Semiconductor Nanocrystals: The Next Thing in Fluorescent Probes
Some of the more shadowy secrets of biology may soon be illuminated through the use of a new type of fluorescent probe developed by scientists with the U.S. Department of Energy's Lawrence Berkeley National Laboratory (LBNL) and the University of California, Berkeley (UCB).
A joint LBNL-UCB research team led by Paul Alivisatos and Shimon Weiss has discovered a way to use nanometer-sized semiconductor crystals—cadmium selenide and cadmium sulfide—as fluorescent probes for studying biological materials. Semiconductor nanocrystals offer a distinct advantage over conventional dye-molecules in that the crystals emit multiple colors of light, which means they can be used to label and measure several biological markers simultaneously. The unique optical properties of semiconductor nanocrystals also hint at the possibility of observing changes that take place over time in labeled biological systems such as living cells.
Alivisatos' expertise lies in the chemical production of semiconductor nanocrystals, simple inorganic solids consisting of a hundred to a hundred thousand atoms; Weiss, a staff scientist at Berkeley Lab's Materials Sciences Division.
"Form follows function" is the golden rule in cell biology, which is why fluorescence-labeled microscopy has been the heart and soul of biological research for so long. In fluorescent labeling, markers, usually antibodies that attach to specific proteins, are tagged with dye-molecules that fluoresce or emit a specific color of light when stimulated by laser light, usually from a confocal microscope.
"Sometimes, in order to fully characterize a sample, a population of cells, for example, you need to look at combinations of markers," says Alivisatos. "Such measurements require multiple-color light emissions which are difficult to obtain with conventional dye molecules. Ideal probes for multi-color experiments should emit at spectrally resolvable energies, should have a narrow, symmetric emission spectrum, and the whole family should be excitable at a single wavelength."
Semiconductor nanocrystals meet these demands, as shown in a dual-emission-from-single-excitation labeling experiment on mouse 3T3 fibroblasts. A core nanocrystal of cadmium selenide was enclosed within a shell of cadmium sulfide to boost the amount of fluorescence and reduce photochemical degradation. This core-shell combo was then enclosed within a shell of silica for water solubility and biocompatibility.
Earlier work by Alivisatos showed shown that the color of light emitted by a semiconductor nanocrystal depends upon its size, which enables labeling mouse cells with two different sizes of core-shell nanocrystals. It was also known that modifying the surface of the silica shell can be used to selectively control its attachment to components within a cell. In this case, the smaller nanocrystals (two nanometers), which fluoresced green, are modified to penetrate the nucleus of each cell; larger nanocrystals (four nanometers), which emitted red light, are modified so that they would attach themselves to actin filaments along the outer cell membrane.
Using wide-field microscopy, the green and red labels are visible to the naked eye and could be photographed in true color with an ordinary camera. Confocal microscopy images show that cell nuclei penetrate with the green probes and the actin fibers stain red. After repeated scans, the nanocrystal labels show far less photobleaching than would have occurred in the control sample labeled with conventional dyes.
"The development of semiconductor nanocrystals for biological labeling gives biologists an entire new class of fluorescent probes for which no small organic molecule equivalent exists," Alivisatos said. "These nanocrystalprobes can be complementary and in some cases may be superior to existing fluorophores."
Compared with conventional fluorophores, semiconductor nanocrystals have a narrow, tunable, symmetric emission spectrum, and are photochemically stable. These features, along with a relatively long fluorescence lifetime (hundreds of nanoseconds) indicate that, in addition to serving as direct probes, semiconductor nanocrystals could also be used as sensitizers for traditional dye-molecule probes, meaning they transfer their excitation energy to the dye.
For more information: Paul Alivisatos, Materials Sciences 11-B62, Lawrence Berkely National Laboratory, One Cyclotron Road, MS 66, Berkeley, CA 94720. Tel: 510-643-7371. Fax: 510-642-6911.
By Angelo DePalma