| STM/AFM: A BROADENING ARRAY OF APPLICATIONS|
The scanning tunneling microscope (STM) was developed by physicists Gerd Binnig and Heinrich Rohrer in 1982 to investigate the surfaces of solids, such as silicon (Physical Review Letters 49:57, 1982). Binnig and Rohrer, who were awarded the Nobel Prize in 1986 for the invention of STM, were quick to recognize the great potential of their instrument in biology and chemistry.
Imaging with STM can be done only on electrically conducting substrates, however, which has presented problems for biological applications. A second instrument, the atomic force microscope (AFM), invented by Binnig in 1986 - this time along with Calvin Quate and Christopher Gerber -- is not hampered by this limitation (Physical Review Letters, 56:930, 1986). Although at present most research using these tools is being conducted in the fields of physics, chemistry, and materials science, applications in biology are growing rapidly.
How they Work
STM and AFM are both scanning probe microscopes -- that is, unlike light microscopes, which must focus light rays and electron microscopes, which focus electrons, there is no beam emitted and nothing to focus. STM technology is based on quantum mechanics theory, which allows that there is an electron cloud that extends beyond the surface of any material. When two electron clouds -- in the case of STM, from the tip and the substrate -- come close enough, the electron can "tunnel" through a potential barrier not allowable under classical physics. STM exploits this effect by bringing a sharp tip close to a conducting surface. If a sample is not itself conducting, it is deposited on a conducting substrate. Because the tunneling current is very sensitive to distance between the tip and surface, an electrical feedback mechanism maintains the tip at a constant height above the sample. By scanning the surface, vertical heights on the surface are displayed on a monitor, giving a magnified, three-dimensional image.
AFM has a tiny, cantilevered tip that acts as a probe to sense atomic forces within the molecules it scans. Because there is no current involved, AFM has an advantage over STM by not requiring a conducting substrate or sample.
Recently, high-resolution, reproducible images of DNA have been obtained by scientists using AFM. And this advance could go a long way toward repairing the techniques' image, tarnished by earlier work that claimed to have produced images of DNA that later turned out to be the graphite substrate the molecules were deposited on.
Pushing the work still further, there are attempts to view biochemical interactions of proteins and DNA as they happen. And attempts are being made to use these micro- scopes for nondestructive DNA sequencing that could be far more rapid than the most automated chemical techniques available today.
Although most research using STM, invented in 1982, and AFM, invented in 1986, has been in physics, chemistry, and materials science, the potential for biological applications has always been recognized. There are indications that this potential is finally being realized. At the spring meeting of the American Chemical Society, for example, several sessions were devoted to "Macromolecules and the New Microscopy." Topics included imaging of DNA and DNA-RNA polymerase complexes, morphology and surface interactions of polymer molecules observed with STM/AFM, and microscopy of a liquid crystal mixture.
Microscopic DNA SequencingEdward Cox of Princeton University, who is funded by the National Institutes of Health to develop methods to sequence DNA using STM and AFM, calls his work "a high-risk, but high-payoff, project," whose difficulty he does not underestimate.
Cox's approach to sequencing involves measuring the tunneling current as the microscope's probe passes .over the molecule, then sweeping back again while illuminating the sample with ultraviolet light and measuring the current again; this is done at two different wavelengths. It has been found that when a molecule is irradiated at its absorption wavelength, it changes the tunneling current. And since the four DNA bases absorb differently in the UV, in principle, it should be possible to tell them apart. According to Cox, the key is that the difference in the signal is a property of the entire purine or pyrimidine molecule, the base components of nucleic acids. Higher resolution may not be necessary or even possible for DNA sequencing. "I am convinced, perhaps wrongly," Cox says, "that trying to get atomic resolution is a complete waste of time."
For the human genome, with 3 billion base pairs of DNA, Cox believes that sequencing must be unambiguous in blocks of at least 20 base pairs. Even with noise interruptions, if the DNA molecule is scanned over and over again, the computer can determine when there is a completely overlapping sequence. If his method has merit, Cox predicts that it will be possible to sequence millions of base pairs a day.
Such a rate would be far more rapid than the 60 million bases per year projected for the Institute for Genomic Research when it gets up to speed. The institute, to be located in Montgomery County, Md., is a nonprofit research facility being set up under the direction of Craig Venter, the former NIH researcher whose applications for patents on thou- sands of partial DNA sequences created a major flap in the genome community (The Scientist, April 27, 1992, page 1).
Surface scientist Thomas Beebe, Jr. of the University of Utah is an- other NIH grantee studying the feasibility of DNA sequencing using STM and AFM. In an exploratory project, his group is looking at ways to attach the molecule to modified gold surfaces and then to do the imaging as a second step. Beebe also sees other important applications of his work.
"There is potential for a whole slew of additional information that can be gotten besides just sequencing," he says. "Some of the modes by which enzymes and proteins recognize DNA have to do with the shape of DNA. In principle, this technique can give information about shapes of molecules as well as things such as their sequence. So, it is not only the primary structure but the secondary and tertiary structure, as well. Since it is a microscopy technique, it might be possible to see these molecules coming up to each other and doing what it is that they do," he says.
