Quantum Dots As Cellular Probes

Annu. Rev. Biomed. Eng. 2005. 7:5576 doi: 10.1146/annurev.bioeng.7.060804.100432 Copyright c 2005 by Annual Reviews.
All rights reserved First published online as a Review in Advance on April 4, 2005
QUANTUM DOTS AS CELLULAR PROBES

A. Paul Alivisatos,1,2 Weiwei Gu,2,3 and Carolyn Larabell2,4
1 Department of Chemistry, University of California, Berkeley, California 94720; email: alivis@uclink4.berkeley.edu 2 Materials Science Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720; email: wwgu@lbl.gov, larabell@lbl.gov 3 Department of Anatomy, University of California, San Francisco, California 94143 4 Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
Annu. Rev. Biomed. Eng. 2005.7:55-76. Downloaded from www.annualreviews.org by Inflibnet N-LIST Programme on 09/03/11. For personal use only.
Key Words semiconductor nanocrystals, nanoparticles, uorescent probing, imaging, biological application Abstract Robust and bright light emitters, semiconductor nanocrystals [quantum dots (QDs)] have been adopted as a new class of uorescent labels. Six years after the rst experiments of their uses in biological applications, there have been dramatic improvements in understanding surface chemistry, biocompatibility, and targeting specicity. Many studies have shown the great potential of using quantum dots as new probes in vitro and in vivo. This review summarizes the recent advances of quantum dot usage at the cellular level, including immunolabeling, cell tracking, in situ hybridization, FRET, in vivo imaging, and other related technologies. Limitations and potential future uses of quantum dot probes are also discussed. CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . QUANTUM DOTS: FROM PHYSICAL CHEMISTRY TO BIOLOGY . . . . . . . . . . Synthesis and Optical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biocompatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bio-Molecule Conjugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Specicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . QUANTUM DOTS AS IN VITRO PROBES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ImmunolabelingMolecular Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ImmunolabelingSignaling Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Live Cell Markers and Cell Lineage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Motility Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluorescence Resonance Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . QUANTUM DOTS AS IN VIVO PROBES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . OTHER APPLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PROSPECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1523-9829/05/0815-0055$20.00 56 56 56 59 60 60 61 61 61 63 64 65 66 66 68 69 70
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INTRODUCTION
Fluorescence probes are widely used in cell biology. Organic uorophores, the most commonly used probes, suffer from fast photobleaching and broad, overlapping emission lines, and therefore are limited in applications involving long-term imaging and multicolor detection. Advances in synthesis and biofunctionalization of colloidal semiconductor nanocrystals during the past decade have generated an increasingly widespread interest among investigators in the elds of biology and medicine. These nanometer-sized crystalline particles, also called quantum dots (QDs), are composed of periodic groups of IIVI (e.g., CdSe) or IIIV (e.g., InP) materials. They are robust uorescence emitters with size-dependent emission wavelengths. Their extreme brightness and resistance to photobleaching enables the use of very low laser intensities over extended time periods, making them especially useful for live-cell imaging, such as consecutive acquisition of z-stacks for high-resolution three-dimensional (3-D) reconstructions over time [four-dimensional (4-D) imaging]. The intense brightness is also particularly helpful for single-particle detection and an increasing number of biomedical assays. The tunable emission wavelength and distinct emission spectra of QDs facilitates data acquisition and analysis of multiple tagged molecules of interest. Quantum dot chemistry and its early biological applications have been reviewed elsewhere (17). In the past six years, the progress in synthesis and optimization of QDs for biological environments has opened the doors to an expanding variety of biological applications, such as serving as specic markers for cellular structures and molecules, tracing cell lineage, monitoring physiological events in live cells, measuring cell motility, and tracking cells in vivo. Therefore, in this review we summarize recent progress in QD biological and biomedical applications.
QUANTUM DOTS: FROM PHYSICAL CHEMISTRY TO BIOLOGY Synthesis and Optical Properties
The synthesis of monodisperse semiconductor nanocrystals, such as CdSe, CdS, or CdTe, can be achieved by injecting liquid precursors into hot (300 C) coordinating organic solvent (8, 9). Adjusting the amount of precursors and crystal growth time generates QDs of specic sizes (9). The quantum yield of the nanocrystal core synthesized as above is relatively low (less than 10%) (8, 10). Usually, a shell of high band-gap semiconductor material, such as ZnS, is epitaxially grown around the core to achieve the quantum yield of up to 80% (10, 11). QDs, which are only a few nanometers in diameter, exhibit discrete sizedependent energy levels. As the size of the nanocrystal increases, the energy gap also increases, yielding a size-dependent rainbow of colors. Extensive tunability, from ultraviolet to infrared (12), can be achieved by varying the size and the
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composition of QDs (Figure 1), enabling simultaneous examination of multiple molecules and events. For example, small nanocrystals (2 nm) made of CdSe emit in the range between 495 to 515 nm, whereas larger CdSe nanocrystals (5 nm) emit between 605 and 630 nm (8, 12). QDs have several dramatically different properties compared to organic uorophores, one of which is their unique optical spectra. As illustrated in Figure 2, organic dyes typically have narrow absorption spectra, which means they can only be excited within a narrow window of wavelengths. Furthermore, they have asymmetric emission spectra broadened by a red-tail. In contrast, QDs have broad absorption spectra, enabling excitation by a wide range of wavelengths, and their emission spectra are symmetric and narrow. Consequently, multicolor nanocrystals of different sizes can be excited by a single wavelength shorter than their emission wavelengths, with minimum signal overlap.
Figure 2 Excitation (dotted line) and uorescence (solid line) spectra of uorescein (top) and a typical water-soluble QD (bottom). The excitation wavelength was 476 nm and 355 nm for uorescein and QD, respectively. The gure is reprinted with permission from Reference 12, copyright 1998 AAAS.
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Figure 3 Comparison of uorescence intensities between QD 608 (emission at 608 nm) and Alexa 488 after continuous illumination. The gure is reproduced with permission from Reference 14.
QDs are also very stable light emitters owing to their inorganic composition, making them less susceptible to photobleaching than organic dye molecules (4, 12). This feature has been demonstrated in a number of biological labeling experiments where the photostability of QDs was compared with commonly used uorophores, such as rhodamine, uorescein, and Alexa-Fluor (Figure 3) (1219). This extreme photostability makes QDs very attractive probes for imaging thick cells and tissues over long time periodsa challenging task that necessitates collection of multiple optical sections without damaging the specimen. In addition, the two-photon crosssection of QDs is signicantly higher than that of organic uorophores (3, 20, 21), making them quite well suited for examination of thick specimens and in vivo imaging using multiphoton excitation. Another interesting characteristic of QDs is their uorescence lifetime of 10 to 40 ns (4, 22, 23), which is signicantly longer than typical organic dyes or auto-uorescent avin proteins that decay on the order of a few nanoseconds (4). Combined with pulsed laser and time-gated detection, the use of QD labels can produce images with greatly reduced levels of background noise. There are also some photophysical properties of QDs that can, in some cases, be disadvantageous. One of these is the property referred to as blinking, that is, QDs randomly alternate between an emitting state and a nonemitting state. This intermittence in emission of QDs is universally observed from single dots, which imposes some limitations in QD applications requiring single-molecule detection. However, there is limited evidence suggesting that QD blinking can be suppressed on some timescale by passivating the QD surface with thiol moieties (24), or when using QDs in free suspension (20). It has also been reported that QD uorescence intensity increases upon excitation, an event referred to as photobrightening (25,
