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Noninvasive Imaging of Quantum Dots in Mice Byron Ballou,*,†,‡ B. Christoffer Lagerholm,† Lauren A. Ernst,† Marcel P. Bruchez,§ and Alan S. Waggoner†,‡ Molecular Biosensor and Imaging Center, and Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213 and Quantum Dot Corporation, 26118 Research Road, Hayward, California 94545. Received August 29, 2003; Revised Manuscript Received November 23, 2003
Quantum dots having four different surface coatings were tested for use in in vivo imaging. Localization was successfully monitored by fluorescence imaging of living animals, by necropsy, by frozen tissue sections for optical microscopy, and by electron microscopy, on scales ranging from centimeters to nanometers, using only quantum dots for detection. Circulating half-lives were found to be less than 12 min for amphiphilic poly(acrylic acid), short-chain (750 Da) methoxy-PEG or long-chain (3400 Da) carboxy-PEG quantum dots, but approximately 70 min for long-chain (5000 Da) methoxy-PEG quantum dots. Surface coatings also determined the in vivo localization of the quantum dots. Long-term experiments demonstrated that these quantum dots remain fluorescent after at least four months in vivo.
INTRODUCTION
Quantum dots (QDs) are stable, bright fluorophores that, under ideal conditions, can have high quantum yields, narrow fluorescence emission bands, high absorbency, very long effective Stokes shifts, high resistance to photobleaching, and can provide excitation of several different emission colors using a single wavelength for excitation (1). Because QDs have size-tunable narrow emission bands, in addition to single-wavelength excitation, QDs are suitable for detecting multiple ligands in a single experiment. Multiplexing using several colors and intensities of quantum dots to code assay supports (“barcoding”) would allow the detection of many distinct ligands in single experiments (2-4). Moreover, QDs may be readily imaged using two-photon microscopy in vitro and in vivo (5). However, commercially available materials do not yet offer many of the potential advantages of QDs. The chief hindrances to routine use have been difficulties in reproducible production of QDs in quantity and the lack of suitable surface coatings that maintain high quantum yields, confer stability in aqueous solution, and can be chemically manipulated for biological applications. Bruchez et al. (6) and Chan and Nie (7) first reported the use of quantum dot conjugates for labeling biological specimens. In these experiments, QDs were coated with silica and mercaptoacetic acid layers, respectively. Both groups showed specific labeling by covalent coupling of ligands to these surfaces. Subsequently, several authors have shown labeling of whole cells and tissue sections using several different surface modifications of QDs (1, 8-11). A variety of colors of Qdot conjugates for immunospecific labeling are now available commercially from Quantum Dot Corporation (www.qdots.com). * To whom correspondence should be addressed. E-mail:
[email protected]. † Molecular Biosensor and Imaging Center, Carnegie Mellon University. ‡ Department of Biological Sciences, Carnegie Mellon University. § Quantum Dot Corporation.
Quantum dot preparations reported to date have not been optimized for in vivo studies. For in vivo imaging, QDs must have adequate circulating lifetime, must show minimal nonspecific deposition, and must retain their fluorescence for a sufficiently long time. We report that QDs coated using an amphiphilic poly(acrylic acid) polymer are stable in vivo, that changing surface chemistry prolongs circulating lifetime and reduces nonspecific deposition in vivo, that targeting to tissues may be observed by noninvasive whole body fluorescence, and that QDs may be visualized in tissue sections by light and electron microscopy many weeks after injection. MATERIALS AND METHODS
Quantum Dots. Quantum dots were supplied by Quantum Dot Corporation, Hayward, CA 94545. Coreshell zinc sulfide-cadmium selenide QDs emitting at 606, 635, 645, and 655 nm were coated with an amphiphilic poly(acrylic acid) polymer (amp QDs (9)) or the same coating conjugated with methoxy- or carboxy-terminated poly(ethylene glycol) amine (mPEG-750 QDs, mPEG5000 QDs, COOH-PEG-3400 QDs). Quantum dots have spectra that exhibit an absorption peak at a wavelength slightly shorter than the emission wavelength (“longest wavelength peak.”) At wavelengths shorter than this peak, absorbance first declines, then rises steeply at shorter wavelengths, with no definite peak. Absorption and excitation spectra are nearly identical; thus QD’s may be excited at any wavelength shorter than the emission maximum (e.g., (6)). QD concentration was determined by absorbance using appropriate extinction coefficients (for example, 800 000 M-1 cm-1 at 638 nm for the 655 materials and 650 000 M-1 cm-1 at the longest wavelength peak for the 605 materials). These values agree with concentrations measured by a variety of distinct techniques including elemental analysis, stoichiometric gel shift assays, and fluorescence correlation spectroscopy. Quantum Yield Determination. Quantum yields were measured by comparing integrated fluorescence intensity of absorbance matched solutions in an intensity
