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First Quantum Dots Applied To Living Organism

Quantum dots are nano-sized crystals that exhibit all the colors of the rainbow due to their unique semiconductor qualities. These exquisitely small, human-made beacons have the power to shine their fluorescent light for months, even years. But in the near-decade since they were first readily produced, quantum dots have excluded themselves from the useful purview of biology. Now, for the first time, this flexible tool has been refined, and delivered to the hands of biologists.

Quantum dots are about to usher in a new plateau of comparative embryology, as well as limitless applications in all other areas of biology.

Two laboratories at The Rockefeller University -- the Laboratory of Condensed Matter Physics, headed by Albert Libchaber, Ph.D., and the Laboratory of Molecular Vertebrate Embryology, headed by Ali Brivanlou, Ph.D. -- teamed up to produce the first quantum dots applied to a living organism, a frog embryo. The results include spectacular three-color visualization of a four-cell embryo.

The scientists' results appear in the Nov. 29 issue of Science.

"We always knew this physics/biology collaboration would bear fruit," says co-author Brivanlou, "we just never knew how sweet it would be. Quantum dots in vivo are the most exciting, and beautiful, scientific images I have ever seen."

To exploit quantum dots' unique potential, the Rockefeller scientists needed to make a crucial modification to existing quantum dot technology. Without it, frog embryos and other living organisms would be fallow ground for the physics-based probes.

"Quite simply, we cannot do this kind of cell labeling with organic fluorophores," says Brivanlou. Organic fluorophores (synthetic molecules such as Oregon Green and Texas Red) don't have the longevity of quantum dots. What's more, organic fluorophores and fluorescent proteins (such as green fluorescent protein, a jellyfish protein, and luciferase, a firefly protein) represent a small number of colors, subject to highly specific conditions for effectiveness. Quantum dots can be made in dozens of colors just by slightly varying their size. The application potential in embryology alone is monumental.

Hydrophobic, but not claustrophobic

Benoit Dubertret, Ph.D., a postdoctoral fellow working with Libchaber, toiled for two years with quantum dots' biggest problem: their hydrophobic (water-fearing) outer shell. This condition, a by-product of quantum dots' synthesis, makes them repellent to the watery environment of a cell, or virtually any other biological context.

Since 1993, scientists have tinkered with the organic ligands, or binding molecules, necessary to the fabrication of quantum dots' outer surface, to make them more friendly, and useful, in biology. Various improvements were successful, but never foolproof. These included substituting a new ligand for the hydrophobic one, and overcoating, like painting over, the existing ligand with human-made surfactants and polymers.

Dubertret is adept at making quantum dots, a volatile process, which he does through a special research collaboration between Libchaber's lab at Rockefeller and NEC, a research institute in Princeton, N.J.

Working at NEC with another postdoctoral fellow in Libchaber's lab, Vincent Noireaux, Ph.D., Dubertret tried a different approach to the hydrophobia problem. Why not encapsulate the entire quantum dot with something untried - a- micelle? The micelle, a simple chemical ligand with two parts -- a hydrophobic head, and a hydrophilic (water-loving) tail - -did the trick. Drug companies use micelles as a coating for drugs that have hydrophobic qualities. But no one had ever thought to use them to contain quantum dots, until Dubertret and Noireaux thought of it.

"As is often the case in science, it is a little detail that makes all the difference," says Libchaber.

"When I saw the micelle engulf the quantum dot in 30 seconds, folding up in a star-shape that exposed its hydrophilic section and sealed off its hydrophobic section, I knew we had succeeded," says Dubertret. "Other kinds of modifications were slow going -- requiring up to five days of synthesis -- and always had flaws."

Rather than modifying the final coat of the quantum dot, Dubertret and his colleagues trapped the quantum dot, including its hydrophobic coating, inside a new outer barrier.

"Now, the outside world does not come into direct contact with the quantum dot," says Libchaber. "Instead, the cell sees only a phospholipid monolayer, an organic type of surface that is normal to its environs, mixed with ethylene glycol (PEG), which can be functionalized easily."

Once the micelles proved stable and durable as a quantum dot "capsule," the improved quantum dots were ready to test in a biology lab.

"Compatible with life"

Quantum dots have numerous potential applications, especially in biology where visualization is so often a component of the "readout" of results. The cell lineage and comparative embryology studies that Brivanlou and his colleagues want to conduct require large numbers of quantum dots. That's because each cell division has to disseminate many quantum dots.

The collaborating team of physicists and molecular biologists, microinject (inject a large quantity) quantum dots to very early frog embryos. Though in their first experiments a single quantum dot was toxic to the embryo, they purified the quantum dots so that a quantity of 10^9, or a billion, quantum dots could be injected to a single cell.

The differentiation processes of their rapidly dividing cells make embryos highly sensitive to physical and chemical changes in their environment. The Rockefeller scientists are pleased that such a large number of quantum dots has proven safe.

"We have made the quantum dot compatible with life," says Brivanlou. "Each safe application in an organism requires experimentation. But we're optimistic that if they work in frog embryos, they will work in other in vivo contexts."

The properties of quantum dots make them suitable for application in many areas from tagging single proteins in cells to diagnostic imaging.

Brivanlou and one of his graduate fellows, Paris Skourides, document the improved quantum dots in their experiments via fluorescence microscopy. In the process, they've noted something new.

"We've seen quantum dots dispersed throughout the cytoplasm of early embryonic cells suddenly relocalize to the nuclei about four hours after fertilization," says Skourides. "This indicates the onset of embryonic transcriptional activity; visualization with quantum dots is the first in vivo marker of this important developmental stage."

During the early stages of Xenopus, or frog, development, there is no RNA synthesis. In other words, there is no active rewriting of the information contained in the embryo's DNA, and thus no manufacture of proteins needed during development. Instead the embryo relies on protein and RNA that the mother provides to the egg. Once the embryo reaches its 12th cell division (4,000 cells) its own RNA appears, and transcriptionally speaking, the embryo can fend for itself.

Time-lapse movies from the Brivanlou lab show that the translocation, the concentration of quantum dots to the nuclei, occurs at the time known to be consistent with earliest gene expression of the embryo. (See http://violin.rockefeller.edu/press)

Brivanlou and Libchaber together will continue testing quantum dots. The two scientists are considering many additional experiments to test their new technology. Still, they each represent different disciplinary perspectives, and ask different questions.

Brivanlou and his lab colleagues will pursue safety and efficacy of quantum dots in vivo, and a variety of other biological applications questions.

Libchaber and his lab colleagues will study the limits of detection of quantum dots, including the application of single quantum dots in biology.

Both lab groups agree, however, that their institutional milieu set the stage for success.

"The physicists who come to Rockefeller want to interact with biologists," says Libchaber. "The program that supports physics on campus was designed to be this way."

"Rockefeller is the place where you can bring not just the technologies, but the thinkers and ideas, together productively," says Brivanlou.

This is true in part because the university is a unique research environment, with no formal departments directing inquiry. But it is also true because a research center for a nascent physics- and biology-oriented approach was created in 1994. The Center for Studies in Physics and Biology has been continuously and enthusiastically supported by the university's administration.

"In very few places would it be possible to do this work so fast," says Skourides. "The infrastructure for doing collaborative science is in place."

Dubertret and Libchaber of the Science publication thank their Rockefeller colleagues Professor Sandy Simon and postdoctoral fellow Jyoti Jaiswal, with whom they discussed quantum dots.