Scientists keen on monitoring cellular events in real time can now add another imaging technique to their repertoire-the use of quantum dots.

Quantum dots (QD) are tiny nanoparticles of fluorescent inorganic semiconductors. About 10 nm in diameter, they are much larger than conventional organic fluorophores, but have superior characteristics, including resistance to degradation and very sharp emission spectra, the latter ensuring that several QDs can be measured simultaneously without cross-interference.

The widespread use of these fluorophores has been hindered, however, by some major obstacles, including difficulty coupling them to biological molecules and the question of toxicity. As reported in today’s Nature Biotechnology, two groups have successfully crossed these hurdles, using CdSe/ZnS nanocrystals to monitor subcellular organelles, and to label individual proteins.

Researchers from The Rockefeller University, New York, and the U.S. Naval Academy, Washington, DC, led by Sanford Simon, show that whole cells, either mammalian HeLa cells or cells from the slime mold Dictyostelium discoideum, can take up quantum dots by a variety of mechanisms including endocytosis, but do not suffer any deleterious effects. Furthermore, the amoeba cells responded normally to developmental stimuli and could be monitored for up to 18 hours with no adverse effects (See the Simon lab for time lapse video-Real Player required). In addition, the stability of quantum dots was demonstrated by the ability to monitor human cells for up to 12 days with no loss of signal.

First author Jyoti Jaiswal et al. were also able to label specific proteins by employing the immunoglobulin-binding power of avidin-conjugated dots. These conjugates were used to detect antibodies bound to the cell surface transporter, P-glycoprotein, in HeLa cells. The specificity of this detection method was proven by comparing quantum dot images with those of P-glycoprotein/green fluorescent protein chimeras in the same cell.

This type of conjugate approach was also used by Marcel Bruchez and colleagues at Quantum Dot Corporation, Hayward, California, and Genentech, San Francisco, to detect the breast cancer marker protein Her2 on live human cell lines, actin filaments in fixed fibroblasts, and nuclear antigens in fixed human epithelial cells.

First author Xingyong Wu et al. also compared the fluorescent intensity of their quantum dots with that of Alexa 568, reputedly the brightest organic fluorophore available. Fluorescence of the quantum dot, measured at the optimum excitation and emission wavelengths for the organic dye, was about four times brighter, while use of the optimum wavelengths for the QD more than doubled this difference. The QD was also much more stable than the organic reporter, which photobleached more rapidly.

"The paper by Wu et al is a lovely study which complements our own," commented Sanford Simon. "In both cases we have been able to show that quantum dots can be used to specifically label proteins for long periods of time." (See also comment below).-Tom Fagan.

References:
Jaiswal JK, Mattoussi H, Mauro JM, Simon SM. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nature Biotechnology. 2002 December 2. Abstract

Wu X, Liu H, Liu J, Haley KN, Treadway JA, Larson JP, Ge N, Peale F, Bruchez MP. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nature Biotechnology. 2002 December 2. Abstract

Comments

Make a Comment

To make a comment you must login or register.

Comments on this content

  1. Biology is at a wonderfully exciting, but vexing point. We have the sequences for all of the human genes. However, the business end of the body occurs [is run by] the products of the genes. Often, to understand how things are functioning, or what happens when they do not function properly, we need to be able to follow these gene products. Many diseases are diseases in trafficking of proteins, i.e., the protein is properly made but goes to the wrong place in a cell, where it may serve another function or may be processed abnormally. Often, it is even more important to know where a protein is being made, where it is going in response to a stimulus, where is it not going, than to know how much protein is being made. For the past 20 years, my lab has focused on such issues as protein targeting and transport. A nice summary of work on protein targeting and transport appears in Gunter Blobel's Nobel Address at the Nobel Web site.

    To study the movement of a protein we need to study it in a living cell. One of the best ways to do that is to label it with something that allows it to be seen with a light microscope (electron microscopes require "fixing" cells, and trying to study protein movement in a "fixed" cell would be like trying to study blood flow in a cadaver). Two of the more powerful approaches have been to chemically attach a fluorophore or to synthesize the protein of interest fused together with the GFP protein from jellyfish so it would glow, or fluoresce. Alas, all these fluorophores have a few limitations: first, they bleach (fade away) very quickly, often in seconds. Second, they emit light over a wide region of the light spectra, thus it is usually possible only to resolve one or two proteins at the same time.

    Quantum dots have been suggested to be a panacea for many of the problems of imaging multiple proteins. However, there have been a few major limitations:

    1) Quantum dots have been studied by physicists in organic solvents. In water, they die.

    2) It has been difficult to develop techniques for linking the quantum dots to probes so the specific molecules could be followed in the cell.

    3) It was not known if quantum dots would damage the viability of the cells.

    The current manuscript (one of six that we have in press) is the result of a few years’ work in which we have tried to develop quantum dots as a tool that can be used by the wider biological community. In the manuscript:

    1) We demonstrate that we can selectively and specifically label proteins in living cells with quantum dots. This is extremely important. In a field of cells, a few were selectively transfected to express a reporter protein that was fused to GFP. Only those cells expressing the jellyfish protein were labeled with the quantum dots. All other attempts have failed to show specificity.

    2) We demonstrate that it is possible to do continuous imaging of cells labeled with quantum dots through their entire development with no adverse effects. We followed Dictyostelium (the common amoeba) for 14 hours through their whole life cycle, and we followed human HeLa cells for 12 days.

    3) We demonstrate that it is possible to follow multiple quantum dot probes simultaneously, fulfilling a long-anticipated goal for these markers.

    The processing of APP is a critical problem in our attempts to understand Alzheimer’s. Thus, the interest of the Alzforum is quite relevant to the work at hand.

  2. It was demonstrated a few years ago that QDs had the potential to become a new and better class of fluorescent labels with advantages over conventional organic dyes. However, since some key technical problems remained to be solved, these advantages were not fully demonstrated in real applications, and QDs were not available for scientists who didn't have a chemistry lab to make QDs.

    Recently we have made a breakthrough in generating QD conjugates. In the article, we demonstrated the advantages of QDs (high intensity, photostability, multiplex flexibility) in real applications with specimens ranging from fixed tissue sections to live cells. In addition, we reported the first true multiplexed detection of specific cellular targets with QDs conjugated to streptavidin and IgGs.
    Our new quantum dot technology allows us to generate QDs at a commercial scale and sell QD conjugates (see www.qdots.com for products information). QD-based probes will have applications in immunocytochemistry, pathological diagnosis, live cell imaging, and multiple target analysis in many biological and biomedical applications. QDs have potential for in-vivo biomedical imaging. For example, QDs can be conjugated to antibodies or drugs, and the specificity of the antibodies and drugs can be monitored in vivo after they are injected into the circulation system.

Comments on Primary Papers for this Article

No Available Comments on Primary Papers for this Article

References

Paper Citations

  1. . Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat Biotechnol. 2003 Jan;21(1):47-51. PubMed.
  2. . Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol. 2003 Jan;21(1):41-6. PubMed.

External Citations

  1. Simon

Further Reading

Papers

  1. . Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat Biotechnol. 2003 Jan;21(1):47-51. PubMed.
  2. . Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol. 2003 Jan;21(1):41-6. PubMed.

Primary Papers

  1. . Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol. 2003 Jan;21(1):41-6. PubMed.