Fluorescence Microscopy/Imaging Flow Cytometry Organic Dyes Quantum Dots Fluorogens
The most seminal work on quantum dot nanocrystals was undertaken by Louis Brus at Bell Laboratories and Alexander Efros and A.I. Ekimov of the Yoffe Institute in St. Petersburg (then Leningrad) in the former Soviet Union. Brus and his team experimented with these semiconductor materials and observed solutions of strikingly different colors made from the same materials. Their work constituted the main understanding of the quantum confinement effect that explains the correlation between nanocrystals size and observed color. This work was continued by Dr. Moungi Bawendi and Dr. Paul Alivisatos whose research teams found ways to make the quantum dots water soluble. In addition, they also discovered that adding a passivating inorganic "shell" around the nanocrystals, and then iluminating them with violet light, caused the different quantum dots solutions to emit bright fluorescence in discrete color bands.
Quantum dots are fluorophores, just like organic dyes, they are substances that absorb photons of light and then re-emit photons at a longer wavelength. There are however some important and distinct differences between them and organic dyes and naturally fluorescent proteins. These 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.
Quantum dots fluoresce in a radically different way from organic fluorophores. In their case, the formation of excitons, or Coulomb correlated electron-hole pairs cause the emitted fluorescence. The exciton can be thought of as similar to the excited state of an organic fluorophore; however, excitons typically have much longer lifetimes (up to microseconds), a property that can be advantage in time-dependent studies.
Yet another distinction arises from the direct, predictable relationship between the physical size of the nanocrystal and the energy of the exciton (ie the wavelength of the emitted fluorescence). This property has been referred to as "tuneability", and is 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 ordinarially lead to the photo-induced deterioration of other types of fluorophores.
Quantum dots can also be conjugated to proteins, oligonucleotides and small molecules, which are then subsequently used to direct the binding of quantum dots to targets of particular interest. Examples of quantum dot bioconjugates include streptavidin and biotin families of conjugates. These bioconjugates are often used as simple replacements for analogous conventional dye conjugates when their unique optical properties and performance characteristics are required to achieve optimal results.
