Quantum dots are useful for biological imaging because they are stable and bright, and their emission depends on their size. Researchers can pick quantum dots of a certain emission to match their application or use several sizes for multiplexing. They may soon have a new choice: self-illuminating quantum dots. Scientists from Stanford University in California recently developed a way to modify quantum dots so that they provide their own excitation light by converting chemical energy into light. Because no excitation light is necessary, the dots virtually eliminate autofluorescence and could provide a way to image deep within the tissue of living animals. Typically, quantum dots consist of a heavy metal or semiconductor core, such as cadmium selenide. Because the core is toxic, it’s sealed, often with a zinc-sulfide shell covered by an organic coating. The use of a shell also has the advantage of rendering the quantum dot water-soluble. The core-shell structure, a few tens of nanometers in diameter, emits in the visible when excited by an external light source of the right wavelength. Competing signals There are some downsides to quantum dots, including questions about their safety and size. Both are problems for in vivo applications. However, one of the biggest disadvantages is one that the researchers experienced firsthand. “We began to work on quantum dot in vivo fluorescence imaging first and ran into the problem of autofluorescence,” said Jianghong Rao of Stanford’s School of Medicine. Self-illuminating quantum dots were used to image C6 glioma cells in vitro and in mice. On the left is a bioluminescence image of a nude mouse injected via the tail with the labeled cells, which accumulated from deep lung tissue. On the right is an overlay of fluorescence and differential interference contrast images of cells labeled with self-illuminating quantum dots, conjugates of a quantum dot that emits at 655 nm and Luc8 luciferase. Courtesy of Jianghong Rao and Stanford University. The external light source used to get quantum dot emission is the same wavelength that excites fluorescence in a host of organic compounds found in abundance in living animal cells. The list includes collagens, porphyrins and flavins. In addition, because tissue scatters and absorbs the excitation light before it gets to the quantum dots, the signal from the dot is weakened and must compete with the autofluorescence of the surrounding tissue. At the same time the researchers were attempting quantum dots fluorescence imaging, they also were working on in vivo bioluminescence imaging. The contrast between results from the two methods was stark. While the quantum dots signal was drowning in a flood of unwanted light, the bioluminescent imaging showed high sensitivity and low background noise. Therefore, they began to work on ways to bioluminescently image quantum dots. The key to achieving this involved the creation of a light source that was part of the probe itself. For this, they turned to bioluminescence resonance energy transfer (BRET), a naturally occurring phenomenon in which a light-emitting protein, the donor, transfers energy to a fluorescent protein, the acceptor, when the two are close together. BRET is similar to Förster resonance energy transfer (FRET); however, unlike FRET, the donor’s energy comes from a chemical reaction. FRET is known to occur between quantum dot donors and organic dye acceptors, but it wasn’t known whether the dots could act as a BRET acceptor. To find out, the investigators selected luciferase from the sea pansy Renilla reniformis because its 480-nm emission peak was bluer than that of other luciferases, an important consideration because quantum dots absorb bluer light more efficiently. This luciferase is typically used in bioluminescence imaging and, like luciferases found in fireflies and elsewhere, it emits light in the presence of oxygen and a substrate, luciferin. In this case, the luciferin was coelenterazine. Through an extensive process of mutagenesis, they created an eight-mutation variant designated Luc8 that was more stable in serum and had improved catalytic efficiency than the wild-type protein. They attached the variant to CdSe/ZnS quantum dots (QD655) from Quantum Dot Corp. of Hayward, Calif., that had a fluorescence emission at 655 nm. “The conjugation of the protein to quantum dots is rather straightforward, a simple amide formation chemistry, but it took a lot of time to optimize the procedures to avoid aggregations of the product,” Rao said. The layer of luciferase increased the size of the quantum dots by only 2 nm, with the diameter of the conjugate at 22 nm. In the presence of a sufficient concentration of coelenterazine, the luciferase emitted light and initiated BRET with the quantum dots. This produced 655-nm fluorescence from the acceptors without an external light source. Thus, there was also minimal autofluorescence. After verifying that the QD655-Luc8 conjugate worked in mouse serum and whole blood, the researchers injected it into mice about 3 mm below the skin, in the muscles. They injected coelenterazine and imaged the mouse, with and without a filter. They used a bioluminescence imaging system from Xenogen Corp. of Alameda, Calif., with a 30-s image acquisition time and found that they could detect the conjugate from millimeters deep within the animal. On the other hand, they spotted the luciferase emission only at the surface. Multiplexing images Using quantum dots of different sizes as the BRET acceptor, they multiplexed the imaging of conjugates at emission peaks of 605, 655, 705 and 800 nm. They were able to unmix the various spectral components, employing an imaging system and specialized software from CRI Inc. of Woburn, Mass. The work was published in the March issue of Nature Biotechnology. The technique could be useful for applications that are difficult for standard quantum dots. “The light emission from the quantum dots can be turned on and off by a biological interaction,” Rao said. That capability could be used to build nanosensors that emit light in response to specific biological activity. Rao readily acknowledged that this approach doesn’t get around any potential problems due to quantum dot size or toxicity, adding that a small and nontoxic probe would be best for in vivo imaging. He noted that ongoing research efforts aim to resolve these issues. However, the researchers are not waiting for such innovations before attempting to realize the potential of quantum dots conjugates for in vivo imaging. “The next step we are taking is to apply it to cancer imaging by attaching tumor cell-specific molecules to the quantum dots, and to image multiple tumor targets at the same time with multiplexed imaging,” Rao said. He also noted that the technique is not limited to a particular luciferase. It could potentially be used with other light-producing proteins.