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Physiology 19: 322-325, 2004; doi:10.1152/physiol.00030.2004
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Physiology, Vol. 19, No. 6, 322-325, December 2004
© 2004 Int. Union Physiol. Sci./Am. Physiol. Soc.

EMERGING TECHNOLOGIES

Imaging Takes a Quantum Leap

Diane S. Lidke and Donna J. Arndt-Jovin

Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany

dlidke{at}gwdg.de


   Abstract
 
Semiconducting nanocrystals, or quantum dots (QDs), have emerged as a new tool in physiological imaging, combining high brilliance, photostability, broad excitation but very narrow emission spectra, and surface chemistry compatible with biomolecular conjugation. In this review, we demonstrate the power of QDs in diverse applications, including long-term in vivo fluorescence imaging.


   Introduction
Top
 Introduction
Live cell imaging
 Single-molecule imaging
 Live animal studies
 Stability and toxicity
 References
 
Fluorescence imaging microscopy complements the wide variety of biochemical, genetic, and physiological techniques for studying cellular function. Structure, localization, and dynamics can be visualized in the live animal, in the single cell, or even at the single-molecule level, thereby yielding information about physiological state and activity. Visible fluorescent protein (VFP) constructs have provided biologists with a tool of immense utility. This technology and that of RNA interference represent (the most) significant technical advances for cell-biological studies in the past decade.

However, organic and biomolecular fluorophores have limitations in their applications. Such fluorophores generally exhibit only moderate Stokes shifts between their excitation and emission spectra, have relatively broad emission spectra (making it difficult to discriminate multiple colors), and undergo photobleaching when monitored over extended periods of time. In addition, the need for adding 27 kDa of a VFP moiety to a protein to generate an intrinsic label may interfere with normal protein function. A promising alternative to conventional fluorophores is quantum dots (QDs).

The core of the QD consists of a semiconductor nanocrystal, typically CdSe, surrounded by a passivation shell of ZnS (FIGURE 1Go). Upon absorption of a photon, an electron-hole pair is generated, the recombination of which in ~10–20 ns leads to the emission of a less-energetic photon. This energy, and therefore the wavelength, is dependent on the size of the core (smaller -> lower wavelength), which can be varied almost at will by controlled-synthesis conditions (2, 3, 9).



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  		FIGURE 1. Structure of a quantum dot
Quantum dots (QDs) consist of a CdSe nanocrystal surrounded by a ZnS passivation shell. The surface is coated with a polymer that protects the QD from water and allows for chemical coupling to biomolecules. The sizes of other common imaging probes are listed for comparison.

 
QDs provide many advantages over conventional labels. The excitation spectra of QDs are a continuum, rising into the UV, and the emission spectra are narrow and slightly red-shifted to the band-gap absorption. Thus QDs with different emissions can be ex-cited with a single excitation wave-length (6, 22). The large extinction coefficient and the relatively high quantum yield of QDs, as well as their extraordinary photostability, permit the use of a low sample irradiance and prolonged imaging with a detection sensitivity extending down to the single-QD level.

The advantages of QDs render them excellent tools for fluorescence microscopy. However, they have only recently found applications in biology following improvements in the shell and surface coating, which isolate the core from water while simultaneously increasing solubility in biological buffers and providing reactive chemical groups for protein conjugation. QDs are commercially available (e.g., http://www.qdots.com and http://www.evidenttech.com) with a variety of conjugated or reactive surfaces, e.g., amino, carboxyl, streptavidin, protein A, biotin, and IgGs (reviewed in Refs. 16, 29, and 31). Recent applications of QDs in biological studies demonstrate that the promises of QD technology are turning into reality. Here we highlight some examples of the "quantum leaps" QDs have provided for imaging in a variety of biological systems.


   Live cell imaging
Top
 Introduction
 Live cell imaging
Single-molecule imaging
 Live animal studies
 Stability and toxicity
 References
 
The observation of protein dynamics and subcellular localization is essential for understanding cellular function. New studies have employed QDs for specific multiplex protein target labeling (32), multicolor tracking over long-term imaging regimes (14), and monitoring signal transduction (20).

Wu et al. (32) demonstrated the means for labeling multiple compartments and specific antigens such as the membrane protein erbB2 (Her2), microtubules, actin, and nuclear antigen with QDs in both live and fixed cells and in tissue sections. By sequential biotin-streptavidin labeling of nuclear antigens with QD630 and microtubules with QD535, the simultaneous detection of the two proteins was possible. Similarly, they detected erbB2 with QD535-IgG and nuclear antigens with QD630-streptavadin. Both Wu et al. (32) and Jaiswal et al. (14) demonstrated the dramatic photostability of the QDs compared with organic dyes.

