Quantum dots for live cell and in vivo imaging

Abstract

In the past few decades, technology has made immeasurable strides to enable visualization, identification, and quantitation in biological systems. Many of these technological advancements are occurring on the nanometer scale, where multiple scientific disciplines are combining to create new materials with enhanced properties. The integration of inorganic synthetic methods with a size reduction to the nano-scale has lead to the creation of a new class of optical reporters, called quantum dots. These semiconductor quantum dot nanocrystals have emerged as an alternative to organic dyes and fluorescent proteins, and are brighter and more stable against photobleaching than standard fluorescent indicators. Quantum dots have tunable optical properties that have proved useful in a wide range of applications from multiplexed analysis such as DNA detection and cell sorting and tracking, to most recently demonstrating promise for in vivo imaging and diagnostics. This review provides an in-depth discussion of past, present, and future trends in quantum dot use with an emphasis on in vivo imaging and its related applications.

Keywords: Quantum dots; applications; biocompatibility; in vivo imaging; nanoparticles.

Figure 1.

(A) Scanning transmission electron micrographs of PbSe quantum dots: low resolution (full scale = 320 nm) showing the ordering of an ensemble of nanocrystals. Reprinted with permission from [18]. (B) Schematic of a quantum dot conjugate used for in vivo imaging. The image shows a cross section where the quantum dot semiconductor core is visible, which is coated with a shell material, comprised of a second semiconductor material. The core/shell quantum dots are coated for biocompatibility and solubility, followed by conjugation to a biological element for recognition or a specific interaction in vivo. In this instance, the quantum dot bioconjugate is shown covered with antibodies. For illustrative purposes, and not drawn to scale.

Figure 2.

Comparison of (A) the excitation and (B) the emission profiles between rhodamine 6G (red) and CdSe quantum dots (black). The quantum dot emission spectrum is nearly symmetric and much narrower in peak width. Its excitation profile is broad and continuous. The quantum dots can be efficiently excited at any wavelength shorter than ∼530 nm. By contrast, the organic dye rhodamine 6G has a broad and asymmetric emission peak and is excited only in a narrow wavelength range. Reprinted with permission from [40].

Figure 3.

(A) Size- and material-dependent emission spectra of several surfactant-coated semiconductor nanocrystals. The blue series represents different sizes of CdSe nanocrystals with diameters of 2.1, 2.4, 3.1, 3.6, and 4.6 nm (from left to right). The green series is of InP nanocrystals with diameters of 3.0, 3.5, and 4.6 nm. The red series is of InAs nanocrystals with diameters of 2.8, 3.6, 4.6, and 6.0 nm. (B, C, D) Size-dependent optical properties of cadmium selenide quantum dots dispersed in chloroform, illustrating size tunable fluorescence emission. (B) Fluorescence image of four vials of monodisperse quantum dots with sizes ranging from 2.2 to 7.3 nm in diameter, obtained with ultraviolet lamp illumination at 365 nm. (C) Fluorescence spectra of the same four quantum dot samples, excited at 400 nm. Narrow emission bands (23–26 nm full-width at half-maximum) indicate narrow particle size distributions. (D) Absorption spectra of the same four samples. Notice that the onset of absorption is slightly blue-shifted from the emission peak for each quantum dot sample, and that the absorption spectra are very broad, so that a wide spectrum can be used for excitation. Both the absorption and emission intensities are plotted in arbitrary units (AU). Adapted with permission from [1,48].

Figure 4.

