by Michael Wilkerson

Shuming Nie, Assistant Professor of Chemistry, Indiana University Bloomington, holds flasks that contain tiny, suspended silver and gold particles in solutions called colloids. He sits next to a multicolor, fully automated digital microscope. --credit

In this quiet environment, Shuming Nie, an assistant professor of chemistry, and his colleagues are making advances in instrumentation and analysis that could soon lead to a variety of startling practical applications, in such diverse areas as vastly enhanced computer performance and the ability to provide instant analyses of a person's entire genetic code. Nie's research involves several different projects linked by an overall emphasis in refining and increasing our ability to see, study, and alter microscopic particles of both organic and inorganic materials.

A native of China and a faculty member since 1994, Nie is part of the university's highly regarded division of analytical chemistry, which is typically ranked in the top three nationally. "Analytical chemistry is half engineering and half science," Nie says. "You study problems, then you have to invent and build the equipment to solve them." In essence, the scientific problems and the advances in instrumentation they require are inseparable. Accordingly, Nie's insights and published papers represent a combination of breakthroughs in the understanding of chemical particles and advances in the instruments needed to study them.
The February 1997 publication in Science magazine of "Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering," by Nie and graduate student Steven R. Emory, achieved almost instant renown for the Nie Group. "We immediately had at least twelve invitations to speak based on that article," Nie says. It's not surprising, considering the remarkably broad implications of the technique Nie and his colleagues have invented.

Previously, the identification of a single molecule had not been possible at the levels of precision Nie's technique allows. Raman scattering, named for its 1930s-era Nobel-prize-winning discoverer, is similar to fluorescence, the phenomenon of particles emitting light as their energy states change. Fluorescence, Nie explains, offers a relatively broad measure of molecular activity. Raman scattering, however, measures each molecule so precisely that scientists can know the composition and identification of that molecule as surely as a fingerprint we can match to a specific person. The confocal microscope Nie and his team constructed to perform the breakthrough experiment combines visual identification with spectroscopy- experimental observation of the dispersion of light to its individual components. Though it resembles a regular microscope attached to computers, it consists of a variety of devices, including an argon laser, a spectrograph, and several different optical elements. The combination of these elements effectively reduces the limitations inherent in each one, allowing for increased stability of the sample being viewed and for much more finely tuned results and analysis than any single element could achieve. In microscopy:

• An oil-immersion microscope objective lens gathers the scattered photons that have been excited by an attached argon laser.
  
• A pinhole camera rejects out-of-focus light.
  
• The light is coupled to a spectrograph by a focusing lens.
  
• A CCD (charge-coupled device) camera films the process.
  
• The captured data are analyzed and processed by attached computers.

The confocal microscope can also be used for fluorescence, depending on the need of the application. Nie points out the confocal microscope is closely related to microscopes produced by Topometrix, a company in Santa Clara, California, founded by Jack Gill, the IU alumnus whose gift made possible the Linda and Jack Gill Center for Instrumentation and Measurement Science.

Structure of silver nanoparticles that exhibit large optical enhancement when excited with a blue (488 nm), green (568 nm), or red (647 nm) laser beam. --credit

Fundamentally, the importance of tracking and identifying single molecules of certain substances is that, contrary to intuition, substances have different properties when the size of the sample changes. "We're used to seeing materials in bulk," Nie says. "But if you take an example of silver or gold, and divide it to a size where the particles are about 50 nanometers (50 billionths of a meter) or less, silver will be yellow in color; at an even smaller size, it appears completely transparent. Similarly, gold in tiny particles is exactly the color of French red wine." Samples in Nie's lab of gold and silver suspended in chemical solutions called colloids, display these colors.

Color, of course, is only the most obvious property that can change when particles are small. Other properties such as conductivity and magnetism have implications that could affect growing and diverse sectors such as nanotechnology, nanoelectronics, catalysis, and biochemical sensing. "People are even thinking of using nanoparticles as lasers, and the work we've done with cadmium selenite, a semiconductor, might change some characteristics of computer chips," Nie says.

The ability to see such small particles is only the first step toward modifying and manipulating them with chemical probes. The infinitesimal nature of nanoparticles makes physical manipulation with instruments impossible in most cases. Instead, chemical probes are used. The nanoparticle's response to the stimulus of the chemical probe results in the desired modification. Nie's techniques could soon lead to advances in the manufacturing of microchips. The Pentium II computer in Nie's office, for example, is powered by a chip with transistors laid out in widths of about 250 nanometers; Nie's Raman scattering process could enable manufacturers to see and manipulate particles well enough to reduce that critical width to 50 nanometers.

Such a change is possible in the future, Nie says. "It would mean that you could pack a lot more elements on a chip, making the density go up twenty-fivefold." That would allow not only vastly improved power and speed but also more redundancies, creating virtually fail-safe computer chips, with much more room for programming and therefore more capability for artificial intelligence.

The Raman technique Nie and his group have pioneered also has the potential to alter other analytical processes traditionally used by scientists. "It turns out that certain kinds of optical processes can be enhanced not by a million- or billionfold, but by a hundred trillionfold!" Nie says. One such process involves measuring of the influence of electromagnetic fields on small particles; the finer the measurement of such influence, the more understanding scientists will have about the properties inherent in, as well as the influences on small particles.

