Specific integrin labeling in living cells using functionalized nanocrystals

We present an integrin labeling method using functionalized quantum dots (QDs). Cyclic Arg-Gly-Asp (RGD) peptides and a biotin-streptavidin linkage are used to specifically couple individual QDs to integrins of living cells. The spacer distance between the RGD sequence and the QD surface is a crucial parameter to ensure specific binding to individual alpha(v)beta(3) integrins of osteoblast cells. Despite blinking, the position of single QDs is tracked with nanometer precision and localized diffusive behavior is observed. We show that blinking events do not prevent the acquisition of quantitative parameters from the QD trajectories.     

Single Chain Epidermal Growth Factor Receptor Antibody Conjugated Nanoparticles for in vivo Tumor Targeting and Imaging

Epidermal growth factor receptor (EGFR) targeted nanoparticle are developed by conjugating a single‐chain anti‐EGFR antibody (ScFvEGFR) to surface functionalized quantum dots (QDs) or magnetic iron oxide (IO) nanoparticles. The results show that ScFvEGFR can be successfully conjugated to the nanoparticles, resulting in compact ScFvEGFR nanoparticles that specifically bind to and are internalized by EGFR‐expressing cancer cells, thereby producing a fluorescent signal or magnetic resonance imaging (MRI) contrast. In vivo tumor targeting and uptake of the nanoparticles in human cancer cells is demonstrated after systemic delivery of ScFvEGFR‐QDs or ScFvEGFR‐IO nanoparticles into an orthotopic pancreatic cancer model. Therefore, ScFvEGFR nanoparticles have potential to be used as a molecular‐targeted in vivo tumor imaging agent. Efficient internalization of ScFvEGFR nanoparticles into tumor cells after systemic delivery suggests that the EGFR‐targeted nanoparticles can also be used for the targeted delivery of therapeutic agents.     

AFM observation of surface acoustic waves emitted from single symmetric SAW transducers

We report the first experimental observation of surface acoustic waves (SAWs) launched from a single symmetric SAW transducer, employing scanning acoustic force microscopy (SAFM). SAFM is a simple technique for the imaging of complex interdigital transducer (IDT) radiation patterns with nanometer lateral resolution. We demonstrate submicron lateral resolution and high sensitivity by investigating a single excitation element on a weakly coupling substrate (GaAs), visualizing the launched wave and second-order effects     

Aqueous‐Phase Synthesis of Highly Luminescent CdTe/ZnTe Core/Shell Quantum Dots Optimized for Targeted Bioimaging

Semiconductor quantum dots (QDs) have traditionally been synthesized in organic phase and transferred to aqueous solution by functionalizing their surface with silica, polymers, short‐chain thiol ligand, or phospholipid micelles. However, these complex steps result in i) a reduction of the quantum yield (QY) of QDs, ii) partial degrdation of the QDs, and iii) a drastic increase in the hydrodynamic size of QDs, which may hinder their biomedical applications. In this work, the fabrication and applications of cysteine‐capped CdTe/ZnTe QDs, which are directly synthesized in aqueous media, as optical probes for specific targeting of pancreatic and esophageal cancer cells in vitro are reported, as well as their capability for in vivo imaging. The CdTe/ZnTe QDs are synthesized in a one‐pot method and capped with amino acid cysteine, which contains both carboxyl and amine functional groups on their surfaces for bioconjugation. The fabricated QDs have an ultrasmall hydrodynamic diameter (3–5 nm), possess high QY (52%), and are non‐toxic to cells at experimental dosages. Confocal imaging is used to demonstrate a receptor‐mediated uptake of antibody‐conjugated QDs into pancreatic cancer cells in vitro. In vitro cytotoxicity studies (MTS‐assay) show that the IC₅₀ value of these QDs is ≈160 µg mL^?1, demonstrating low toxicity. In addition, the QDs are used for small‐animal imaging where the in vivo biocompatiblity of these QDs and their clearance following systemic injection is studied.     