Sensitivity and multicolor capability of QD imaging in live animals. (A,B) Sensitivity and spectral comparison between QD-tagged and GFP transfected cancer cells (A), and simultaneous in vivo imaging of multicolor QD-encoded microbeads (B). The right-hand images in A show QD-tagged cancer cells (orange, upper) and GFP-labeled cells (green, lower). Approximately 1,000 of the QD-labeled cells were injected on the right flank of a mouse, while the same number of GFP-labeled cells was injected on the left flank (circle) of the same animal. Similarly, the right-hand images in B show QD-encoded microbeads (0.5 μm diameter) emitting green, yellow or red light. Approximately 1–2 million beads in each color were injected subcutaneously at three adjacent locations on a host animal. In both A and B, cell and animal imaging data were acquired with tungsten or mercury lamp excitation, a filter set designed for GFP fluorescence and true color digital cameras. Transfected cancer cell lines for high level expression of GFP were developed by using retroviral vectors, but the exact copy numbers of GFP per cell have not been determined quantitatively. Reprinted with permission from [23].

Figure 5.

Applications of quantum dots as multimodal contrast agents in bio-imaging. Reprinted with permission from [61].

Figure 6.

Schematic illustration of CdSe/ZnSe nanocrystals possible solubilization methods and stabilization procedure. Reprinted with permission from [52].

Figure 7.

Sketch of the silanization method. The TOPO-capped CdSeZnS core/shell particles are dissolved in pure MPS, drawn in its hydrolyzed form for convenience in the figure. After basification, the MPS replaces the TOPO molecules on the surface. The methoxysilane groups (Si-OCH₃) hydrolyze into silanol groups (Si-OH), and form a primary polymerization layer. Heat strengthens the silanol-silanol bridges by converting them into siloxane bonds and releasing water molecules. Then, fresh silane precursors containing a functional group (F = −SH, −NH₂; −PO−(O−)CH₃) are incorporated into the shell and may tailor the nanocrystal surface functionality. In a last step (not shown) the remaining hydroxyl groups are converted in methyl groups; this last step blocks further silica growth. Reprinted with permission from [62].

Figure 8.

Schematic representation of the surface coating chemistry of CdSe/ZnS nanocrystals with phytochelatin-related R-peptides. The peptide C-terminal adhesive domain binds to the ZnS shell of CdSe/ZnS nanocrystals after exchange with the TOPO surfactant. A polar and negatively charged hydrophilic linker domain in the peptide sequence provides aqueous buffer solubility to the nanocrystals. TMAOH: Tetramethylammonium hydroxide; Cha: 3-cyclohexylalanine. Reprinted with permission from [51].

Figure 9.

Schematic diagram showing direct exchange reactions between the monovalent capping ligand octadecylamine and the multivalent copolymer ligands. Reprinted with permission from [39].

Figure 10.

Sequential lymph nodes (1 and 2) and the lymphatic channel between them were imaged (C, D) in a rat by white light and NIR fluorescence five minutes after injection of the quantum dots (A, B). Reprinted with permission from [75].

Figure 11.

No fluorescence was seen in the interstitial fluid surrounding the incision in the rat model with DHLA-coated (InAs) ZnSe quantum dots (A, B). With DHLA-PEG, however, fluorescence was observed from extravasated quantum dots (C, D). Reprinted with permission from [75].

Figure 12.

Schematic illustration of the quantum dot complex, the quantum dot complex entered into the circulation, extravasated into the interstitial space from the vascular space, bound to the tumor cells through the interstitial region, and having reached the perinuclear region after traveling on the intracellular rail protein. All processes exhibit a characteristic “stop-and-go” movement. Reprinted with permission from [78].

Figure 13.

The percent of the initial dose of cadmium that remained at the site of injection of the CdSe quantum dot. The skin that contained the site of injection was removed, completely digested using HNO₃, and the total cadmium was detected using ICP-MS (mean ± SE). Values indicated by an asterisk (*) were significantly different (p _ 0.05) than the value at 0 h, while values indicated with the cross (†) were not significantly different from each other (p > 0.05). Reprinted with permission from [79].

Figure 14.

Distribution of cadmium in organs following intradermal injection of CdSe quantum dot. The data are expressed as mean ± SE for: liver (solid circles); regional lymph nodes (open circles); and kidney (open diamonds). Values indicated by an asterisk (*) were significantly different (p < 0.05) than the value in the same organ at 0 h. Reprinted with permission from [79].

