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11/14/94

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Tracking molecules: one by one

STANFORD -- A simple and inexpensive way to study individual molecules as they move and react in a chemical solution has been developed by scientists at Stanford University.

"We are able to see a single molecule so well that we can follow its behavior directly in real time rather than use some complicated statistical analysis to prove that we saw it," says chemistry Professor Richard N. Zare.

The capability of tracking individual molecules as they participate in chemical reactions can provide scientists with potentially valuable new information about complex chemical and biological processes, such as the way enzymes operate and the manner in which viruses infect cells.

The new method is reported in the Nov. 11 issue of the journal Science by Zare; Shuming Nie, who recently left Stanford to take a faculty position at Indiana University in Bloomington; and Stanford graduate student Daniel Chiu.

Traditionally, chemists have studied the properties of chemical reactions by looking at the average behavior of millions of molecules. That method is a little like studying a large crowd of people from such a high vantage point that an observer cannot pick out individuals, but can observe large-scale movements. While there are a number of valid conclusions one can make about what is going on in the crowd, the ability to see individuals would give important additional insights.

For that reason, scientists have developed a number of techniques in recent years to detect individual molecules. But most of these methods require either that the molecule be fixed to a surface or that its motion be slowed down by cooling it to temperatures near absolute zero.

The new Stanford technique does not have either of these limitations. It can detect individual molecules moving at room temperature. Currently, however, the method works only with fluorescent molecules. To return to the crowd analogy, the distant observer can follow an individual's movement only when he or she is wearing a bright flashing light.

"The method is wonderfully simple. Anyone with a $25,000 equipment budget can do it," says Nie.

It requires a special kind of microscope - called a confocal microscope - a laser, highly sensitive light detector and fluorescent dye molecules. The microscope allows the researchers to focus on an extremely small volume of a liquid sample: half a femtoliter. For a sense of just how small this is, if a liter of water were equivalent to all the water in the world¹s oceans, then a half femtoliter would be equivalent to the contents of 10 large bathtubs.

For detection purposes, this volume is so small that the scientists, by picking the correct concentration, can ensure that only one dye molecule at a time passes through it. This tiny volume is illuminated with laser light that causes an individual dye molecule to fluoresce when it swims into the volume. That is, the molecule absorbs a photon at the laser wavelength and re-emits another photon with a wavelength characteristic of the dye molecule. This absorption, re-emission cycle happens repeatedly during the millisecond or so that the molecule remains in the volume. Using the light detector, the researchers can directly detect these microscopic flashes and use them to monitor the molecule's activity.

"When a molecule moves into this volume, it's as if it begins jumping up and down and shoutinging, 'I'm here. I'm here,¹ ² Zare says. "Because we can make real time measurements of an individual molecule for an extended period of time, we can look for a rare event, an event that before would have been hidden in the statistics. We don't know exactly what we will find."

Nie said that ³there are some quite interesting applications of this technique in studying chemical reactions, but I think the most important uses will be in biological sciences and biotechnology.²

It should be possible, for example, to use the technique to learn more about how individual enzymes work. These biological catalysts convert miscellaneous molecules into compounds essential for life. By immobilizing the enzyme within the field of view of the microscope and feeding it special compounds that it converts into fluorescent molecules, the scientists say that they should be able to determine the rate at which individual enzyme molecules work, whether they work continuously or discontinuously, and if there are differences among them.

Nie also intends to use the method to study the structure of viruses, such as the hepatitis and the AIDS virus. "We should be able to detect individual viruses and monitor how virus particles enter and infect an individual biological cell in real time," he says.

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