In this laboratory, Lidke et al. (20) used a combination of QD-labeled ligands and VFP-fusion proteins to study the ligand-induced activation as well as homo- and heterodimerization of members of the erbB family. QDs were coupled to a natural cell surface receptor ligand, EGF, resulting in a QD-ligand complex (EGF-QD) that was physiologically active, i.e., capable of inducing activation and internalization of the EGF receptor erbB1. Due to the photostability of the QDs, their signal could be monitored continuously for long periods (>60 min). In this way and for the first time, activated receptors could be tracked from the cell surface, through endocytosis, on to vesicular trafficking and fusion in living cells (FIGURE 2Go). As little as 50 pM ligand-QDs were easily detectable as single and small clusters of QD-bound receptor, and a new retrograde mechanism for the transport of the receptors on cellular filopodia was discovered. By studying the behavior of transgenic cell lines expressing VFP-erbB proteins in the presence of EGF-QDs, we were able to determine that erbB2, but not erbB3, can influence the endocytosis of activated erbB1.



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  		FIGURE 2. Tracking activated EGF receptors from the cell surface with QDs
Selected images from a time series of Chinese hamster ovary cells expressing erbB1-GFP (green) upon addition of 250 pM EGF-QDs (red) are shown. Images are maximum-intensity projections of 12 optical sections taken at 30-s intervals ({Delta}x,y,z: 0.14, 0.14, 0.64 µm) (20).

 
Many of the problems in cellular physiology require information about the exact localization of a macromolecule in time, its local environment, and the partners with which it may interact. Thus a variety of ratiometric dyes or transgenic proteins have been developed to sense pH, ionic flux, and small-molecule or macromolecule concentrations in vivo (24, 33). A powerful tool used by some of these probes is Förster Resonance Energy Transfer (FRET) for assessing the direct interaction of two or more molecular species (15). The unique spectral properties of QDs with their broad excitation spectrum make them ideal donors for fluorescence energy transfer experiments, as has been demonstrated in vitro (8, 23) and can easily be extended to in vivo studies. For example, using quenching of QD fluorescence by an appropriate acceptor, we were able to demonstrate that the retrograde transport of the erbB1 discussed above occurs on the surface of filopodia and that the receptor is internalized after reaching the cell body (unpublished observations).


   Single-molecule imaging
Top
 Introduction
 Live cell imaging
 Single-molecule imaging
Live animal studies
 Stability and toxicity
 References
 
The standard approach for tracking single molecules on the cell membrane involves the use of particles (gold particles of ~40 nm or latex beads of ~500 nm) that may interfere with protein dynamics or small fluorescent labels that suffer from photobleaching. Many tracking assays have underestimated the diffusion rates due to low signal-to-noise ratios that precluded very fast data acquisition (25). QDs are smaller (10–20 nm) than traditional beads and, as stated above, are more photostable than conventional dyes. Dahan et al. (10) exploited these qualities for tracking the glycine receptor at the single-molecule level in living cells using QDs conjugated to antibodies. The photostability of the QDs allowed the observation of individual receptors for up to 20 min, compared with 5 s achieved with Cy3-labeled antibodies. The brightness of the QDs increased the signal-to-noise ratio by a factor of 10 and led to an improvement in lateral resolution by more than a factor of 4. The authors showed that the diffusion constant obtained by tracking QDs was 25% of the value obtained with micrometer-sized beads, indicating that attachment of relatively large beads to the receptor may influence its diffusion properties.

Another advantage of QDs is that the CdSe core is electron dense and thus amenable to detection by transmission electron microscopy (TEM), including energy-filtering techniques (26). Dahan et al. (10) demonstrated the ability to monitor dynamics with single-molecule fluorescence and confirm localization with TEM on QD-labeled samples. The comparison of light and TEM images provides the means for integrating dynamic measurements and high-resolution localization.


   Live animal studies
Top
 Introduction
 Live cell imaging
 Single-molecule imaging
 Live animal studies
Stability and toxicity
 References
 
The photophysical properties of QDs make them excellent probes for in vivo imaging when single-photon infrared (IR) or two-photon sources are used to extend deep into tissues or whole animals. In particular, IR-emitting QDs (4) are now available, and modifications to the surface-coating chemistry have increased the circulating half-lives (5) as well as provided a means of organ targeting (1).