Figure 15.

Anatomy of the lymphatic system in the upper body of the mouse and a schematic of five-color spectral fluorescence imaging, with a graph of the emission spectra of each of the five carboxyl quantum dots used (Qdot 565, blue; Qdot 605, green; Qdot 655, yellow; Qdot 705, magenta; Qdot 800, red). The colored lymph nodes are the draining lymph nodes visualized in this study. Reprinted with permission from [81].

Figure 16.

Quantum dot surfaces, measured in 0.01 M sodium borate buffer, pH 8.5. The hydrodynamic diameters are expected to be roughly similar to those of the 655 nm quantum dots. Reprinted with permission from [80].

Figure 17.

(A) Fluorescence images of rats injected with quantum dot-Cys incubated in FBS at the indicated pH, 4 hours postinjection (Bl, bladder; Ki, kidneys; Li, liver; RP, retroperitoneum; AW, abdominal wall, and Sp, spleen.): color image (left), 565 nm fluorescence (middle), merged (right). (B) GFC analysis of QD-Cys incubated in FBS at various pH (left) and in urine (right) 4 hours postinjection (fluorescence detection at 565 nm). MW markers M1 (thyroglobulin, 670 kDa), M2 (ç-globulin, 158 kDa), M3 (ovalbumin, 44 kDa), and M4 (myoglobin, 17 kDa) are shown by arrows. Reprinted with permission from [85].

Figure 18.

Schematic illustration of bioconjugated quantum dots for multiplexed in situ molecular profiling. (A) Multicolor quantum dot bioconjugates prepared with SMCC activated quantum dots and chemically reduced antibodies. (B) Cell staining using multicolor quantum dot-bioconjugates. (C) Quantification of tumor biomarker expression using wavelength-resolved spectroscopy. Reprinted with permission from [92].

Figure 19.

Surface coating chemistry and structure of polymer-encapsulated quantum dots (CdSe/CdS/ZnS). Schematic diagram showing conversion of carboxylated quantum dots (coated with poly(acrylic acid) octylamine) to hydroxylated and cross-linked quantum dots. The small-molecule agent for hydroxylation is 1,3-diamino-2-propanol. Reprinted with permission from [90].

Figure 20.

CCFF barcodes. (A) The CCFF process includes the concentration reactor and the production nozzle. An enlargement is shown in the top right with a cross-sectional diagram of the flow-focusing nozzle in which a quantum dot fluorophore solution in chloroform with 4% dissolved polymer is introduced through the top (yellow). The flow-focusing fluid (deionized water) is introduced from the right (blue). A close-up view of the quantum-dot–polymer solution being focused and “pinched off ” into microscopic droplets by the water flow is shown on the bottom right corner. Each small droplet formed is an active polymer microbead with homogeneous quantum dot encoding. (B) UV illuminated picture of the five stock solutions of quantum dots used to generate a 105-barcode library. The number printed on each bottle represents the fluorescence emission wavelength in nanometers of the respective quantum dots. Reprinted with permission from [57].

Figure 21.

Multiplexed protein and DNA assays. Six DNA and six proteomic multiplexed assays were performed on the QD-barcoded beads. Triplicates were performed for each experiment and computed standard deviation was less than 10% in all cases. Reprinted with permission from [57].

Figure 22.

Scanning electron micrograph images of the beads’ surfaces: original polystyrene beads (A) and the Si quantum dots coated beads (B) The insets are magnified 50,000 times, and the images were taken under the same conditions. Reprinted with permission from [111].

Figure 23.

Internalization of nanoworms and nanospheres conjugated with peptides into cells. A conceptual scheme illustrating the increased multivalent interactions expected between receptors on a cell surface and targeting ligands on a nanoworm compared with a nanosphere. Adapted with permission from [122].