10.1021/bc034153y CCC: $27.50 © 2004 American Chemical Society Published on Web 12/30/2003
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Table 1. Optical Properties and Circulating Half-Lives of Surface-Modified QDs. See Methods for Details QD surface coating molar extinction, 450 nma emission maximum, nm quantum yield circulating half-life, imaging (min) circulating half-life, venipuncture (min)
amp
mPEG-750
COOH-PEG-3400
mPEG-5000
4.5 × 106 655 0.52 ND 4.6 ( 1.0
4.1 × 106 645 0.28 6.1 ( 2.9b 3.2 ( 0.6
4.6 × 106 655 0.38 ND 12.6 ( 4.9
6.8 × 106 655 0.89 141 ( 45 71 ( 20
a See the discussion of QD optical properties at http://www.qdots.com. These data are from the QD preparations used in this paper and are not corrected for scatter. b Weighted average of two-exponential fits to data; in the original fits, the fast component was 2.2 ( 2.5 min; the slow component was 9.8 ( 9.1 min.
calibrated Ocean Optics S2000 fluorimeter. The absorbance of the solutions was fixed at 0.03 ( 0.003 AU at the excitation wavelength. Red QDs (576-655) were compared to Rhodamine 101 in methanol (QY 0.95) with excitation at 530 nm. The integrated fluorescent intensities were scaled according to the absolute absorbance of the sample and the standard, and the ratio of these values multiplied by the reported standard quantum yield gives the absolute sample quantum yield. Properties of the quantum dots used in this paper are shown in Table 1. PEG Conjugation. Poly(ethylene glycol) derivatives were acquired from Shearwater Polymers Corp (Huntsville, AL 35806; COOH-PEG-3400-amine, mPEG-5000amine) and from Sigma-Aldrich (Milwaukee, WI 53201; mPEG-750-amine). Other reagents were supplied by Sigma. These reactions have been carried out at a variety of scales, but the 5 mL scale reaction is described here for the mPEG-750 amine. Quantum dots were dispersed at 1.0 uM in 50 mM sodium borate, pH 8.0, with a molar ratio of 2000:1 of PEG-amine (7.5 mg). These materials were then reacted with a 1500:1 molar ratio of Ndimethylaminopropyl-N′-ethylcarbodiimide (EDC, 1.4 mg) for 2 h followed by purification using a 100 kDa centrifugal ultrafiltration device (Vivascience, Edgewood, NY). The number of PEG-amine molecules coupled to each quantum dot was determined by conducting the coupling reaction in the presence of an excess of PEG-amine, then reacting the residual amines with fluorescamine reagent. This reagent produced a blue fluorescent species that was monitored at 490 nm and compared to the signal produced from the materials without EDC activation. The % retained signal corresponds to the remaining excess amine that did not react with the quantum dots. The difference was assumed to have completely reacted with the quantum dots, so the concentration of consumed amines (initial concn × (final signal/initial signal)) divided by the concentration of quantum dots yielded the number of amines reacted per quantum dot. For all PEG-substituents, we attempted to saturate available carboxyl groups. The levels of substitution were approximately 300 residues/QD for mPEG-750, and 100 residues/QD for COOH-PEG-3400 and mPEG-5000. The final materials were filtered through a 0.2 µm filter unit and prepared at an appropriate concentration for tail vein injection prior to use. Each dose was adjusted to 0.14 M NaCl by addition of 1.4 M NaCl just prior to use. Mice. Balb/C and athymic nude mice were obtained from Harlan Sprague Dawley (Indianapolis, IN, 46229); mice were used at ages from 6 to 12 weeks without any noticeable effect of age. For all results shown in this paper, QDs (360 pmol) in 200 µL normal saline were injected into the tail veins of mice (typically 18 g; thus 180 nM QD, 20 pmol QD/g animal weight). In other experiments, doses ranging from 50 to 500 pmol in 50-
