Seminal developments in the story of nanocrystal technology emerged in the early 1980s from the labs of Louis Brus at Bell Laboratories and of Alexander Efros and A.I. Ekimov of the Yoffe Institute in St. Petersburg (then Leningrad) in the former Soviet Union. Dr. Brus and his collaborators experimented with nanocrystal semiconductor materials and observed solutions of strikingly different colors made from the same substance. This work contributed to the understanding of the quantum confinement effect that explains the correlation between size and color for these nanocrystals. Two scientists from Bell Labs—Dr. Moungi Bawendi and Dr. Paul Alivisatos—moved to MIT and UC Berkeley, respectively, and continued investigating quantum dot optical properties. These researchers found ways to make the quantum dots water soluble. They also discovered that adding a passivating inorganic "shell" around the nanocrystals, and then shining blue light on them, caused the quantum dots to light up brightly. Invitrogen is the exclusive licensee of several of their discoveries.
The Fluorescence Basics module (within the Molecular Probes School of Fluorescence) provides 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 probes.
Qdot probes structure
Fundamentally, Qdot probes 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 probes 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.
Relative size of Qdot probes
Qdot probes are roughly protein-sized clusters of semiconductor material.
At the heart of the fluorescence of Qdot probes is the formation of excitons, or Coulomb correlated electron-hole pairs. The exciton can be thought of as analogous to the excited state of traditional fluorophores; however, excitons typically have much longer lifetimes (up to ~µseconds), a property that can be advantageous in certain types of "time-gated detection" studies.
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 probes 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 probes 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 probes. Five different Qdot probes solutions are shown excited with the same long-wavelength UV lamp; the size of the probe determines the color.
Qdot probes bioconjugates
Qdot bioconjugate is a generic term to describe Qdot probes coupled to proteins, oligonucleotides, small molecules, etc., which are used to direct binding of the quantum dots to targets of interest. Examples of Qdot bioconjugates include streptavidin, protein A, and biotin families of conjugates. Qdot bioconjugates are often used as simple replacements for analogous conventional dye conjugates when their unique performance characteristics are required to achieve optimal results.
Most dye conjugates are synthesized by attaching one or more fluorophores to a single biomolecule; however, the large surface area afforded by the Qdot probe fluorophore allows simultaneous conjugation of many biomolecules to a single Qdot probe. 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 probes efficiently absorb white light using broad excitation filters, and the outstanding photostability of Qdot bioconjugates allows the microscopist more time for image optimization.
Applications for Qdot probes
Multicolor, multiplexed assays are a particular strength of Qdot bioconjugates. The emission from Qdot probes is narrow and symmetric; therefore, overlap with other colors is minimal, yielding less bleed through into adjacent detection channels and attenuated crosstalk and allowing many more colors to be used simultaneously. Since each bioconjugate color is based upon the same underlying material (they differ only in size), the conjugation and use methods for one color are easily extrapolated to all of the different colors, simplifying and speeding assay development. Furthermore, every Qdot probe can be excited using a single light source—narrow laser and broad lamp excitation are both useful. Three- or four-color detection no longer requires multiple lasers or laborious alignments and compensations.
Qdot probes and bioconjugates are ideal for experiments requiring long-term photostability or single-excitation, multicolor analysis.
Multicolor immunofluorescence imaging with Qdot secondary antibody conjugates. 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.