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26). Although in most cases this property can be advantageous, it is problematic in uorescence quantization studies. Both blinking and photobrightening are linked to mobile charges on the surfaces of the dots, and although the prospects are good that they can be eliminated, for the time being these should be considered as limitations of QDs.
Biocompatibility
The core and core-shell QDs synthesized as described are only soluble in nonpolar solvents because of their hydrophobic surface layer. For QDs to be useful probes for examination of biological specimens, the surface must be hydrophilic. Several strategies have been used to stabilize core-shell nanocrystals in aqueous solutions. The easiest approach is to exchange the hydrophobic surfactant molecules with bifunctional molecules, which are hydrophilic on one side and hydrophobic on the other side, which bind to the ZnS shell. Most often, thiols (-SH) are used as anchoring groups on the ZnS surface and carboxyl (-COOH) groups are used as the hydrophilic ends. Many biological applications of QDs have been achieved by using mercaptohydrocarbonic acids (SH-. . .-COOH) to make QDs water soluble (13, 2732). The long-term stability of the QDs depends on the bond between thiol- and ZnS, which is not strong although it can be enhanced by using two thiol groups instead of one (33, 34). Therefore, the water solubility of the core-shell QDs capped in mercaptocarbonic acids is limited. An alternative approach is to grow a silica shell around the particle, also called surface silanization (12, 25). The rst step of this process involves exchanging the surface ligand with thiol-derived silane such as mercapto-trimethoxysilane (MPS). The trimethoxysilane groups can be cross-linked by the formation of siloxane bonds. During further silica shell growth, other types of silanes can be added to render a different charge and provide functional groups on the surface. Those most frequently used are aminopropyl-silanes (APS), phospho-silanes, and polyethylene glycol (PEG)-silane (2, 25). Because the silica shells are highly cross-linked, silanized QDs are extremely stable. This methods drawbacks are that it is more laborious and the silica shell may eventually be hydrolyzed (4). Recently other solubilization methods have been reported, one of which involves coating the surface with amphiphilic polymers (14, 35). Instead of exchanging the hydrophobic surfactant, the particles in this case are coated with a cross-linked amphiphilic polymer. The hydrophobic tails of the polymer intercalate with the surfactant molecules and the hydrophilic groups stick out to ensure water solubility of the particle. Although this is a general method for nanocrystals grown in different organic solvents, the nal size of the particles after coating is rather large. For CdSe/ZnS QDs, the diameter is between 19 and 25 nm (35), which could place restrictions on many biological applications. Other approaches, such as coating the QDs with phospholipid micelles (19), dithiothreitol (36), organic dendron (37, 38), and oligomeric ligands (39), have also been reported.
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Bio-Molecule Conjugation
To make QDs more useful for probing live cells and other biological applications, QDs need to be conjugated to biological molecules without disturbing the biological function of these molecules. Several successful approaches have been used to link biological molecules to QDs, including adsorption, electrostatic interaction, mercapto (-SH) exchange, and covalent linkage. It has been reported that simple small molecules, such as oligonucleotides (40, 41) and various serum albumins (18), are readily adsorbed to the surface of watersoluble QDs. This adsorption is nonspecic and depends on ionic strength, pH, temperature, and the surface charge of the molecule. Mattoussi and coworkers presented a method of conjugating proteins to QD surfaces using electrostatic interactions. The protein of interest was engineered to fuse with a positively charged domain, which in turn interacted electrostatically with the negatively charged surface of the QD. The protein-QD conjugates prepared in this way were very stable and the uorescence yield was even higher than that from the nonconjugated dots (33, 34). Biological molecules containing thiol groups can be conjugated to the QD surface through a mercapto exchange process (28, 31, 32, 4244). Unfortunately, the same problem of using thiol as anchoring group on a ZnS surface occurs because the bond between Zn and thiol is not very strong and is dynamic. As a result, biomolecules can readily detach from the surface, causing QDs to precipitate from the solution. A more stable linkage is obtained by covalently linking biomolecules to the functional groups on the QD surface using cross-linker molecules (6, 12, 13, 25, 36, 38, 45). If the surface of the nanocrystal bears -COOH, -SH, or -NH2 , it is easy to link it to biological molecules that also have these reaction groups. For example, the cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is commonly used to link -NH2 and -COOH groups, whereas 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC) can be used to cross-link -SH and -NH2 groups (46). Using the above methods, there have been numerous reports of conjugating QDs with various biological molecules, including biotin (12); oligonucleotides (19, 40, 41, 45); peptides (42, 44, 47); and proteins, including avidin/streptavidin (14, 38), albumin (48), and antibodies (13, 14, 17, 44, 49, 50). In most cases, the biological functions of these molecules have not been affected by linkage of QDs.
Toxicity
Concerns have been raised about the toxicity of QDs, especially when they are used to study live cells and animals because they contain elements such as cadmium and selenium. When properly capped by both ZnS and hydrophilic shells, no acute and obvious CdSe QD toxicity has been detected in studies of cell proliferation and viability in live cells (17, 44, 51, 52) and animal (20, 42) models. The most sensitive
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environment in which to test QD toxicity is that of the live embryo because even slight cellular perturbances are manifested as measurable biological phenotypes. The toxicity of QDs was examined in Xenopus embryos (19), where injection of low concentrations of QDs had no negative effects. Although some abnormalities were noted with increasing concentrations, it was not clear whether they resulted from the QDs or changes in the osmotic equilibrium of the cell. In another case, primary hepatocyte cultures were examined because the liver is the major target of cadmium injury (52). Under standard conditions of synthesis and water solubilization, QDs were not toxic. However, cytotoxicity was observed when Cd2+ was released (52). This happened when the QD surface coating was not stable, exposing the CdSe to oxidization by air or UV damage. Therefore, strategies to protect the QD surfaces from oxidative environments are critical for live cell and animal experiments. Coating the QD surface with ZnS eliminates toxicity owing to air exposure but provides only partial protection of the core from UV exposure. Larger molecules, such as bovine serum albumin (BSA) and polymer/streptavidin, further slow down the photooxidation process. However, the potential effect of Cd2+ release over time has not been examined. There remains a pressing need for further investigations into QD toxicity in live animals and safety for diagnostic applications.
Specicity
Specicity is one of the most critical criteria for measuring the value of cellular probes. When QDs were used in early applications, nonspecic binding was reported (12, 36). However, as the surface chemistry has been rened, QD specicity has greatly improved and the number of biological applications has dramatically increased. Because the surface molecule and surface charge of the QDs play an important role in their binding properties (14, 36, 38, 51), manipulating QD coating and surface charge affects their stickiness. Both silica- and mercaptohydrocarbonic acidcoated particles bind nonspecically to cells (12, 36). Alternatively, the use of hydroxyl groups, which have low nonspecic binding to biomolecules, has improved specicity of QD-tagged probes (36, 38). Coating the QDs with a mixture of phospholipids and PEG polymer also prevents aggregation of dots and nonspecic binding, because of the low nonspecic adsorption offered by PEG (19).
QUANTUM DOTS AS IN VITRO PROBES ImmunolabelingMolecular Localization