200 µL normal saline were administered, with no significant dose effect on circulating time or distribution. Mice were anesthesized for imaging either by pentobarbital intraperitoneally to effect, or by inhalation anesthesia using isofluorane (12). For timed measurements, animals were first anesthetized, then injected while under the camera. To compare whole-blood lifetimes to lifetimes derived from images, mice were bled by saphenous venipuncture (13). All operations on animals were in accord with institutional animal use and care regulations. Macro Imaging. Whole body images were acquired on a Photometrics (Tucson, AR) C258 cooled CCD camera equipped with a custom filter holder and lens adapter (Bioptechs, Butler, PA 16002). Animals were illuminated using four 250-W quartz-halogen illuminators (Cuda Products, Jacksonville, FL 32217) equipped with halfinch fiber optic bundles terminating in filter holders (Edmund Industrial Optics, Barrington, NJ 08007). Excitation filters were 460/50 nm, and emission filters were 20 nm wide band-pass filters, centered on the emission maxima of the QDs. All filters were provided by Chroma Technologies (Brattleboro, VT. 05301). Imaging times varied with time postinjection and were adjusted on the fly to give maximum dynamic range, but were typically 0.5 s; for time series, three images were acquired in rapid succession at 1-min intervals; acquisition times of 0.2, 1.0, and 5 s were normally used to bracket the large range in intensities between different sites of deposition. Microscopy. Light and fluorescence images were acquired on an inverted Zeiss Axiovert 135TV microscope (Zeiss, Thornwood, NY 10594) equipped with a Hamamatsu ORCA II cooled CCD camera (Hamamatsu, Bridgewater, NJ 08807). Fluorescence images of quantum dots were acquired with a 440/20 nm excitation filter and appropriately selected 20 nm wide band-pass emission filters, centered on the emission maxima of the quantum dots. Tissue samples for light microscopy were 10 µm frozen sections. Sections were cut on a cryostat, fixed for 10 s in methanol, dipped for 1 min in Gill’s 2X hematoxylin, briefly rinsed and then developed in Scott’s solution. Electron Microscopy. Tissue specimens were fixed in 2% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.4. The tissue was washed with 3 changes of 0.1 M sodium cacodylate buffer, pH 7.4, and postfixed for 1 h in a solution containing 1% OsO4 buffered with 0.1 M sodium cacodylate, pH 7.4, then washed in three changes of dH2O and dehydrated in EtOH. Propylene oxide was used as a transitional solvent. The tissue was infiltrated overnight in a 1:1 mixture of Epon-Araldite and propylene oxide. The following day, the 1:1 mixture was removed and replaced with 100% Epon-Araldite. The specimens were infiltrated an additional 8 h, placed in embedding capsules, and polymerized for 48 h at 60 °C.
Quantum Dots in Mice
The embedded specimens were thin sectioned (0.1 µm), then stained with 1% uranyl acetate and Reynolds lead citrate. The sections were viewed on a Hitachi H-7100 TEM operating at 50 kV. Digital images were obtained using an AMT Advantage 10 CCD Camera System and NIH Image software. Image Analysis. All image analysis was performed in ImageJ (http://rsb.info.nih.gov/ij/) while curve fitting was done using Mathematica (Wolfram Research, Champaign, IL; 61820; http://www.wolfram.com/). To analyze circulating lifetimes from bleeds, macro fluorescence images were acquired using ∼20 µL blood samples in 1 mm × 0.1 mm bore rectangular glass capillary tubes (VitroCom, Mountain Lakes, NJ 07046). Fluorescence intensity was measured using a rectangular region of interest over the body of the capillaries; background was determined using an identical region of interest over an adjacent noncapillary area. The mean fluorescence intensity was determined and the resulting background corrected data was curve fit to single exponentials. To obtain circulating lifetimes by whole mouse imaging, mice were imaged at 1 min intervals postinjection for 1-3 h (Figure 1). Lifetime data were then generated by selecting nine 2 × 2 pixel regions of interest per mouse, where the regions of interest were selected to coincide with conspicuous blood vessels, and by tracking them through time. Background due to intrinsic fluorescence was normally negligible; subtraction of fluorescence in adjacent tissue made little or no difference to the slope of the intensity versus time plot. The resulting background-corrected data was fitted to one- and twoexponential curves with the two-exponential curve providing a better fit for mPEG-750 QDs, as evidenced by F-statistics. However, mPEG-5000 QD data showed no improvement using a two-exponential fit as compared to a one-exponential fit. RESULTS
