Scanning Tunneling Microscopy (Molecular Biology)

1. Principle of Operation

The scanning tunneling microscope (STM), invented by the Nobel prize winners Binnig and Rohrer, is a scanning probe technique that provides information at atomic resolution about structures that are electrically conducting. STM is based on the quantum mechanical principle that electrons travel across the classically forbidden energy potential zone (i.e., tunnel) from one surface to another without physical contact if there is overlap in their probabilities of finding their positions, as determined by their electron density functions at the two surfaces. In such a situation, applying a small potential difference between the corresponding surfaces causes electrons to tunnel through the potential barrier that exists between the surfaces of the sample and the probe, which gives rise to the tunneling current. The tunneling current density i through a planar potential barrier at small voltages V is inversely proportional to the exponential power of distance and can be approximated by

In this expression, f is proportional to the average potential barrier height of the two surfaces, and s is the tunnel distance in angstroms. The constant A contains several important features, such as the applied bias voltage V. For a greater applied bias voltage V, the effective barrier height becomes a function of V. For a nonplanar barrier, like the barrier between a conducting surface and an STM tip, the tunneling current cannot be described so easily because, in addition in these circumstances, the current is extremely sensitive to the tip-sample separation and is manifested by an exponential dependence on the tunnel distance s. Generally, the possibility of finding any tunneling current beyond a few angstroms from a weakly conducting surface, such as a biological material, and for a small bias potential, is negligible.

2. Modes of operation

The most commonly used STM operating mode is "constant current topography" imaging. In this mode of imaging, the tunneling current is kept at a constant value by feedback electronics, and the change in the scanner height necessary to maintain constant tunneling current is registered as topographic information (see Fig. 1 of Scanning Probe Techniques). Such height change is registered at each lateral x-y coordinate as the tip scans over the surface. This mode is most suitable to measuring the topography of nonflat surfaces. Because of the finite reaction time of the feedback, however, the scan speed is limited. Another way of operating the STM is the "constant height mode." For this mode, the height of the probe is fixed, and the tunneling current is measured at each x-y coordinate during the scanning. This mode allows much faster scanning of atomically flat surfaces because the feedback does not have to respond to surface features. This is important because fast scanning allows dynamic studies. It also minimizes data acquisition time, and image distortion caused by thermal drift and creep in the piezo scanner.

STM images give pure topographic information about surfaces only when the surface has uniform electronic properties because it actually measures the electronic structure of the sample and probe via barrier heights. The resolution is limited primarily by the size, shape, and mechanical stability of the tip and is on the order of 1 A laterally, and 0.1 A vertically.

STM can be operated in a more spectroscopic mode, measuring current-voltage characteristics. For each lateral position of the probe, the feedback is temporarily disabled after which the tunneling current is measured while ramping the bias voltage. The variation in the tunneling current at specific bias voltages can be visualized by constructing real-space images of surface electronic states. Spatially resolved current-voltage curves have been used to examine the electronic structure of semiconductor and metallic surfaces. The energy resolution of the spectroscopic imaging mode is influenced by several factors. Some of these factors are the thermal limit, which depends on the width of the edge of the Fermi distribution of the electrons in the electrode surfaces; an energy-dependent lifetime broadening of electronic states; and the uncertainty principle, which plays a role because the lateral resolution and tip radius are both of the order of angstroms.

3. Mechanical design

The mechanical design of a STM system can be modified for specific experimental needs. The most general criteria that must be satisfied by a STM setup are (1) an x, y, and z scanning range that allows for a scanning window sufficiently large to find and identify features of interest; (2) a resolution of 0.1 A laterally and 0.01 A in the z-direction; and (3) rigid construction of the tip-sample junction part with high resonance frequency to minimize vibrational disturbances. The scanners use mostly polycrystalline ceramic materials, like lead zirconate titanate and barium titanate.

The operating environment of STM studies depends on the nature of the contamination. Most inert samples are imaged in air, but for more accurate and reliable studies on clean surfaces, a STM situated in a vacuum chamber may be necessary. The mechanical construction in this case is more complicated because of the need for vibration isolation in the vacuum chamber, for in situ cleaning, and for possible use of Auger electron spectroscopy in the same chamber which is low-energy electron diffraction. Another approach that imposes harsher requirements on a piezo scanner is STM imaging in cryogenic conditions because the low temperature reduces the piezoelectric response of the ceramics.

4. Probe preparation

The physical shape, size, and chemical identity of the STM probe employed influences the resolution obtained in a STM scan and also the electronic structure. Three important properties make a "good" probe for STM. First, a large blunt macrostructure ensures high mechanical resonance frequency, leading to low hysteresis and an increase in the data acquisition rate. Second, the microstructure of the tip at the very apex needs a single site of atomic approach to the sample. If this is not met, multiple tip imaging occurs because electrons tunnel through several points on the tip apex. Third, the purity of the tip is important because any impurities may lead to barriers through which electrons do not tunnel. Most STM tips are made of metal wires, such as tungsten, platinum-iridium, or gold. Platinum-iridium tips are used most because platinum is inert to oxidation, produces a pure tip, and iridium adds stiffness to the probe. They are cut and sharpened in different ways, from simply using a wire cutter to ion milling or electrochemical etching.

5. Applications

The first STM work was done on conducting or semiconducting surfaces. A precise image of the atomic arrangements of those surfaces was obtained, mostly with the STM running in ultrahigh vacuum, primarily to avoid contamination from gases, mainly oxygen, and organic materials present under ambient conditions. Although it is difficult to image thicker biological samples with STM, small molecules like aromatic molecules, fatty acids, hydrocarbons, and liquid crystal molecules are routinely imaged. Energy levels between the various electron orbitals can be precisely calculated in some cases, and the use of a substrate like graphite can shift these orbitals in the adsorbed liquid crystalline molecules. Numerous similar applications of STM are described elsewhere.

Although attempts have been made to obtain structural information about larger biological macromolecular specimens, which are nonconducting, the mechanisms of image generation and their interpretation are unclear. Some of the early imaging of large molecules, such as DNA, with STM were later proven to be artifacts, when it was found that features that look very similar to a double-helical DNA strand could be obtained from a clean sample of graphite. More recently, a new way of imaging DNA and possibly other macromolecules with STM was described. The technique imaged DNA and tobacco mosaic virus on mica surfaces under a relative humidity of 65% and found clear images with a tunneling current in the subpicoampere range. The electrical conductivity is explained by a layer of water on the surface that has conductivity five times higher than bulk water.