Petroleum Research Funds, administered by the American Chemical Society (a petroleum research foundation that supports pure research) initially funded the Raman enhancement project. Nie's work in optical enhancement has now attracted the attention of the National Institutes of Health. NIH seeks major improvements in medical diagnostics, an area with which Nie became familiar during a postdoctoral residency at Georgia Institute of Technology after his education at Nankai University in China and Northwestern University.

A pressing current problem in medicine, for example, is diagnosis of the AIDS virus; at present, treatment with a triple antiviral drug therapy has been known to reduce the incidence of the virus below detectable levels. But, Nie points out, the virus is still present in the body, and if a patient stops the drug regimen, the virus regains its strength. With Nie's technique, a single virus could be found, and researchers might discover ways to destroy the weakened virus. Many other diseases, such as prostate cancer, could be found before they have a chance to take hold in the body. "I think we should be able to detect viral RNA and disease-specific antibodies at the single-molecule level," Nie says.

Nie stands next to a laser confocal microscope with William E. Haskins, a doctoral student in chemistry, discussing how the laser beam should be aligned with the microscope. - credit

Nie and his associates also have constructed a hybrid instrument that combines both optical and atomic force microscopy, enabling workers to correlate the morphology of the object with its atomic properties. In effect, the microscope uses two different techniques simultaneously on the same sample. Thus, both the spectra from a Raman or fluorescent, light-based process can be correlated with a structural examination conducted by the atomic-force microscope. "The rationale here is that if we can find out what is unique about the structure of a particle, we can develop ways to modify that structure so as to produce more of the desired structures." Such a technique, only possible with the sophisticated and expensive equipment Nie and his associates have built, could make possible the alteration of damaging viruses and infectious agents.

His group's other major area of inquiry begins with microscopy and culminates in the manufacture of microchip-like devices. It involves collaboration with researchers at the IU School of Medicine and is broadly related to the ambitious Human Genome Project, through which teams of scientists seek to map every aspect of human DNA. "One strand of DNA is very complex," Nie points out, and it's normally twisted and folded. If stretched out flat, the DNA containing a person's entire human genome would be a full meter long. Nie's team has discovered a way to stretch and stabilize the DNA so it can be viewed. The next step is to use chemical probes--DNA-binding proteins and synthetic oligonucleotides--to identify specific sites where unique genes are located.

They use two coverslips, one positively charged and one negatively charged. A minute amount of solution, in which the DNA is suspended, floats between the two pieces of glass. Since DNA carries a negative charge, the end of the strand will attach to the positively charged coverslip, holding it in place. As they apply minute amounts of force to the coverslips, effectively squeezing them together, the DNA strand flows with the liquid as the slips come together. Because one end is attached to the inside of the coverslip, however, the twisted strand of DNA stretches out and remains fixed, where it can be viewed under a microscope.

"The idea that you can stretch a molecule is amazing," Nie says, "but it works. Because the liquid is so thin, there is no turbulence. Thus, you can look at real human DNA in real time." The viewable fragments of DNA are short, less than 0.1 millimeter long, but each would have many genes along its length. (The entire human genome has somewhere between 50,000 and 80,000 genes.)
The Nie group's work might enable scientists to speed up the mapping and sequencing of the human genome, which Nie believes could be completed in five to ten years, thanks to an accelerating series of breakthroughs. IU's DNA-stretching and tagging project could conceivably help accelerate the analysis of human genetic material by up to a thousandfold.

Schematic diagram of the integrated optical and atomic force microscope developed at IUB. This instrument is capable of (a) confocal stationary monitoring of single molecules and nanoparticles, (b) video-rate (30 frames per second) wide-field imaging, (c) single-particle fluorescence and Raman spectroscopy, and (d) nanometer-scale topographical imaging. --credit

Genetic mapping and ultrasensitive microscopy have enabled Nie and other scientists to create a new kind of micro chips, called the "DNA chip." Nie's group has built a prototype machine, "the IU Chip-maker," which deposits bits of genetic material on a piece of glass for analysis by computerized microscopes. He envisions a system where a DNA chip with a very small active area, analogous to the magnetic strip on a credit card, would be inserted into a computerized "reader." The reader, connected to powerful computer databases, could identify all the genetic pairs in the sample material and flag the defective ones, recommending genetic modification or medical treatment. Such an advance may seem like science fiction, but "It's not Star Trek!" Nie says. Already, graduates of his work group are employed by a thriving biotechnology company in California that is manufacturing DNA chips for clinical use. "They're doing the commercial development, while we're interested in the fundamental aspects: how the probes recognize specific gene sequences, how to make the spots smaller and smaller on the chip, and how to improve the sensitivity of the detection equipment. We're also interested in looking at the fundamental process of how proteins recognize specific DNA sequences." It's an ambitious agenda, but one sure to reap significant dividends. These dividends are not only for Shuming Nie, the division of analytical chemistry, and IU, but also for a broad array of medical providers and consumers, well into the next generation.

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