Although the ultimate goal would be to achieve atomic resolution of biological molecules, Beebe predicts that "for most systems, that probably won't be possible in the next five or 10 years."
According to Helen Hansma, a biologist doing DNA sequencing feasibility studies under an NSF grant at University of California, Santa Barbara, "The ultimate goal is to be able to see DNA at a level where we can sequence it. It all depends on technology that isn't quite here yet."
One of the main problems has been finding ways to attach the sample to a substrate so that it is held rigidly and does not move when the scanning tip passes over its surface. Because the tip exerts a force, the molecule is frequently pushed around, causing blurred images. As a result, imaging DNA at sufficient resolution to be really useful has remained elusive.
Collaborative BreakthroughA major breakthrough in imaging of biological macromolecules came out of the recent collaboration between biophysicist Carlos Bustamante at the University of Oregon in Eugene and physicist Paul Hansma at UC-Santa Barbara, who used AFM to reliably and reproducibly image molecules of DNA, deposited on mica, using "super tips" ( Science, 256:1180, 1992).
According to Bustamante, the resolution they achieved is "state of the art." Since the force between the tip and the sample is a function of the area of contact, the size of the probe is what limits the resolution. Better resolution will require finer tips.
Paul Hansma agrees. "The problem with the tips is so great that it eclipses our interest in the substrate," he says.
Right now, the tips they are using have radii of curvature of 60-100 angstroms. The goal is to develop tips with radii of curvature of 10, 15, or 20 angstroms.
Movement of molecules as a result of humidity is another problem that has plagued researchers. In Hansma and Bustamante's work, this was circumvented by imaging under propanol.
Bustamante acknowledges that propanol is not ideal, since it does not provide a physiological environment. It is also not conducive to his studies of protein-DNA complexes.
Helen Hansma, who did the imaging for the Science paper and is working on physiological solvents like water and buffers, still thinks working with propanol can yield valuable results. "People worry about propanol," she says. "But the miracle of molecular biology is that you can take apart the whole cell and throw it all in a test tube and change so much, and you still get exciting, really biologically relevant information."
Bustamante views this work as an area that is only beginning to develop, with techniques "that completely redefine the methods of imaging.., a kind of Braille way of looking at molecules." He says that if they can improve the tips and the means of attachment so that imaging can be done under physiological conditions, it will be possible to follow processes such as the interactions of molecules with their specific receptors. Helen Hansma concurs, saying it might be possible even to make movies of processes involving DNA. "We are just on the edge of that," she says.
For Bustamante, part of the excitement of this field has been working with a heterogeneous group. He looks to the physicists for modifications in the instruments, to the chemists to develop new imaging substrates, and to the biologists for biological relevance and the choice of biological systems.
Repairing A Tarnished ImageThe fact that the DNA imagining work done in Bustamante's Oregon laboratory was reproducible by the Hansma group in California should bring some respectability back to the field. In a 1991 paper, Carol Clemmer and Beebe (Science, 251:640) demonstrated that even if nothing was deposited on highly ordered pyrolytic graphite (an imaging substrate being used by many researchers), what looked like DNA images were observed.
"Tunneling AFM has a fairly bad reputation at the moment for [DNA] sequencing projects because most of die STM images that have been published turned out to be artifactual," says Princeton's Cox. But, he adds, "I don't know that that says anything about the intrinsic worth of the technique."
Criticism of STM/AFM has also come from scientists who question whether it is serious research or simply a way of taking pretty pictures. In response, Cox points to die Hansma-Bustamante work as a "real success," and says, "I am convinced that that's DNA."
Researchers continue to emphasize that the field is still in its infancy, with these microscopes having been developed only about 10 years ago. The electron microscope, by comparison, was used as a research tool for decades before serious pictures of biological samples such as DNA were obtained.
It is to the well-established community of electron microscopists, in fact, that scientists like Beebe feel they have to prove themselves. "They know an awful lot about how to prepare samples, and how to interpret images. We can learn a lot from that field," he says.
The electron microscopy community has actually been quite receptive to the new microscopes, with scientists getting the instruments to supplement what they already have.
Additional criticism is expressed by those who are content with existing chemical methods of sequencing DNA, who question whether the use of STM/AFM will ever be accurate enough to be useful. The answer to this should come within the next decade and possibly much sooner, according to Beebe and Helen Hansma. Within five years, Hansma thinks it will be possible to count the bases without necessarily knowing which is which. "I would hope that within 10 years the sequencing could be at the end of the tunnel, or that we could be near to sequencing DNA," she says.
Several laboratories are currently working very hard to develop instruments that will be ideal for biological applications. This, coupled with the success of imaging DNA molecules with better substructure resolution, as reported in the Hansma-Bustamante paper, has researchers upbeat about the possibility of solving some very basic problems in structural biology using scanning probe microscopes.
Sara Brudnoy is a chemistry professor at Russell Sage College in Albany, N.Y.