Fluorescence immunolabeling is widely used in cell biology for probing structure and locating signal transductionrelated molecules. Owing to their robust optical properties, QDs are ideal probes in this area. Since the rst experiment where semiconductor CdSe/ZnS nanocrystals were used to stain F-actin in xed cells (12), investigators have performed a variety of
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Figure 4 Immunouorescence labeling of microtubules for mouse embryo broblast cells (NIH/3T3) using streptavidin-coated QD 605 (left) and streptavidin conjugated Alexa 568 (right). The microtubules were rst incubated with mouse biotinylated anti-tubulin antibody and then streptavidin conjugated QDs or Alexa. The excitation wavelength 488 nm was used to excite QD 605, whereas 543 nm was used for Alexa 568. The emission lter was LP 560 nm for both. As compared to Alexa, QD labeling shows the same labeling pattern of microtubules. The scale bar is 20 m.
experiments in which QDs have been used to localize molecules in cells and tissues, both in live and xed specimens (Figure 4). Kaul and colleagues reported immunouorescence labeling of the heat shock 70 protein, mortalin, using QDs to show different staining patterns in normal and transformed cells (53). Consequently, this protein has been suggested as a reliable marker for normal versus transformed cells. Taking advantage of the high photostability of QDs, Tokumasu & Dvorak were able to collect 40 consecutive optical sections using confocal microscopy and generated a 3-D reconstructed, high-resolution image of the membrane domain band 3 in erythrocytes (54). Ness et al. developed an immunohistochemical (IHC) protocol that combines conventional enzymatic signal amplication and QD labeling to detect intracellular antigens in rat and mouse brain tissue sections. Their study showed that QD IHC labeling had greater sensitivity than similar IHC approaches using conventional dyes (16). Wu and coworkers developed reliable and specic QD probes to localize the breast cancer cell surface marker Her2, cytoskeleton bers, and nuclear antigens in xed cells, live cells, and tissue sections, with a substantial increase in brightness and photostability as compared to organic dyes (14). Minet and colleagues also examined breast tumor cells, using QDs to label membrane glycoproteins to study heat stress effect (55). They found alterations of plasma membrane organization and integrity upon heat stress. All these studies show that QDs have moved beyond the demonstration stage and are excellent probes with enhanced signal-to-noise ratio, extremely high stability, and improved specicity suitable for studying important biological problems.
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ImmunolabelingSignaling Pathways
Research of signaling pathways between and within cells also relies heavily on bright and sensitive uorophores. QDs have begun to play a major role in this eld. Rosenthal et al. reported using serotonin-linked QDs to target the neurotransmitter receptor on the cell surface. The QD probes not only recognized and labeled serotonin-specic neurotransmitters on cell membranes, but also inhibited the serotonin transportation in a dose-dependent manner (43). Although one to two orders of magnitude less potent at inhibiting the receptor than free serotonin, the behavior of QD conjugates was similar to that of free serotonin, making QDs a valuable probe for exploring the serotonin transportation mechanism. Dahan and coworkers studied the dynamics of individual glycine receptors (GlyRs) in neuronal membranes using QDs as uorescent probes (56). The GlyRs were linked to QDs through primary antibody and secondary F(ab ) fragment bridges. The QD labeling enabled the collection of sequential images for up to 20 min, whereas a comparable Cy3 probe lasted only approximately 5 s. By tracking individual dots and analyzing the trajectories, three different locations of GlyRs were characterized and corresponding diffusion coefcients were obtained. It is not surprising that the diffusion coefcients obtained are much larger than those measured with uorescent beads because the QDs are extremely small (1015 nm) compared with the beads (1 micron). The difference in size can signicantly affect the molecules motion. Being more photostable than organic dyes and much smaller than beads, QDs proved to be a suitable probe for single-molecule experiments in living cells, bridging the gap between large beads and small organic uorophores. Other signaling pathways, such as erbB/HER receptor-mediated signal transduction, have also been examined using QDs (57). Epidermal growth factor (EGF) conjugated to QDs is still capable of binding to and activating its receptor, the erbB1 receptor, which triggers internalization of both EGF-QD and its receptor via endocytosis. In this case, examination of single QDs enabled discovery of a retrograde transport process in which the EGF-QD, after binding to the lopodium of the cell, moves toward the cell body at a velocity of 10 nm/s. Owing to the photostability of QD, EGF-QD binding and internalization kinetics were obtained, the latter being the rate-limiting process (57). Such quantitative understanding of the transduction mechanism is essential for receptor-targeted therapeutics. QDs will be a valuable reagent for this kind of investigation. The process of cell endocytosis has also been studied using QDs (58). In this experiment, the endocytosis efciency of 15 nm QD conjugated sugar balls was compared with the efciency of 5 nm and 50 nm particles, revealing that endocytosis is highly size dependent. The 15 nm QDs were less effective at triggering endocytosis than were 50 nm particles, but more effective than 5 nm particles (58). The QD conjugated sugar ball was an excellent size marker for studying the size effect of endocytosis in the viral size region, and thus provided useful information for the design of articial molecule delivery systems for gene and drug delivery.
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Live Cell Markers and Cell Lineage

