is very small - of the order of nanometers. In STM, a small, yet sharp, metallic probe is positioned a few nanometres above a sample adsorbed onto a very flat conducting substrate. To scan the surface the tip is pushed toward the sample until the electron clouds of each gently touch. The application of a voltage between the tip and sample causes electrons to flow through a narrow channel in the electron clouds. This flow is called the tunnelling current. Since the density of an electron cloud falls exponentially with distance, the tunnelling current is extremely sensitive to the distance between the tip and the surface. A change in the distance by an amount equal to the diameter of a single atom causes the tunnelling current to change by a factor of as much as 1000. It is this sensitivity that is the key to the operation of the STM (Figure 3.5).

Figure 3.5: Schematic representation of a conducting STM tip over a sample adsorbed onto a conducting graphite substrate.

Piezoelectronics provide the answer to positioning the tip. Compression of a sample of material that has piezoelectric properties generates a potential difference across the material. Conversely, the application of electrical signals across piezoelectric materials can be used to control the expansion or contraction of these materials to a high degree of accuracy. The effect is used to position, and also scan, the conducting probe of the STM over the surface of the sample. Rather than simply monitoring the tunnel current during a scan, it is usually more convenient to control the vertical displacement of the probe by means of a feedback circuit designed to maintain a constant tunnelling current. The motions of the probe can then be amplified so as to visualise the surface features of the sample. Maintaining a constant tunnelling current makes it possible for the probe to lift over rather than 'crash into' samples deposited on the conducting substrate. STM has limited applications in biology because the material under study has to be conducting.

In 1985 Binnig, together with Calvin Quate and Christopher Gerber introduced the atomic force microscope (AFM), a scanned-probe device that does not need a conducting specimen (Binnig et al., 1986). In the AFM, the probe is mounted on a 'soft' spring, and is brought into contact with a surface such that it experiences a very small interaction force, usually of the order of nano-newtons. The probe is then raster scanned across the surface, while maintaining a constant force between the tip and the sample (Figure 3.6). These deflections of the cantilever, which are caused by changes in surface stiffness or topography, allow the AFM to record topographic contours of a surface. The forces are governed by the interaction potentials between atoms. The interaction is attractive at large distances due to the van der Waals interaction. At short distances the repulsive forces have their origin in the quantum mechanical exclusion principle, which states that no two fermions can be in exactly the same state, that is, have the same spin, angular momentum, and location. This principle allows different modes of imaging in AFM. The basic mode of operation is known as the contact or DC mode, in which the tip is held a few angstroms above the surface. The surface and the tip interact by repulsive forces. The alternative method of measurement is known as the non-contact or attractive mode. In such measurements, the tip is held some tens of angstroms above the surface and is oscillated at a frequency above its natural resonant frequency. Although the non-contact mode may be recommended for the study of soft materials such as biological and certain polymeric specimens, however, the disadvantage of this method is a loss in lateral resolution as compared to the contact mode.

Figure 3.6: A schematic representation of imaging using atomic force


MATERIALS AND METHODS

Sample preparation

Gliadin

Sigma gliadin was dissolved in 50% (v/v) ethanol and centrifuged as described. The protein concentration in the supernatant was determined by the method of Lowry (1951). The protein concentration used was 0.5mg/ml.

Deamidated gliadin

Deamidated gliadin samples were prepared as described. Samples were then diluted with distilled water or mixed with 80ml beta-mercaptoethanol and 8M urea as required. The concentration of protein used was 0.625mg/ml.

Mounting of samples on silicon substrate

Each sample (40ml) was placed onto a silicon substrate previously cleaned with (95%v/v) ethanol solution in a ventilation hood and dried at room temperature. The silicon substrate, mounted with the protein samples was transferred into petri dishes and covered to protect the samples from dust contamination.

Method

Imaging was undertaken on a Nanoscope II SPM from Digital Instruments, USA, equipped with microfabricated silicon nitride Cantilever (100-200mm). The silicon substrate holding the protein sample was glued to a stell disk on top of an XYZ translator. The AFM tip was held stationary and the sample was scanned in contact mode using piezoelectric transducers. The scanning was activated by applying voltages across the piezo translator which was controlled by software from Nanoscope II SPM.