By Saren Johnston
Ed Yeung and Xiaohong Nancy Xu are analytical chemists, but lately they've also become quite the movie buffs, producing and directing mini-documentaries of sorts. Making what they call "chemical movies," the two scientists are unraveling the mysteries of what transpires in a single-molecule environment.
Yeung, Ames Lab program director for
Physical and Biological Chemistry and a distinguished professor
of chemistry at Iowa State University (ISU), and Xu, a
postdoctoral fellow, have developed a technique that not only
allows them to detect single molecules, but also lets them
monitor molecular movements in chemical solutions, providing
critical information about the intermediate steps in chemical
reactions. This knowledge may eventually make possible the early
diagnosis of diseases, such as Alzheimer's and muscular
dystrophy, and lead to improved treatments for AIDS and cancer.
"The ability to observe one molecule react with another molecule may have great implications for the fields of medicine, catalysis and biotechnology," says Yeung, enthusiastically. "Looking ahead, maybe we'll be able to detect a single HIV virus or one copy of a specific gene in DNA."
Scientists have been able to detect single molecules in solution before, but Yeung's and Xu's technique is distinctive because it is the first to offer continuous monitoring over a period of time. Now zeroing in on single molecules will no longer require that they be immobilized on a solid. Researchers will be able to track their meanderings in their natural environment -- liquid solution.
Yeung and Xu have been successful in tracking the motion and photodecomposition lifetime of single molecules of the compound, rhodamine, in water. In addition, because rhodamine is highly fluorescent, they have used it to tag and track individual DNA molecules in water.
The ability to follow single molecules as they move through liquids will make it possible to gather more detailed information about their individual behaviors and physical and chemical properties. This represents a significant advancement over traditional methods, which can only determine the characteristics of molecules based on population averages from bulk studies.
Learning more about the properties of individual molecules may also lead scientists to the discovery of new fundamental principles. "We may find that many of today's fundamental principles for macroscopic-scale studies are no longer true at the single-molecule level," says Xu.
Yeung describes the new technique for single molecule detection (SMD) as a relatively simple combination of an optical microscope and an extremely sensitive intensified charge-coupled device (ICCD) camera, both stock commercial instruments that he and Xu use along with a blue-green-argon-ion laser to track molecules in motion.
The ability to use standard laboratory equipment is an attractive feature of the SMD experiments that Yeung and Xu do. "They don't require very expensive equipment. It's affordable research," says Xu.
To begin the process of making their chemical
movies, Yeung and Xu carefully adjust the laser power to excite a
molecule in solution, inducing it to vibrate and emit light in a
particular way. Different molecules vary in the length of time it
takes them to photodecompose, but their individual lifetimes are
usually inversely proportional to the laser excitation power. A
lower laser power allows molecules to continue to emit light over
a longer time, providing Yeung and Xu more time to monitor
molecular dynamics. However, longer observation times either
allow more and different molecules to approach the observation
region, or they create greater diffusion distances for each
molecule. So great care is required to select the optimal
combination of laser power and exposure time to achieve the high
signal-over-background ratio for observing the molecule of
interest.
Once Yeung and Xu have selected the desired laser excitation power, they pass the laser beam through the right-angle prism located on the microscope stage. This process creates a total internal reflection spot called the excitation zone, where Yeung and Xu can place one drop of a sample solution. The light emitted in the excitation zone is collected by the objective of the microscope and focused on the ICCD camera, which is controlled by a computer and is sensitive enough to detect a single photon. By opening the shutter of the camera for a long period time, Yeung and Xu can image either a liquid-surface interface or a thin layer of solution. Molecular activity is recorded continuously in the ICCD camera and captured in a single photograph in which the image of the molecule appears as a streak representing its lifetime and motion.
"An ICCD camera is just a sophisticated camcorder," says Yeung, "but there's a special way of operating it that gives you time resolution that's maybe a hundred times, or even more, faster than prior techniques, which can't look at molecules in solution at that kind of speed. And speed in data collection is essential, since molecules in solution cannot be kept in a confined space."
The entire process of molecule-tracking and data-gathering is accomplished with submillisecond time resolution. The information collected shows that each single molecule has its own unique traits and reaction rate, which deviate from measurements previously acquired by looking at millions of molecules and averaging their properties.
"People have been able to monitor single molecules in the gas phase for a long time," says Yeung, "because in gases you have better control and fewer interfering species. Now we're bringing that kind of study to the solution phase, where most biochemical reactions occur. This allows us to actually follow the behavior of molecules to obtain molecular motion and other kinetic information."
For more information:
Ed Yeung, 515-294-8062, yeung@ameslab.gov
Current research funded by:
DOE Basic Energy Sciences Office
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Last revision: 4/17/98 sd
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