The procedure for this experiment included: construction of a tip, preparation of the sample, and operation of the STM.
The construction of the tip is, perhaps, the most crucial aspect of the experiment. As stated in the discussion of the theory of the STM, construction of a fine (one atom) tip is essential for resolving images. The tip, which was made from a platinum-iridium alloy wire, was constructed using two methods: Mechanical Construction and AC Etching.
Mechanical Construction consists of cutting the end of the Pt-Ir wire with wire cutters. The cut should be made at approximately a 45 degree and as the wire is being cut the wire cutters should be pulled away. This will have the effect of drawing out a fine tip. Once the sharp end is cut, flatly cut the wire approximately 1 cm away and place in the STM tip mount.
The mechanical construction method proves to be most efficient and was used to acquire about half of the DNA images in this experiment.
AC Etching is another more elaborate way of constructing a tip for the STM. However, if performed careful, the AC Etching method can provide even higher resolution. This method uses an AC power supply at 20 volts through a platinum electrode and Pt-Ir wire in a 5M KOH solution. The Pt-Ir wire is mounted on a micrometer so it may be raised and lowered. The entire apparatus is placed on a sturdy lab bench to dampen external vibrations.
Once the etching apparatus was constructed the Pt-Ir wire was lowered approximately 2 mm into the 5M KOH. The AC power supply was then turned on and gas evolved from the solution. The power supply was turned off once the current discontinued its flow. (Basically, the bubbling stopped.) Then, using the micrometer, the wire was lowered approximately 100 microns into the solution. The power was then turned on for approximately five seconds. The wire was carefully removed from the apparatus and cleaned with distilled water and methanol. The wire was then flatly cut with wire cutters approximately 1 cm from the end. The tip was then placed in the STM tip mount.
Preparation of the Sample
The substrate for this experiment was highly ordered pyrolytic graphite (HOPG). The graphite sample was mounted on the STM sample carriage and cleaned using Scotch tape. The HOPG sample provide a suitable substrate for the DNA because of it flatness and regular hexagonal carbon lattice.
The linear DNA (450bp in length) dissolved in a 100 ng/l aqueous solution was obtained from the Davidson College Biology department. Approximately 3 ul of the100 ng/l DNA solution was pipetted onto the substrate and allowed to dry in a fume hood. (Note: Various concentrations of DNA were applied throughout the course of the experiment ranging from 10-100 ng/l. It was found that 100 ng/l provided enough DNA on the surface that extensive searching was unnecessary.) Once the water had evaporated, a small ring formed on the substrate. The inside of this ring provided the starting place for the search for DNA.
Operation of the STM
The Scanning Tunneling Microscope used in this experiment was a Burleigh Instructional STM.
The apparatus was connected to a computer which interpreted the output from the STM. Once the tip and sample were mounted in the microscope images could be made. For this experiment, a constant current mode was used to collect data. Therefore, the tip scanned the surface of the sample maintaining a constant tunneling current. The constant tunneling current was maintained by a piezoelectric device which allowed for atomic-sized movement in the X, Y, and Z directions. For this experiment the time constant was set to a minimum, and the gain and filter where set to a maximum. The bias voltage ranged from 0.15-0.9 mV, and the reference current ranged from 1.0-3.0 nA depending on the magnification and stability of the image. Images from 3750 x 3750 Angstroms to 150 x 150 Angstroms were collected and analyzed using the Burleigh ISTM software.
The basic technique used for scanning was: