Although quantum dots are increasingly finding their way into biological research as imaging agents, concern remains high about their heavy metal content and possible toxic side effects.Researchers at Clemson University in South Carolina are developing an alternative contrast agent made from carbon. The material appears to avoid the safety issues associated with quantum dots, while maintaining bright photoluminescence with two-photon excitation in the near IR, and to have potential applications in cancer and other biomedical imaging. Principal investigator Ya-Ping Sun’s team prepared samples from nanosize (below 10 nm) pure carbon particles that were surface-passivated with organic or other molecules. The investigators used a Leica confocal fluorescence microscope equipped with an argon-ion laser and a separate Spectra-Physics femtosecond-pulsed Ti:sapphire laser to image the carbon particles. They found that the carbon dots were strongly emissive when excited in the visible with the argon-ion laser (operating at 458 nm) and in the near-IR (800 nm) with the Ti:sapphire laser using two-photon excitation. The carbon dots exposed to two-photon excitation also retained their photostability. The same microscope setup was used to measure the two-photon spectra. The researchers observed that the spectra varied slightly from spot to spot, a reflection of the inhomogeneous sample. Shown is a two-photon image of MCF-7 human breast cancer cells internalized with carbon dots. Courtesy of Ya-Ping Sun.They determined that carbon dots are comparable in performance to other two-photon luminescent nanomaterials, including quantum dots. The two-photon absorption cross section of carbon dots at 800 nm was 39,000 ±5000 Goeppert-Mayer units. By comparison, cadmium-selenium quantum dots with a zinc-sulfide shell (with fluorescence at 605 nm) have a two-photon absorption cross section of ~50,000 Goeppert-Mayer units. To determine how well carbon dots could image cancer cells, the researchers incubated human breast cancer MCF-7 cells with the imaging agent. When excited by 800-nm laser pulses, both the cell membranes and the cytoplasm became brightly illuminated. Although the nontoxicity of the carbon dots is their main attraction, the material also is abundant, relatively inexpensive and easy to conjugate. One shortcoming is that it does not appear to have the same wide color tunability as quantum dots.Journal of the American Chemical Society, Sept. 19, 2007, pp. 11318-11319.