Core-shell cadmium selenide-zinc sulfide quantum dots having maximum emission wavelengths at 606, 630, 645, and 655 nm were supplied by Quantum Dot Corporation. The core-shell size (and hence emission wavelengths) of the QDs had no significant effects on tissue deposition or circulating lifetimes. Four different surface coatings were used: an amphiphilic poly(acrylic acid) primary coat (amp) that stabilizes the quantum dots in aqueous solution (9), and quantum dots having the amp coating further coupled to methoxy-terminated poly(ethylene glycol), molecular weight 750 (mPEG-750), carboxy-terminated poly(ethylene glycol), molecular weight 3400 (COOH-PEG-3400), or methoxy-terminated poly(ethylene glycol), molecular weight 5000 (mPEG-5000). We compared the amp-QDs to the three differently substituted PEG-QDs by noninvasive imaging and repetitive bleeds to determine whether PEG-substitution would affect the circulating lifetime. Immediately after intravenous injection, fluorescence from all QD emission wavelengths and surface coatings is easily seen in the superficial vasculature. Subsequently, QDs are seen to deposit in liver, skin, and bone marrow in a surface coating dependent manner. By 1 h postinjection, QDs having amp-, mPEG-750-, and COOH-PEG-3400 coatings are cleared from the circulation, while mPEG-5000 QDs remain visible in circulation for at least 3 h (Figures 1 and 2, and see Supporting Information, which includes a Quicktime movie of in vivo distribution of the mPEG750 and mPEG-5000 quantum dots). Circulating lifetimes (Table 1) were 12 min or less for the amp, mPEG-750,
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and COOH-PEG-3400 QDs, while the mPEG-5000 QDs had a much longer circulating lifetime (50-100 min by venipuncture, 140 min by imaging). There was no statistically significant difference between nude and normal mice in the circulating lifetime of QDs. The circulating lifetime obtained by noninvasive imaging of whole animals was consistently longer than that obtained by venipuncture; this is caused mostly by the presence of extravasated QD’s in surrounding tissue (data not shown.) Due to an erratic pattern of early deposition in the skin by amp and COOH-PEG-3400 QDs, only early lifetime data (four months) postinjection. QDs remained for at least one month in liver, lymph nodes, and bone marrow. By one month, fluorescence was substantially decreased, but the overall distribution of fluorescence remained similar to that at 24 h after injection. In follow-up experiments at 133 days postinjection, QD fluorescence was still visible on necropsy, though background fluorescence from intestinal contents was now higher than that in lymph nodes. The liver had no grossly visible fluorescence; however, QD’s were still grossly visible in lymph nodes and bone marrow (Figure 5), and visible by fluorescence microscopy in liver, lymph nodes, and spleen (data not shown). Cellular sites of uptake were identified by microscopy of tissue sections. Imaging of whole spleen by fluorescence 24 h postinjection of mPEG-750 QDs showed a freckled pattern of dark centers having bright haloes. Figure 6 shows one frozen section from a spleen taken 24 h postinjection, stained using hematoxylin and visualized in white light and by fluorescence. Deposition is perifollicular, with no accumulation in germinal centers. The QDs are concentrated in internal vacuoles of phagocytic cells. Sections of lymph nodes showed a similar pattern. QDs were further localized by electron microscopy. Figure 7 shows transmission electron microscope images of a mouse spleen cell 24 h postinjection. Localization in endosomes is confirmed by these images, which also demonstrate that QDs deposited in tissues may be imaged easily using standard electron microscopic meth-
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Figure 1. Noninvasive imaging using 645 nm mPEG-750 QDs (left, A, C, E) and 655 nm-emitting mPEG-5000 QDs (right, B, D, F, G). Nude mice were imaged at 1min (A, B), 10 min (C, D) 1 h (E, F), and 3 h (G) postinjection. Note that even at 1 min, significant liver uptake is visible using mPEG-750 QDs (A), while even at 10 min there is little or no visible liver uptake using mPEG-5000 QDs (D). At 1 h, the difference between mPEG-750 QDs (E) and mPEG-5000 QDs is even more marked, with PEG-750 QDs completely cleared from the circulation, while mPEG-5000 QDs persist (F); even 3 h postinjection, mPEG-5000 QDs remain in circulation (G). Since essentially all fluorescence is cleared from the circulation by 1 h, imaging of mPEG-750 QDs was terminated at 1 h after injection. In other experiments, we found that the pattern of deposition of mPEG-750 QDs seen at 1 h remained stable for at least 24 h.
ods. At one month postinjection, electron microscope images show subcellular localization in the spleen similar to that seen 24 h postinjection. Whether QDs remain in the cells that originally took them up is not yet known.
DISCUSSION
We have shown that quantum dots are well suited for fluorescence visualization in whole animals. Because of
Quantum Dots in Mice
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their high quantum yields and high absorbency, the fluorescence of QDs in the superficial vasculature was readily visible to the naked eye just after injection; even the orange 605 nm-emitting QDs can be imaged noninvasively in liver and bone marrow. QDs in tissue can be imaged on scales that range from the whole animal to the electron microscope level. Moreover, the QDs used in these experiments are stable and fluorescent in vivo for at least four months. Microscopy showed no signs of
localized necrosis at the sites of deposition, and there was no obvious sign of breakdown of the QDs as seen by electron microscopy. This stability is presumably due to the amp coating, which preserves fluorescence and protects the QDs from degradation in vivo. We cannot exclude the possibility that some QDs are no longer fluorescent after prolonged residence in tissue, but their structure is preserved as revealed by electron microscopy. QDs can be excited by any light wavelength shorter than their emission wavelength. We used blue excitation because of the higher absorbency of QDs in the blue, and the ability to use QDs with emission wavelengths from green through infrared with a single excitation wavelength. Red light excitation should improve visualization and resolution at increased tissue depth, at some cost in efficiency of excitation. Because of the resistance of QDs to photobleaching, higher intensity illumination or a long integration time should make up for any decrease in absorbency in the red. However, in our hands, blue excitation with a wideband filter proved quite satisfactory in mice, and use of red laser illumination produced no significant improvement. The small size of QDs and their conjugates should allow efficient tissue perfusion and good targeting to specific sites in tissues, provided that the QDs remain in circulation long enough. For many uses in vivo, reducing Qdot uptake by the reticuloendothelial system and increasing circulating lifetime will be essential. PEGylation of nanoparticles and liposomes to prolong serum lifetime and aid in targeting is an area of active research; second-generation PEG derivatives may offer longer lifetimes and enhanced tissue perfusion (14-24.) Our results recall those of Gref et al. (23) and Mosqueria et al. (24), who found that increasing PEG density and chain length-reduced nanoparticle binding to cells and
Figure 3. Mouse imaged 24 h postinjection of 645 nm mPEG750 QDs. Left, light images; Right, fluorescence images. A, whole animal; B, skin and peritoneum removed, sternum pinned back showing marrow staining, but concealing cervical nodes. C, sternum, heart, lungs, liver, and digestive tract removed. Note the visibility of axillary and inguinal lymph nodes through the skin (A), and the retention of QDs by bone marrow as well as by lymph nodes and liver (B, C).
Figure 4. Mouse imaged 24 h postinjection of 655 nm mPEG5000 QDs. Left, light images; Right, fluorescence images. A, whole animal; B, skin and peritoneum removed, sternum pinned back showing marrow staining, but concealing cervical nodes. C, sternum, heart, lungs, liver, and digestive tract removed. Note the minimal labeling of lymph nodes compared to mPEG750 (Figure 3). Note the high relative brightness of the spleen, displayed on the upper right of the dissection (C).
Figure 2. Regions of interest (2 × 2 pixels) over several different surface vessels of the animals in Figure 1 were used to determine the circulating lifetime of the QDs. Mean fluorescence intensities from nine regions of interest on each animal were measured, normalized to maximum initial values, and plotted versus time after injection (triangles, mPEG-5000 QDs; squares, mPEG-750 QDs.) Bars indicate standard deviations. Exponential fits of the fluorescence intensities as a function of time yielded average circulating half-lives of 6.1 ( 2.9 min for mPEG-750 QDs and 141 ( 45 min for mPEG-5000 QDs (Table 1).