Because QDs are extremely bright and photostable, they can be used as cell markers for long-term studies such as cell-cell interactions, cell differentiation, and cell lineage tracking. Most experiments conducted to date have shown that QDs do not interfere with normal cell physiology and cell differentiation (19, 51, 59). The idea to use QDs as cell markers is based on the discovery that QDs can be internalized by cells, by either receptor-mediated (13) or nonspecic endocytosis (4, 17, 18, 51). The exact pathway of incorporating QDs into cells under endocytosis is not understood. However, after QDs enter cells, they accumulate in vesicles in the perinuclear region (17, 18, 51). Perinuclear vesicles lled with nanocrystals were rst seen by multiphoton microscopy (51). Later, with the use of an endosome-specic marker, such as uorescein-labeled dextran (18) or pECFP (17), it was conrmed that QDs were indeed accumulated in the endosomes or lysosomes and not coating them on the basis of colocalization of QDs and endosome markers. Once internalized, QDs divided with the daughter cells at cell division (18, 19, 51). There are several other approaches for delivering QDs into live cells. One of these was developed by Matteakis and coworkers (59), who used a peptidemediated transportation method to incorporate different colored QDs into a variety of live mammalian cells, generating a unique and spectrally resolvable code for each cell type. The carrier for delivering QDs into the cells is the protein translocation domain Pep-1, an engineered 21-residue peptide composed of a hydrophobic sequence for QD binding and a hydrophilic lysine-rich sequence from the nuclear localization sequence for penetrating cells (59). Cells can also be loaded with QDs by microinjection. Dubertret et al. (19) microinjected phospholipid-coated QDs into early-stage Xenopus embryos and tracked the embryogenesis using uorescence visualization. Compared to nonspecic endocytosis or peptide delivery, microinjection is more laborious and less efcient. However, it is worthwhile noting that microinjected QDs were homogenously distributed throughout the cell and were not compartmentalized (19). Internalized QDs are powerful probes for long-term studies of cell-cell interactions. Our group examined the interactions of human mammary epithelial tumor cells with normal cells growing in a 3-D culture system using QDs as cell-type identication markers. With different color-emitting QDs, we were able to unequivocally identify the tumor cells and normal cells in a coculture system. When cultured in the 3-D matrix, human mammary epithelial cells form acini, polarized clusters of cells that resemble natural glandular tissue. Each acinus develops from a single cell during a 1014 day time span. If the original cell was preloaded with QDs, all cells in the acinus will subsequently contain a subset of those QDs. This enabled us to add tumor cells tagged with QDs of a different emission wavelength to the acini and clearly distinguish normal cells from tumor cells during two-week culture periods. We observed that tumor cells migrate toward the acini, extend invadopodia to contact the acini, and subsequently undergo programmed cell death (60) (Figure 5). Death is always contact dependent and is induced by polarized
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cells, those organized into acini, but not individual unpolarized cells. The high photostability of QDs enabled us to track and image these cocultures for up to two weeks, which cannot be achieved by organic dyes.
Cell Motility Assay

Motility and migration of cancer cells are measurable properties that are associated with metastases and the formation of secondary tumors. Present methods for measuring cell patterns and the extent of cell movement suffer from a number of practical limitations. We developed an assay based on our nding that cells nonspecically incorporate QDs as they crawl over them, leaving behind QD-free zones representing the pattern of phagokinetic uptake of QDs (Figure 6) (51). Similar phagokinetic cell motility assays using gold colloids or polystyrene micro beads have been used with some degree of success (61, 62), but typically require xation of the cells. The use of QDs facilitates monitoring live cells, both before and after perturbations, such as the addition of potential chemotherapeutic agents to monitor
Figure 6 QDs were used to study cell motility. Human mammary epithelial tumor cells, MDA-MB-231, were grown on top of a collagen layer that had been coated with a thin layer of silanized QDs. While cells migrated across the layer, they engulfed the nanocrystals and left areas that were uorescence-free. The scale bar is 200 m. The gure was reproduced with permission from Reference 51.
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changes in motility. We also found that the quantity of QDs incorporated varies among different cell types. For example, MDA-MB-231 tumor cells uptake more QDs than nontumorigenic MCF 10A cells (51). The QD assay provides a useful measure of the invasive potential of cells (63), and it is much more sensitive than the conventional Boyden chamber assay with signicantly reduced specimen processing (6466). Unlike the Boyden chamber assay, which is time-consuming and uses xed cells, the QD-based assay requires no xation, using uorescent detection in live cells. These features make it a powerful new tool for cell motility studies.
In Situ Hybridization
Fluorescent in situ hybridization, also called FISH, utilizes uorescently labeled DNA probes for gene mapping and the identication of chromosomal abnormalities. FISH provides researchers with a method to visualize and map the genetic material in cells, including specic genes or portions of genes, and a way to quantify gene copy numbers within tumor cells that have abnormal gene amplication. However, use of the rapidly photobleaching organic probes compromises the experiments and requires immediate imaging. The use of organic dyes in multicolor FISH also has limitations, including color overlap and differential intensity of the uorophores. These drawbacks can be resolved with the use of QDs. The in situ hybridization procedure requires DNA-linked probes. Typical probes that have been used include DNA conjugated to uorescent molecules (67), gold (45, 68), and microbeads (69, 70). As might be expected, DNA or oligonucleotides can also be conjugated to QDs (19, 28, 36, 40, 41, 45, 51) and in vitro testing demonstrates that these conjugates retain their ability to form complementary sequences of Watson-Crick base pairs (51). In 2001, Pathak and coworkers used QD-based FISH labeling to detect Ychromosome-specic repeats in xed human sperm cells. Although the results show the success of hybridization using QD probes, high salt conditions inside the cells caused partial aggregation of the nanocrystals, which gave rise to varied signal intensities (36). Later in 2004, Xiao et al. reported using a QD FISH probe to analyze human metaphase chromosomes. The experiment was performed with total genomic DNA as the probe, human metaphase lymphocytes as the hybridization substrate, and the ebrB2/Her2/neu gene as the target of hybridization. They compared QD, Texas-Red, and FITC probe detection, demonstrating that the QD probe was more photostable and provided higher signal-to-noise ratio than the organic probes.
Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) is a process in which energy is transferred from an excited donor to an acceptor via a resonant, near-eld dipoledipole interaction (71, 72). FRET is very sensitive to the distance between donor and acceptor and has been used to study biomolecule conformation, dynamics, and interactions. Some problems of using conventional dyes for FRET include
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fast photobleaching and signicant emission overlap between donor and acceptor. QDs provide a potential solution to these problems. In 1996, resonance energy transfer between closely packed CdSe QD solids was reported by Kagan et al. (73, 74). Quenching the luminescence of small dots was accompanied by an enhancement of the luminescence of large dots. This was consistent with resonance energy transfer from the small dots to the large dots. Later experiments demonstrated resonance energy transfer between QD donor/acceptor and organic dye acceptor/donor (75, 76). One potential limitation of using QDs for FRET involves the physical dimensions of nanocrystals. Owing to the size of QDs, the distance between donor and acceptor is usually greater than 3 nm (77). In addition, caution must be taken with single-molecule experiments that might be complicated by the QDs blinking. There are several examples of QDs used for FRET in biological systems. Willard et al. reported using QDs as a FRET donor in a protein-protein binding assay (31). QDs were conjugated to BSA as the FRET donors and tetramethylrhodamine (TMR) was linked to the protein as the acceptor. Enhanced TMR uorescence was observed as energy transferred between the QDs and the TMR. Medintz and coworkers engineered a histidine tag on maltose binding protein (MBP) that nally bound QDs, which served as the FRET donor (77). With a quencher bound to the maltose-binding site, QD uorescence was inhibited but was recovered by adding maltose to displace the quencher. In 2004, the same group did an in-depth study using the MBP system by varying the QD size and the quantity of acceptor dye (78). Increasing the amount of acceptor dye, or the degree of spectral overlap (by changing QD size), caused a substantial enhancement in energy transfer efciency. This study demonstrated that QDs can be used as efcient energy donors in the FRET system and showed that by tuning their size, QDs can transfer energy to a number of organic dye molecules. In another paper, these same researchers demonstrated reversible modulation of QD uorescence using FRET with the photochromic molecule BIPS (1,2,3-dihydro-1 2-(2-carboxyethyl)-3,3-dimethyl6-nitrospiro-[2H-1-benzopyran-2,2,2-(2H)-indoline]) (79). BIPS was converted to colored merocyanine when exposed to UV light and quenched QD emission by acting as a FRET acceptor. When exposed to white light, BIPS was converted back to colorless spiropyran and QD emission was recovered. By switching the QD emission on and off, it is possible to use this type of system as a nanosensor device for sensing photochromical changes. In addition to protein binding assays, other processes like DNA replication and telomerization can also be probed by QD-based FRET as reported by Patolsky and coworkers (80). In DNA telomerization, QDs were conjugated to a DNA template molecule and mixed with dNTP (N = A, C, G) and Texas-ReddUTP. After adding telomerase, FRET transition occurred between the QD donor and Texas-Red acceptor. A similar FRET process occurs when using QD conjugated primers to initiate DNA replication. These results indicate the potential use of the QD FRET process in fast and sensitive DNA detection and DNA array analyses.
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QUANTUM DOTS AS IN VIVO PROBES

Colloidal semiconductor nanocrystals have a two-photon cross-section that is two to three orders of magnitude greater than organic dyes (20). A two-photon process requires about twice the excitation wavelength of a single-photon excitation. Longer wavelengths, extending into the infrared region, can be utilized to excite chromophores in a single quantum event. Therefore, multiphoton microscopy enables the imaging of structures deep within biological tissues with minimum photobleaching and photodamage. The high two-photon cross-section of QDs allows for more efcient probing of thick specimens and in vivo imaging when using multiphoton excitation microscopy. Two in vivo experiments appeared almost simultaneously in 2002. In one paper, Akerman and colleagues used QDs to target tumor vasculature in mice (42). The nanoparticles, conjugated to several peptides that differentially recognized blood vessels located in the lung, tumor blood vessels and tumor lymphatic vessels, were injected into mice. Histological staining revealed that QDs were delivered to the appropriate site in vivo, guided by the peptides. In the other paper, Dubertret et al. microinjected phospholipid-coated QDs into early-stage Xenopus embryos to study the behavior of specic cells during embryogenesis (19). In both cases, QDs provided a stable, robust uorescent probe that could be used in vivo over extended periods of time. Live animal imaging using QD uorescence with multiphoton microscopy was achieved by Larson et al. in 2003 (20). QDs, intravenously injected into mice, were detectable through intact skin at the base of the dermis (100 micron) using an excitation wavelength of 900 nm. To optimize the conditions of in vivo experiments, Ballou et al. tested QDs with different polymer coating in vivo using various imaging techniques, including light and electron microscopy on tissue sections and noninvasive whole-body uorescence imaging (81). Amazingly, these QDs maintained their uorescence even after four months in vivo. Although the QDs showed no deleterious effects upon the animals in these studies, a more detailed evaluation of potential QD toxicity in the body is warranted prior to their long-term usage in higher organisms. Recently, Gao et al. reported in vivo cancer targeting and imaging using QDs (21). They conjugated QDs to the antibody specic for the prostate cancer cell marker PSMA. After injection into mice that had been transplanted with human prostate cancer cells, the QD-tagged PSMA antibodies recognized and bound at the tumor site and were clearly imaged in vivo. Owing to the QDs large absorption coefcient and long lifetime, in vivo imaging of QDs was much brighter and more sensitive than imaging with green uorescence protein (GFP) (Figure 7), which was totally buried in autouorescence and background. The use of QDs during surgical procedures was demonstrated by Kim and coworkers (82), who mapped sentinel lymph nodes (SLN) with near-infrared uorescent QDs in mouse and pigs. QDs were injected intradermally into the animal,
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entered the lymphatic system, and were followed using an intraoperative imaging system. The surgeon followed the ow of QDs in real time with NIR QD image guidance, and quickly identied the position of the SLN in a precise and rapid surgical procedure. The use of QDs as cell tracers after transplantation into mice has also been reported (83). The above studies have shown the great potential of using QDs as in vivo probes for cancer studies, drug delivery, and noninvasive whole-body imaging. It is worth noting that the degradation and metabolism of QDs in the body remains to be investigated and there are reports that QDs injected can accumulate in the kidney, liver, and spleen (21, 42). Whether the QDs can ultimately be cleared from the body is not known. More research in this area needs to be completed before QDs can be used as probes in the human body.
OTHER APPLICATIONS
QDs have also been found to be useful in the study of microorganisms. Kloepfer et al. reported targeting QD conjugates to surface glycoproteins of bacteria and fungi (26). QDs were also used as cell membrane permeable indicators for Escherichia coli (84). The luminescence of QDs can be affected by ionic environments and pH (8588). Cadmium, zinc, and manganese ions increase the luminescence of CdS nanocrystals in basic solution, whereas copper ions quench their emission (89). Chen et al. reported that by changing the capping layer, the luminescence of CdS QDs selectively responded to the presence of zinc or copper ions. Combined with magnetic nanoparticles, QDs have been demonstrated to be useful for cell detection and separation, as reported by Wang et al. The nanocomposite particles, made up of magnetic Fe2 O3 superparamagnetic cores and CdSe/ZnS QD shells, were used to magnetically separate breast cancer cells, which were then detected by uorescence (90). With some technical improvements, this new technique might be useful for in vitro cancer diagnosis in the future. QDs have also been investigated for use in drug delivery. Lai et al. used surfacemodied CdS QDs as chemically removable caps to keep pharmaceutical drug molecules and neurotransmitters inside a mesoporous silica nanosphere-based system. The CdS cap ensures the drug is inside the system until triggered by disulde bondreducing reagents. It is interesting to note that QDs here play a role as a size-dened cap and not as a uorescent molecule (91). Localization of specic biomolecules in cells and tissues at a high resolution provides both structural and quantitative information for molecular cell biology. However, popular immunouorescence labeling is limited in spatial resolution. Because QDs are composed of heavy elements such as cadmium, it is possible to use them as contrast or labeling probes in transmission electron and X-ray microscopy for high-resolution imaging of cells. Nisman et al. used CdSe QDs as probes in both conventional and energy-ltered TEM, demonstrating the feasibility of