量子点在细胞以及体内生物中成像的研究进展

量子点是一种荧光半导体纳米材料,与生物分子结合成一种高亮度而稳定的荧光探针应用于生物成像。通过生物成像可观察量子点标记分子与其靶标的相互作用,实时观测其在活细胞及活体中的运行轨迹,实现对细胞水平及在活体层次的研究。利用这种生物成像技术还可以研究疾病的发生发展过程。介绍了量子点的光学特性,重点综述了量子点在细胞、体内生物成像中的应用,并展望了其发展前景。     

Surface modification of CdSe quantum dots for biosensing applications: Role of ligands

For an optimum performance of colloidal nanocrystal devices for a variety of applications such as photonic devices, solar cells and biological labelling, the determining factors are the nanocrystal surface and size. In this work, these two factors have been tuned via wet chemistry to tailor the material properties: The absorption and emission spectra have been tailored by choice of the nanocrystal size; nanocrystal shape by surface modification and photoluminescence (PL) efficiency determined by surface traps, has been tuned via appropriate selection of the nanocrystal capping ligands. Here, we have shown that through ligand-exchange process, the surface of the CdSe quantum dots (QDs) can be modified by replacing the longer-chain ligands of conventional trioctyl phosphine oxide (TOPO) or oleic acid (OA) with shorter-chain ligand of butyl amine. This imparts colloidal stability and water solubility to CdSe QDs for its potential applications in biosensors and biological imaging. It is conjectured that crystallite sizes, oxidation potential of CdSe QDs and stereochemical compatibility of ligands (TOPO, oleic acid and butyl amine) greatly influences the photophysics and photochemistry of CdSe QDs.     

Imaging of resonant quenching of surface plasmons by quantum dots

In scanning tunneling microscopy (STM), confinement of surface plasmons to the optical cavity formed at the metallic tunneling gap stimulates the emission of light. We demonstrate that quantum dots (QDs) found in such a cavity give rise to discrete, observable transitions in the tunneling luminescence spectrum due to the resonant extinction of the plasmon. The observed resonances represent a fingerprint of the QD and occur at the optical band gap owing to the nearly simultaneous transfer of carriers from both sides of the tunneling gap to the QD. The resonant quenching of surface plasmons enables a new imaging technique, dubbed plasmon resonance imaging, with a spatial resolution potentially similar to that of STM and the energy resolution of optical spectroscopies. This detection and imaging strategy is not restricted to QDs, being of great interest to an entire spectrum of nanostructures, from molecular assemblies and biomolecules to carbon nanotubes.     

Quantum dots as multimodal photoacoustic and photothermal contrast agents

Quantum dots (QDs) have primarily been developed as fluorescent probes with unique optical properties. We herein demonstrate an extension of these QD utilities to photoacoustic (PA) and photothermal (PT) microscopy, using a nanosecond pulse laser excitation (420-900 nm, 8 ns, 10(-3)-10 J/cm(2)). The laser-induced PA, PT and accompanying bubble formation phenomena were studied with an advanced multifunctional microscope, which integrates fluorescence, PA, PT imaging, and PT thermolens modules. It was demonstrated that QDs, in addition to being excellent fluorescent probes, can be used as PA and PT contrast agents and sensitizers, thereby providing an opportunity for multimodal high resolution (300 nm) PA-PT-fluorescent imaging as well as PT therapy. Further improvements for this technology are suggested by increasing the conversion of laser energy in PT, PA, and bubble phenomena in hybrid multilayer QDs that have optimized absorption, thermal, and acoustic properties.     

