Our online fluorescence tutorials provide a general overview of the fundamental concepts of fluorescence. What follows here is a brief discussion of some of the physical processes behind the unique fluorescence properties of Qdot® nanocrystals.
Fundamentally, Qdot® nanocrystals are fluorophores—substances that absorb photons of light, then re-emit photons at a different wavelength. However, they exhibit some important differences as compared to traditional fluorophores such as organic fluorescent dyes and naturally fluorescent proteins, ends there. Qdot® nanocrystals are nanometer-scale (roughly protein-sized) atom clusters, containing from a few hundred to a few thousand atoms of a semiconductor material (cadmium mixed with selenium or tellurium), which has been coated with an additional semiconductor shell (zinc sulfide) to improve the optical properties of the material. These particles fluoresce in a completely different way than do traditional fluorophores, without the involvement of ->* electronic transitions.
Schematic of the overall structure of a Qdot® nanocrystal conjugate. The layers represent the distinct structural elements, and are drawn roughly to scale.
Qdot® nanocrystals are roughly protein-sized clusters of semiconductor material.
Yet another distinction arises from the direct, predictable relationship between the physical size of the quantum dot and the energy of the exciton (therefore, the wavelength of emitted fluorescence). This property has been referred to as "tuneability", and is being widely exploited in the development of multicolor assays. Qdot® nanocrystals are also extremely efficient machines for generating fluorescence; their intrinsic brightness is often many times that observed for other classes of fluorophores. Another practical benefit of achieving fluorescence without involving conjugated double-bond systems is that the photostability of Qdot® nanocrystals is many orders of magnitude greater than that associated with traditional fluorescent molecules; this property enables long-term imaging experiments under conditions that would lead to the photo-induced deterioration of other types of fluorophores.
Tuneability of Qdot® nanocrystals. Five different nanocrystal solutions are shown excited with the same long-wavelength UV lamp; the size of the nanocrystal determines the color.
Most dye conjugates are synthesized by attaching one or more fluorophores to a single biomolecule; however, the large surface area afforded by the nanocrystal fluorophore allows simultaneous conjugation of many biomolecules to a single Qdot® nanocrystal. Advantages conferred by this approach include increased avidity for targets, the potential for cooperative binding in some cases, and the use of efficient signal amplification methodologies. For example, combining biotin-functionalized products with the streptavidin labels allows for successive enhancements in signal via "sandwiching" (streptavidin/biotin/streptavidin/etc.) following an initial labeling step.
Standard fluorescence microscopes are an excellent and widely available tool for the detection of Qdot® bioconjugates. These microscopes are often fitted with bright white light lamps and filter arrangements; Qdot® nanocrystals efficiently absorb white light using broad excitation filters, and the outstanding photostability of Qdot® bioconjugates allows the microscopist more time for image optimization.
Laminin in a mouse kidney section was labeled with an anti-laminin primary antibody and visualized using green-fluorescent Qdot® 565 IgG. PECAM (platelet/endothelial cell adhesion molecule; CD31) was labeled with an anti–PECAM-1 primary antibody and visualized using red-fluorescent Qdot® 655 IgG. Nuclei were stained with blue-fluorescent Hoechst 33342.