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Figure 8 (A) Transmission electron microscopy detection of nuclear promyelocytic leukemia protein (PML) within a Hep-2 PML I cell section with QD labeling. QDs are highlighted with arrows. (B) Electron spectroscopic imaging shows the distribution of discrete QDs within the PML body. The image was generated by dividing a cadmium postedge (510 eV) image by its preedge (415 eV) image after alignment by cross-correlation. The scale bar is 50 nm. A and B were reproduced with permission from Reference 92. (C) Soft X-ray microscopy image of QDs in liposomes. QDs are highlighted with arrows. The X-ray image was taken as described in Reference 93.
labeling the nuclear promyelocytic leukemia (PML) protein on cell sections to obtain correlative uorescence and TEM images. To acquire chemical and structural information and to get better signal-to-noise ratio, electron spectroscopic imaging (ESI) was used to identify the elemental map of cadmium without interference from other elements such as nitrogen (Figure 8). Also, we have demonstrated the ability to detect liposome-encapsulated QDs using soft X-ray microscopy (C. Larabell, unpublished data).
PROSPECTIVE
There are numerous unexplored possibilities to expand the repertoire of QD labeling in the future. One area of biological research that has not yet been addressed with these novel nanostructures is molecular polarity. Semiconductor nanorods (quantum rods), which demonstrate polarized emission properties, might be potent tools for such experiments. The multilevel imaging on both the medical and cellular scales demonstrates the need for development of nanoparticles that can be used in all kinds of imaging techniques, such as magnetic resonance imaging, PET or SPECT, optical confocal microscopy, X-ray microscopy, and TEM. With the completion of the Human Genome Project, it is timely to try to identify specic genes involved in disease and the relationship of gene regulation to cell function. Cellular probes that can identify a single gene, RNA, or protein at a specic time and location within a cell will facilitate an understanding of cellular function at the molecular level. QDs, with their intense luminosity and high photostability, are the best candidate for this research. Tuning the chemistry to avoid
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sample aggregation within cells and optimizing imaging and detection conditions for single QD experiments will be the key to achieve this goal. Recent studies have shown the potential for the use of QDs in cancer diagnosis and therapy. Thus, there is an urgent need to understand QD toxicity and metabolism in the body. Although it is possible to modify QD surfaces so that QDs are cleared from the body within a reasonable time, it will be more difcult to replace the toxic elements of QDs while keeping their desirable optical properties intact. ACKNOWLEDGMENTS We thank Dr. Yi Cui, Aihua Fu, and Christine Micheel for helpful discussions. We are grateful to Rosanne Boudreau and Benjamin Engel for proofreading the manuscript. This work was supported (in part) by NIH National Center for Research Resources through the University of California, Los Angeles, subaward agreement 0980GFD623 through the U.S. Dept. of Energy under Contract No. DE-AC0376SF00098. The Annual Review of Biomedical Engineering is online at http://bioeng.annualreviews.org LITERATURE CITED
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Figure 1 Emission spectra of several semiconductor nanocrystals showing their size- and composition-dependent emission character. Red, green, and blue series represent different-sized InAs, InP, and CdSe nanocrystals, respectively. The sizes of the nanocrystals are indicated above their corresponding spectra. The figure is reprinted with permission from Reference 12, copyright 1998 AAAS.
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Figure 5 QDs were used to study mixed cell interactions in a 3-D matrigel culture system. (A) Human mammalian epithelial MCF 10A cells (tagged with green-emitting silica coated QDs) form acini structures after growing in growth-factor reduced matrigel for 10 days. (B) After the acini were formed, human breast tumor MDAMB-231 cells (tagged with red-emitting silica coated QDs) were added to the culture. After 1416 h of incubation, the tumor cells had attached to the acinis basement membrane. (C) The contact was fatal to the tumor cells, which were found dead surrounding the MCF 10A organoid. Most of the tumor cells had lysed, leaving transparent ghosts loosely attached to the organoid, but a few newly attached cells still retained red-emitting QDs. (D) The MCF-10A organoid and all invading tumor cells; it is a superimposition of all sections, displaying the sharp edge of each cell followed by a projection of color-coded depth information so that red is the uninvolved lower portion of the MCF-10A organoid and the tumor cells are shades of orange through green. The scale bar is 10 m. The figure was reproduced with permission from Reference 60.
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Figure 7 In vivo imaging using QDs. (a) Sensitivity comparison between QD-tagged and green fluorescence protein (GFP) transfected cancer cells. QD-labeled cells (orange) were injected on the right flank of a mouse, whereas the same number of GFP-labeled cells (green) were injected on the left flank (circle) of the same animal. (b) Simultaneous in vivo imaging of multicolor QD-encoded microbeads, which were injected at three adjacent locations on a host animal. The figure was reprinted with permission from Reference 21.
Annual Review of Biomedical Engineering Volume 7, 2005
CONTENTS
FRONTISPIECE, Werner Goldsmith WERNER GOLDSMITH: LIFE AND WORK (19242003), Stanley A. Berger,
Albert I. King, and Jack L. Lewis
xii 1 21 55 77 105
DNA MECHANICS, Craig J. Benham and Steven P. Mielke QUANTUM DOTS AS CELLULAR PROBES, A. Paul Alivisatos, Weiwei Gu,
and Carolyn Larabell
BLOOD-ON-A-CHIP, Mehmet Toner and Daniel Irimia BIOCHEMISTRY AND BIOMECHANICS OF CELL MOTILITY, Song Li,
Jun-Lin Guan, and Shu Chien
MOLECULAR MECHANICS AND DYNAMICS OF LEUKOCYTE RECRUITMENT DURING INFLAMMATION, Scott I. Simon
and Chad E. Green 151 187 223 255 287 327 361
DETERMINISTIC AND STOCHASTIC ELEMENTS OF AXONAL GUIDANCE,