Intravital Multiphoton Imaging of the Selective Uptake of Water‐Dispersible Quantum Dots into Sinusoidal Liver Cells

Although many studies reporting the organ‐level biodistribution of nanoparticles (NPs) in animals, very few have addressed the fate of NPs in organs at the cellular level. The liver appears to be the main organ for accumulation of NPs after intravenous injection. In this study, for the first time, the in vivo spatiotemporal disposition of recently developed mercaptosuccinic acid (MSA)‐capped cadmium telluride/cadmium sulfide (CdTe/CdS) quantum dots (QDs) is explored in rat liver using multiphoton microscopy (MPM) coupled with fluorescence lifetime imaging (FLIM), with subcellular resolution (～1 μm). With high fluorescence efficiency and largely improved stability in the biological environment, these QDs show a distinct distribution pattern in the liver compared to organic dyes, rhodamine 123 and fluorescein. After intravenous injection, fluorescent molecules are taken up by hepatocytes and excreted into the bile, while negatively charged QDs are retained in the sinusoids and selectively taken up by sinusoidal cells (Kupffer cells and liver sinusoidal endothelial cells), but not by hepatocytes within 3 h. The results could help design NPs targeting the specific types of liver cells and choose the fluorescent markers for appropriate cellular imaging.     

CdTe量子点的微波合成及其在双光子显微成像中的应用

采用微波辐射加热的方法,以亚碲酸钠(Na2TeO3)作碲源,以谷胱甘肽(GSH)作稳定剂,在水相中合成出高质量的CdTe量子点。所合成量子点的发射波长从515～630nm可调,荧光量子产率(PLQYs)最高达95%。利用X射线粉末衍射(XRD)、高分辨透射电镜(HRTEM)、紫外-可见吸收光谱(UV-Vis)和荧光发射光谱(PL)等技术表征产物的物相结构和光学性质。用双光子激发荧光法研究CdTe量子点的双光子吸收性质。用双光子激发荧光成像技术,以发红光的CdTe量子点作为双光子荧光探针成功标记了人肺腺癌细胞(A549)。     

Viral Coat Proteins as Flexible Nano‐Building‐Blocks for Nanoparticle Encapsulation

Viral capsid–nanoparticle hybrid structures offer new opportunities for nanobiotechnology. We previously generated virus‐based nanoparticles (VNPs) of simian virus 40 (SV40) containing quantum dots (QDs) for cellular imaging. However, as an interesting issue of nano‐bio interfaces, the mechanism of nanoparticle (NP) encapsulation by viral coat proteins remains unclear. Here, four kinds of QDs with the same core/shell but different surface coatings are tested for encapsulation. All the QDs can be encapsulated efficiently and there is no correlation between the encapsulation efficiency and the surface charge of the QDs. All the SV40 VNPs encapsulating differently modified QDs show similar structures, fluorescence properties, and activity in entering living cells. These results demonstrate the flexibility of SV40 major capsid protein VP1 in NP encapsulation and provide new clues to the mechanism of NP packaging by viral shells.     

Extracellular biosynthesis of biocompatible CdSe quantum dots

An extracellular biosynthesis method has been developed to prepare cadmium selenide (CdSe) quantum dots (QDs) with strong fluorescence emission by incubating cheap Cd and Se inorganic salts with Escherichia coli (E.coli) bacteria. Ultraviolet–visible absorption spectra, photoluminescence (PL) spectra, and high‐resolution transmission electron microscopy analysis showed that the biosynthesised CdSe QDs have an average size of 3.1 nm, the excellent optical properties with fluorescence emission around 494 nm, and the good crystallinity. It was found that addition of 80 mg of mercaptosuccinic acid resulted in the formation of CdSe QDs with highest PL intensity. Furthermore, Fourier‐transform infrared spectra of as‐synthesised CdSe QDs confirmed the presence of a surface protein capping layer. The biosynthesised CdSe QDs were incorporated into the yeast cells as illustrated by laser confocal scanning microscopy images, showing a great potential in bio‐imaging and bio‐labelling application.Inspec keywords: microorganisms, molecular biophysics, fluorescence, visible spectra, nanofabrication, nanobiotechnology, proteins, cellular biophysics, nanostructured materials, wide band gap semiconductors, cadmium compounds, semiconductor quantum dots, II‐VI semiconductors, transmission electron microscopy, photoluminescence, optical microscopy, ultraviolet spectra, Fourier transform infrared spectra, biological techniques, semiconductor growthOther keywords: biocompatible CdSe quantum dots, extracellular biosynthesis method, cadmium selenide quantum dots, high‐resolution transmission electron microscopy analysis, biosynthesised CdSe QDs, Fourier‐transform infrared spectra, Escherichia coli, ultraviolet‐visible absorption spectra, PL intensity, fluorescence emission, photoluminescence spectra, optical properties, surface protein capping layer, laser confocal scanning microscopy images, bioimaging, biolabelling application, yeast cells, f mercaptosuccinic acid, CdSe     