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Figure 5. Long-term retention of QD fluorescence in vivo. A. Mouse imaged 133 days postinjection of 360 pmol of 655 nm mPEG750 QDs. The mouse was dissected, and most internal organs were removed to show continuing fluorescence of axillary, inguinal, and lumbar lymph nodes as well as weak fluorescence of bone marrow. B. Same dissection of control (uninjected) mouse. Mice were imaged under identical conditions, and images are displayed using identical brightness functions.
Figure 6. Spleen, frozen section 24 h postinjection of 630 nm-emitting mPEG-750 QDs. A. Section stained using Gill’s hematoxylin, white light image; B. Fluorescence image of A. Note concentration of QDs in perifollicular areas, but not in germinal centers. Bar ) 200 µm.
Figure 7. mPEG-750 QDs in mouse spleen imaged by electron microscopy 24 h postinjection. A. Perifollicular cell, presumably macrophage, bar ) 10 µm; arrows indicate endosomes. B. Same cell, bar ) 1 µm. C, Enlarged single endosome, showing individual QDs, bar ) 100 nm. Glutaraldehyde fixation, Epon embedding, osmium stain.
plasma components and increased circulating lifetime. We anticipate that circulating lifetimes will be further improved by adjusting the surface composition of QDs. Other groups have used QDs in vivo. Dubertret et al. (25) microinjected QDs enclosed in PEG-conjugated lecithin micelles; these QDs persisted in Xenopus embryos from blastula at least until tadpole stage, with little or no indication of toxicity. Akerman et al. (26) explored the deposition of peptide conjugated and PEGylated QDs in mice using fluorescence microscopy of tissue sections rather than whole-body visualization. By using a high
level of PEG-substitution on QD-peptide conjugates directed to vascular and tumor targets, this group found that uptake in RES sites could be reduced, while selective targeting to organ-specific vasculature and to tumors could be achieved. These results, taken with ours, demonstrate the usefulness of QDs for targeting in vivo and whole-body visualization. Several groups, including ours, have investigated organic fluorochromes as labels for targeting in vivo; all have concluded that far-red and near-infrared fluorophores are more effective than shorter wavelengths (27-
Quantum Dots in Mice
33). For good visualization through a significant thickness of tissue (>1 mm), stable near-infrared-emitting QDs will be necessary (34). Difficulties in the manufacture and stabilization of infrared-emitting QDs are being resolved; this should enable visualization in whole animals at greater depth and with increased sensitivity. That QD surfaces can control serum lifetime and pattern of deposition suggests many medical uses. Rapid deposition in the reticuloendothelial system is potentially useful for defining some tumor types and detecting sentinel lymph nodes. Tumor targeting by QD conjugates is an exciting prospect, as tumor sites and margins could be readily imaged during surgery (26), provided that heavy-metal toxicity due to the QD’s can be appropriately limited or controlled. As with antibody conjugates, the key to good targeting in vivo is the use of surface coatings that reduce nontargeted uptake. We have shown considerable increase in circulating lifetimes and reduction in nonspecific uptake of QDs by the use of long-chain PEG surfaces. At this point, the serum lifetime of mPEG-5000 QDs is similar to that of some of our cyanine fluorochrome antibody conjugates that were successfully used for tumor labeling (28, 29). Finally, thanks to the high stability of amp-coated QDs, experiments using molecules and cells tagged with QDs and injected into animals may permit analysis of antigen trafficking and cellular migration over very long time scales, with unprecedented sensitivity and resolution. ACKNOWLEDGMENT
We thank Joseph P. Suhan (Department of Biological Sciences, Carnegie Mellon University) for excellent technical assistance in electron microscopy, Joyce Horner and Lisa McGaw (Department of Biological Sciences, Carnegie Mellon University) for assistance with animal handling and anesthesia. This work was supported by NIH grant number R01 EB 000364. The work at Quantum Dot Corporation was partly funded by NIST-ATP 70NANB0H3000. Supporting Information Available: A Quicktime movie showing fluorescence images of whole animal distribution of the mPEG-750 and mPEG-5000 quantum dots directly after injection. This material is available free of charge via the Internet at http://pubs.acs.org. NOTE ADDED IN PROOF
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