Susan Maskery and Troy Shinbrot
STRUCTURE AND MECHANICS OF HEALING MYOCARDIAL INFARCTS,

Jeffrey W. Holmes, Thomas K. Borg, and James W. Covell
INSTRUMENTATION ASPECTS OF ANIMAL PET, Yuan-Chuan Tai

and Richard Laforest
IN VIVO MAGNETIC RESONANCE SPECTROSCOPY IN CANCER,

Robert J. Gillies and David L. Morse
FUNCTIONAL ELECTRICAL STIMULATION FOR NEUROMUSCULAR APPLICATIONS, P. Hunter Peckham and Jayme S. Knutson RETINAL PROSTHESIS, James D. Weiland, Wentai Liu, and Mark S. Humayun INDEXES
Subject Index Cumulative Index of Contributing Authors, Volumes 17 Cumulative Index of Chapter Titles, Volumes 17
403 413 416
ERRATA
An online log of corrections to Annual Review of Biomedical Engineering chapters may be found at http://bioeng.annualreviews.org/ v

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Annu. Rev. Biomed. Eng. 2005. 7:5576 doi: 10.1146/annurev.bioeng.7.060804.100432 Copyright c 2005 by Annual Reviews.

QUANTUM DOTS AS CELLULAR PROBES

QUANTUM DOTS AS CELLULAR PROBES

QUANTUM DOTS AS CELLULAR PROBES

QUANTUM DOTS AS CELLULAR PROBES

QUANTUM DOTS AS IN VITRO PROBES ImmunolabelingMolecular Localization

QUANTUM DOTS AS CELLULAR PROBES

Live Cell Markers and Cell Lineage

QUANTUM DOTS AS CELLULAR PROBES

Cell Motility Assay

Fluorescence Resonance Energy Transfer

QUANTUM DOTS AS CELLULAR PROBES

QUANTUM DOTS AS IN VIVO PROBES

QUANTUM DOTS AS CELLULAR PROBES

QUANTUM DOTS AS CELLULAR PROBES

QUANTUM DOTS AS CELLULAR PROBES

QUANTUM DOTS AS CELLULAR PROBES

Annual Review of Biomedical Engineering Volume 7, 2005

DETERMINISTIC AND STOCHASTIC ELEMENTS OF AXONAL GUIDANCE,

STRUCTURE AND MECHANICS OF HEALING MYOCARDIAL INFARCTS,

INSTRUMENTATION ASPECTS OF ANIMAL PET, Yuan-Chuan Tai

IN VIVO MAGNETIC RESONANCE SPECTROSCOPY IN CANCER,

403 413 416

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