Two-photon 3D FIONA of individual quantum dots in an aqueous environment

We report the first two-photon (2P) microscopy of individual quantum dots (QDs) in an aqueous environment with both widefield and point-scan excitations at nanometer accuracy. Thiol-containing reductants suppress QD blinking and enable measurement of the 36 nm step size of individual Myosin V motors in vitro. We localize QDs with an accuracy of 2-3 nm in all three dimensions by using a 9 × 9 matrix excitation hologram and an array detector, which also increases the 3D scan imaging rate by 80-fold. With this 3D microscopy we validate the LamB receptor distribution on E. coli and the endocytosis of EGF-receptors in breast cancer cells.     

Tracking individual kinesin motors in living cells using single quantum-dot imaging

We report a simple method using semiconductor quantum dots (QDs) to track the motion of intracellular proteins with a high sensitivity. We characterized the in vivo motion of individual QD-tagged kinesin motors in living HeLa cells. Single-molecule measurements provided important parameters of the motor, such as its velocity and processivity, as well as an estimate of the force necessary to carry a QD. Our measurements demonstrate the importance of single-molecule experiments in the investigation of intracellular transport as well as the potential of single quantum-dot imaging for the study of important processes such as cellular trafficking, cell polarization, and division.     

Exploring feasibility of multicolored CdTe quantum dots for in vitro and in vivo fluorescent imaging

We report the use of novel multicolored CdTe quantum dots (QDs) as fluorophores for biological fluorescence imaging. The CdTe QDs were prepared to exhibit emission wavelengths in the green, yellow, and red range by using trifluoroacetic acid (TFA), L-cysteine and thioglycolic acid (TGA) as surface stabilizers, respectively. The particles have good water solubility and photostability. Fluorescence imaging potential was evaluated in vitro and in vivo using a multispectral Maestro CRI Fluorescence Imaging system. The results show that different colored CdTe QDs allow sensitive detection simultaneously or separately both in vitro and in vivo against background fluorescence. The studies indicate that CdTe QDs can provide alternative fluorescent probes for biological imaging.     

Label‐Free Tracking of Single Organelle Transportation in Cells with Nanometer Precision Using a Plasmonic Imaging Technique

Imaging and tracking of nano‐ and micrometer‐sized organelles in cells with nanometer precision is crucial for understanding cellular behaviors at the molecular scale. Because of the fast intracellular dynamic processes, the imaging and tracking method must also be fast. In addition, to ensure that the observed dynamics is relevant to the native functions, it is critical to keep the cells under their native states. Here, a plasmonics‐based imaging technique is demonstrated for studying the dynamics of organelles in 3D with high localization precision (5 nm) and temporal (10 ms) resolution. The technique is label‐free and can track subcellular structures in the native state of the cells. Using the technique, nanometer steps of organelle (e.g., mitochondria) transportation are observed along neurite microtubules in primary neurons, and the 3D structure of neurite microtubule bundles is reconstructed at the nanometer scale from the tracks of the moving organelles.     

