Over the last few months I have been using Qdot streptavidin conjugates from Quantum Dot Corporation (QDC) for a variety of biophysical and immuno-staining applications. Quantum dots are a relatively new technology for the selective fluorescent labeling of proteins, nucleic acids, lipids, cells and tissues. Until recently, nearly all fluorescent labeling was achieved with small organic molecules such as fluorescein, rhodamines and cyanines. The cloning of green fluorescent protein and the subsequent development of GFP derivatives and homologues offered the alternative of genetically encoding fluorescence with a protein of interest. Quantum dots further expand the repertoire of fluorescent probes that can be used to label biologically relevant players. Quantum dots, however, possess certain advantages over pre-existing fluorescent molecules - these properties allow quantum dots to be applied to certain situations much more successfully than organic fluorophores.
Quantum Dot Corporation (QDC) is one of only a handful of companies that has commercialized these nano-particles. The development of quantum dots began in the mid-1970s and two of the pioneering scientists, Paul Alivisatos (UC Berkeley) and Moungi Bawendi (MIT), are now on the Scientific Advisory Board of QDC. Quantum dots can be produced with a variety of different compositions although Qdots from QDC all have the same general structure: a nano-sized crystal core is formed from a semiconductor material such as cadmium selenide, the core is coated with another layer of semiconductor such as zinc sulfide to improve the optical properties and finally a polymer shell is added to aid solubility and permit conjugation of ‘biologically compatible’ molecules to the core. The resulting diameter of a Qdot, which governs the emission wavelength, is 10–20 nm, much larger than that of an organic fluorophore, but not really that much larger than a GFP moiety.
In comparison to organic fluorescent molecules, Qdots have quite different spectral properties. The dots confine electrons that can be excited by light, as these electrons return to lower energy levels they fluoresce. Quantum dots have extremely broad excitation spectra and narrow emission spectra. In addition, absorbance increases at lower wavelengths resulting in an overlap of the excitation spectra of various Qdots (excitation and emission spectra can be downloaded from www.qdots.com). Qdots consequently can have very large apparent Stokes’ shifts, allowing fluorescent emissions to be distinguished very easily. The overlapping Qdot excitation profiles also enable Qdots with different emission wavelengths to be simultaneously excited, a property that is exploited in other QDC products. The excitation and emission profiles of Qdots must be considered when choosing filter sets, but several companies, notably Chroma and Omega, now market optimized sets and pre-existing filters in your lab may suffice. The standout features of Qdots are their extreme intensity and photostability - no organic fluorophore can come close to Qdots with respect to these parameters.
Our lab has been using Qdot streptavidin conjugates for both immuno-staining and biophysical studies. Streptavidin conjugated Qdots are one of the bioconjugated range of fluorophores available from QDC; biotin and protein A conjugates are also available. I chose to use the 605 nm Qdot streptavidin conjugates because their emission is far from where you might expect cellular auto-fluorescence. Other emission wavelengths cover visible (525 nm, 565 nm, 585 nm, 605 nm, 655 nm) and infrared spectra (705 nm and 800 nm, made with a CdTe core). Streptavidin conjugated Qdots can be integrated into protocols to illuminate a protein of interest by using widely available biotinylated-antibodies. Initially, the qualities of signal intensity and photostability attracted us to Qdots. Unlike other streptavidin conjugated fluorescent beads tested, Qdot conjugates showed no non-specific binding in our cell culture systems. This may not be the case for all situations, but the technique may be varied using Qdot labeling strategies or avidin/biotin blocking kits. In terms of photostability, Qdots expanded my temporal observation window by over 100-fold compared to organic fluorophores. The intensity of Qdots meant that I had shorter acquisition times with high power objectives while still generating images with excellent signal-to-noise ratios. In addition, I could also view stained cells with lower power objectives enabling me to see a larger field of view. It is worth noting that Qdots can illuminate very low abundance proteins and produce signal from deeper within tissues because of the signal intensity.
I have been really impressed with the performance of Qdot streptavidin conjugates. They are bright, remain fluorescent even after a long illumination and bind very selectively - these features have been of great benefit to my studies. Although the heavy metals used in the protocol might suggest toxicity, published reports indicate that this is not the case. The particulate nature of Qdots means that staining is also inherently ‘dot-like’, potentially obscuring clustered or punctated protein arrangements in immuno-staining. Cost is certainly not an issue, I used Qdot streptavidin conjugates at very low concentrations thus, lasting for a long time. Perhaps the only real issues with Qdots are size and illumination light. For biophysical studies the size of Qdots needs to be considered when size can be a limiting factor. To get the most efficient illumination of Qdots blue light is required, potentially causing more cellular auto-fluorescence and photo-damage – to date, though, my signal-to-noise has been excellent and my cells seem happy!
Peter Haggie, Ph.D.
University of California, San Francisco