Byron Ballou was irritated. The biochemist had just injected some fluorescent particles into the tail of a restrained and anesthetized nude mouse, but he had botched the job.

Carnegie Mellon University
In this image, quantum dot fluorescence is clearly visible in an anesthetized mouse, indicating major sites of quantum dot deposition in the lymph system.
Click photo for larger image.

Now, way too many of the particles, called quantum dots, were circulating and the mouse's tail was glowing brightly. It was only later, reviewing video of the experiment, that Ballou realized something remarkable had occurred.

First, some of the quantum dots migrated into lymph nodes at the mouse's hips, causing them to glow. Then, the dots could be seen flowing through lymphatic vessels up either side of the mouse, eventually reaching the axillary lymph nodes near the front legs.

"I don't think anyone has seen that before," Ballou said later, still a little awed as he reviewed the video with a visitor. Other techniques have produced static images of the delicate lymphatic system, he noted, but hadn't captured this dynamic flow in a living animal. What else the camera might eventually have salvaged from a seeming screw-up will never be known: "We ended the experiment too soon."

For Ballou, the episode provided a hint of just what a powerful tool quantum dots could become for biologists and, eventually, for medical scientists in their attempts to visualize biological processes right down to the molecular level. As a research scientist at Carnegie Mellon University's Molecular Biosensor and Imaging Center, he has more than a little experience in sizing up such technologies; the CMU center is a leader in developing fluorescent dyes and microscopy techniques that allow scientists to peer into living cells.

Quantum dots, also called nanocrystals, are tiny clumps of semiconductor material, amounting to a few hundreds or thousands of atoms each. In bulk, these materials don't fluoresce at all, but in the form of these tiny particles -- each only a few billionths of a meter, or nanometers, in diameter -- they glow brightly when illuminated by a light source. Depending on the size and composition of each particle, they can fluoresce in a variety of colors.

"These are like little nanolights, in a way," said Alan Seadler, president and CEO of Crystalplex Corp., a Pittsburgh company that makes nanocrystal-encoded microbeads.

Cornell University
The branched capillary structure feeding fatty tissue in a living mouse is revealed as quantum dots circulate in the bloodstream.
Click photo for larger image.

The quantum dots are both brighter and longer lasting than conventional fluorescent dyes and potentially could be useful for a variety of applications, from tracking individual cells within tissues, diagnosing cancers and other diseases and highlighting lymph nodes to be removed for biopsies. Work by Ballou and others has shown that they can be used for visualizing processes in living animals.

"Do you know how new all of this is?" said Ballou, who began working with quantum dots a year and a half ago. Though physicists have studied the optical properties of nanocrystals for decades, biologists have only begun using them since the late 1990s.

But they already are causing a stir. The journal Science identified the use of quantum dots in imaging as one of the top 10 scientific advances of 2003 and "perhaps the most exciting new technique to emerge from the collaboration of physicists and biologists."

In the current issue of the journal Bioconjugate Chemistry, Ballou and colleagues from both CMU and the Quantum Dot Corp. report they have been able to keep quantum dots circulating in the blood for hours and glowing brightly for months, thanks to special coatings they've developed. The particles have continued to fluoresce in tissue samples for eight months thus far, suggesting they would be practical for long-term studies in animals.

"This is a solid and careful piece of work," said Shuming Nie, a biochemist at Emory University and a pioneer in the biological use of quantum dots. It could have broad implications, he said, both for molecular imaging and for targeted therapy for various diseases.

By tagging quantum dots with antibodies or peptides, researchers can target quantum dots to attach themselves to certain types of tissues or certain proteins on a cell. Potentially, they might be used to detect cancer tumors, for instance.

They are so bright, they can even be used to guide a surgeon's scalpel. Last month, researchers at Harvard University and MIT reported that animal studies showed that the particles could be used to map "sentinel" lymph nodes -- the lymph node nearest a breast cancer. Surgeons routinely check lymph nodes to see if a cancer has spread. Increasingly, they have begun to biopsy the sentinel node first, which allows them to leave other nodes in place and reduce trauma to the patient if the sentinel node turns out to be "clean."

Because the particles collect light energy and then release it as fluorescence, they also might serve as an energy source for certain therapies. Inactivated drugs might be attached to quantum dots and, once they've reached a tumor or other therapy site, be activated by a flash of light.

While Quantum Dot Corp. of Hayward, Calif., has a proprietary coating that makes quantum dots water soluble, Pittsburgh's Crystalplex uses a different technology, developed by Emory's Nie, that packs large numbers of insoluble nanocrystals of varying colors inside a bead the size of a virus. As a result, each microbead has a distinctive optical signature that can be used much like a barcode to label individual cells or proteins. These are used for a variety of tests or pathology studies performed outside the body.

For instance, Crystalplex is working with the University of Pittsburgh Cancer Institute to develop a test to identify proteins on cells that are markers for prostate cancer, Seadler said.

Ballou said CMU's work thus far has focused on similar uses of quantum dots to label individual cells, a tool that could be used by tissue engineering researchers to monitor the behavior of cells in tissues they are trying to build.

Though the particles sold by Quantum Dot Corp., called QDots, have soluble coatings, he found that they were easily identified by the immune system and rapidly excreted after injection.

With the first QDots, "I might as well have been injecting bricks," Ballou said, noting they were eliminated in a matter of seconds. But he and his CMU colleagues, including Lauren Ernst, Christoffer Lagerholm and the center director, Alan Waggoner, found they could hide the nanocrystals from the immune system by making them "hairy."

It's an old trick that pharmaceutical companies have used for years to make drugs last longer after being ingested -- hanging long strands of a polymer, polyethylene glycol, to the outer coat.

"The hairier it is, the harder it is for the [immune] system to get a handle on it," Ballou explained. Most are still excreted within a day, but the hairy coats allow some of the particles to continue circulating, giving them time to attach themselves to targeted tissues.

Quantum dots, Ballou said, are in a "delightful stage of development," a stage at which their promise appears great, but that surprises and technical barriers still abound. Many of their applications, he added, are still unimagined.

"It's safe to assume we're only scratching the surface," Seadler agreed.

First Published: February 2, 2004, 5:00 a.m.