Quantum dots have several advantageous characteristics that have made them the fluorophore of choice in many applications. Unfortunately they are not particularly suited to whole-animal in vivo imaging, an application that is becoming increasingly popular. Now Jianghong Rao, Sanjiv Gambhir and colleagues from the Molecular Imaging and Bio-X Program for interdisciplinary research at Stanford describe the synthesis and the use of bioluminescent quantum dots for in vivo imaging.

Although quantum dots can emit light in the red and infrared regions of the spectrum that have good tissue penetration, they all require blue light for efficient excitation. Blue light, however, does not penetrate tissue well and also produces high background owing to excitation of endogenous fluorophores. During in vivo imaging, this results in low excitation efficiency and high background. Rao and colleagues thought there must be a way to solve this problem.

Rao says they decided to try using luminescent light to excite quantum dots. This light can come from a bioluminescent protein that produces light via a chemical reaction. “We tried firefly luciferase first, and it was very hard to do the conjugation,” says Rao. “The reason was probably that the firefly luciferase is very fragile and sensitive to any chemical modification. This caused it to dramatically loose its luciferase activity.”

The Gambhir laboratory has been developing methods to use BRET imaging in living mice and has also developed a variant of Renilla reniformis luciferase (Luc8) that showed greater stability and improved efficiency compared to other luciferases. Furthermore, Luc8 emits shorter-wavelength light than firefly luciferase, which overlaps better with the absorption spectrum of quantum dots and its activity is not dependent on the use of ATP. Rao tried using this new Luc8 variant, and it worked. He says, “The beauty of this Luc8 is that it is very tolerant of chemical modifications, and this makes it very easy to do the conjugation and retain the luciferase activity.”

Concerns have been voiced about the ability of quantum dots to function as fluorescence resonance energy transfer acceptors for organic fluorophores. After testing both noncovalent and covalent methods to conjugate Luc8 to quantum dots, however, Rao found that covalent conjugation allowed very efficient bioluminescence energy transfer from Luc8 to the quantum dot (Fig. 1).

Figure 1: A schematic showing the design and function of the bioluminescent quantum dot.
figure 1

Oxidation of coelenterazine by Luc8 conjugated to a quantum dot releases bioluminescence energy. Some of this energy excites the quantum dot, resulting in the release of red light. Reprinted with permission from Nature Biotechnology.

When injected into mice, these bioluminescent quantum dot conjugates produced good signals with no background after injection of the luciferase substrate coelenterazine into the bloodstream. Furthermore, Rao and colleagues were able to exploit the fact that quantum dots with distinct emission spectra all have similar absorption profiles allowing all of them to function as luciferase energy acceptors. Quantum dots with emission peaks from 605 nm to 800 nm all performed well and permitted multiplex detection of signals in vivo.

The properties of these conjugates should make them suitable for use with animals larger than mice, and Rao says they are testing these conjugates for use for functional imaging in vivo. Gambhir says that these probes will allow multiplexing so that multiple events can be monitored in the same living subject. They are also exploring ways to exploit modulation of the BRET signal to detect enzyme or protein function.