For example, Larson et al. (19) used multiphoton excitation of QDs to dynamically image blood flow in capillaries through the skin of live mice. These authors found the two-photon cross-section of CdSe/ZnS QDs to be an order of magnitude higher than the best two-photon-absorbing fluorophores. After QDs were intravenously injected into mice, it was possible to visualize the vasculature at a depth of 100 µm. Line scans were sensitive enough to image the QDs in capillaries, where both the rate of blood flow and the ripples caused by the mouse’s heartbeat could be detected. The authors determined that detection was possible with 1/40 of the fluorophore concentration and 1/5 the power compared with similar measurements with fluorescein.

By adjusting the surface chemistry, Ballou et al. (5) were able to vary the circulating half-lives of QDs. The QDs were easily seen in the superficial vasculature just after intravenous injection. Depending on the coating, the QDs localized to the liver, skin, or bone marrow, and fluorescence could be observed up to 4 mo after injection. The ability to "fine-tune" QD targets based on surface-coating chemistry (30) portends increased use in both diagnostics and, eventually, therapeutics. Basic studies of metastasis or other "homing" phenomena should profit from the ability to label cells with QDs, reintroduce them into living animals, and study them for days and weeks (13).

Deep-tissue imaging is optimized in the near IR (NIR) by reducing tissue-induced light scattering. Kim et al. (17) have developed an NIR fluorescent QD useful for noninvasive imaging in live animals. These NIR QDs were successfully tracked through the sentinel lymph nodes in live mice and pigs, demonstrating improved sentinel lymph node mapping, a prerequisite for cancer surgery resection. Using a low dose of QDs (400 pmol) and low irradiance (5 mW/cm2), the authors were able to image sentinel lymph nodes 1 cm below the skin in real time [FIGURE 3Go; reproduced with permission from the author (17)].



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  		FIGURE 3. Near-infrared QD sentinel lymph node mapping in a live pig
Images of the surgical field in a pig injected intradermally with 400 pmol of near-infrared (NIR) QDs in the right groin. Four time points are shown from top to bottom: before injection (autofluorescence), 30 s after injection, 4 min after injection, and during image-guided resection. For each time point, color video (left), NIR fluorescence (middle), and color-NIR merge (right) images are shown (17).

 
All of the published QD data in live cell and animal imaging are relatively new, because the major breakthrough in the field for biologists has been the recent improvement in biocompatibility of the nanocrystals. Furthermore, the problems of nonspecific binding to cells and aggregation in divalent buffers have been reduced by improved surface coatings. There is still a need for control of the valency of the coupling agents as well as the development of more bioaffinity QD probes. Several basic research laboratories as well as commercial companies are actively pursuing improvements in all of these areas, such that the biologist can expect reagents with even better properties in the near future.


   Stability and toxicity
Top
 Introduction
 Live cell imaging
 Single-molecule imaging
 Live animal studies
 Stability and toxicity
References
 
Tissue culture cells loaded with QDs survive for weeks without diminished growth or division, and the QDs are visible over this entire period (14). In the live animal studies cited here, some of the mice were observed to maintain the QDs over months without obvious deleterious effects (5, 17). In addition, Dubertret et al. (11) report that injection of QD-micelles into Xenopus embryos did not alter the subsequent phenotype and that the QDs could be imaged throughout development. These observations indicate that QDs are safe for long-term imaging in live cells or animals. However, the available data on stability and toxicity is still anecdotal and more studies are required.

It is clear from the above references that QDs are already a valuable tool for in vivo studies of cellular function and live animal imaging. However, many other biomedical and technological areas are benefiting from the use of QDs. For example, by mixing QDs of various colors in different ratios and capturing them in microspheres, an almost infinite number of specific probes can be constructed and targeted to cells, genes, or tissues, thereby establishing a "barcode" identity (12). An interesting application of the CdSe-core QDs in environmental studies is for strain- and metabolism-specific microbial labeling. In this case, targeting was achieved by specific receptors on the organism surface, whereas internal labeling revealed that bacteria are able to extract Cd and Se from QDs in a fashion dependent on the QD surface conjugate, leading to spectral changes in the QD emission properties (18). Single-molecule imaging of physiologically active structures such as myosin-induced actin sliding may be enhanced and long-term recording may be facilitated by the use of QDs (21). QDs are not limited to biological studies, also finding usefulness in such diverse fields as quantum computing (7, 28) and solar cell development (27). We conclude that the future can only be "brighter" in all of these applications.


   Acknowledgments
 
We thank our colleagues for valuable suggestions and discussions and the Max Planck Society and the EU 5th Framework Program (Grant No. QLG2-CT-2001-02278) for financial support.


   References
Top
 Introduction
 Live cell imaging
 Single-molecule imaging
 Live animal studies
 Stability and toxicity
 References
 

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