Cell uptake and intracellular visualization using quantum dots or nuclear localization signal-modified quantum dots with gold nanoparticles as quenchers

Understanding and controlling the interactions between nanoscale objects and living cells is of great importance for diagnostic imaging and therapeutic applications. Quantum dots (QDs) have remarkable optical characteristics, such as uniquely feature bright, photostable, tunable and narrow fluorescence emissions, as well as broad absorption spectra. Here we report a platform of using quantum dots to investigate the cell uptake and the interactions between nanoscale objects and cells. QDs are uptaken by BHK cells easily through endocytosis. We could clearly differentiate the QDs outside the cell or inside the cell by quenching the QDs with similar sized gold nanoparticles and reduce the noise of fluorescent image. Microscopic images show that QDs are homogeneously distributed within the whole cell except the nucleus. However, unmodified QDs could not penetrate the nuclear membrane and move into the nucleus. Coupling QDs with Nuclear Localization Signal (NLS, CGGGPKKKRKVGG) can significantly enhance the translocation amount of QDs into the cell and cell nucleus. This method combined with microscopy imaging system can visualize the particle delivery routes and provide valuable information in the drug/gene delivery and tumor diagnosis.     

Controlled Synthesis of Ag2Te@Ag2S Core–Shell Quantum Dots with Enhanced and Tunable Fluorescence in the Second Near‐Infrared Window

Fluorescence in the second near‐infrared window (NIR‐II, 900–1700 nm) has drawn great interest for bioimaging, owing to its high tissue penetration depth and high spatiotemporal resolution. NIR‐II fluorophores with high photoluminescence quantum yield (PLQY) and stability along with high biocompatibility are urgently pursued. In this work, a Ag‐rich Ag₂Te quantum dots (QDs) surface with sulfur source is successfully engineered to prepare a larger bandgap of Ag₂S shell to passivate the Ag₂Te core via a facile colloidal route, which greatly enhances the PLQY of Ag₂Te QDs and significantly improves the stability of Ag₂Te QDs. This strategy works well with different sized core Ag₂Te QDs so that the NIR‐II PL can be tuned in a wide range. In vivo imaging using the as‐prepared Ag₂Te@Ag₂S QDs presents much higher spatial resolution images of organs and vascular structures as compared with the same dose of Ag₂Te nanoprobes administrated, suggesting the success of the core–shell synthetic strategy and the potential biomedical applications of core–shell NIR‐II nanoprobes.     

A Quantum Dot Photoswitch for DNA Detection,Gene Transfection,and Live‐Cell Imaging

Quantum dots (QDs) coated with an albumin‐derived copolymer shell exhibit significant photoresponsiveness to DNA loading and have great potential for investigating gene delivery processes. The QDs reported herein are positively charged, have attractive optical properties, and are noncytotoxic and notably stable in live cells. Their complex formation with plasmid DNA leads to proportionally decreased photoluminescence and efficient gene transfection is observed. Therefore, they are suitable for live‐cell bioimaging and mechanistic studies of nonviral gene delivery. Fluorescence correlation spectroscopy is applied for the first time to investigate individual QDs diffusing in large endosomes inside living cells, and serves as a valuable tool to study the physical properties of QDs inside live cells. The data obtained in this study strongly support the notable stability of these QDs, even in cell endosomes.     

Targeted tumor cell internalization and imaging of multifunctional quantum dot-conjugated immunoliposomes in vitro and in vivo

Targeted drug delivery systems that combine imaging and therapeutic modalities in a single macromolecular construct may offer advantages in the development and application of nanomedicines. To incorporate the unique optical properties of luminescent quantum dots (QDs) into immunoliposomes for cancer diagnosis and treatment, we describe the synthesis, biophysical characterization, tumor cell-selective internalization, and anticancer drug delivery of QD-conjugated immunoliposome-based nanoparticles (QD-ILs). Pharmacokinetic properties and in vivo imaging capability of QD-ILs were also investigated. Freeze-fracture electron microscopy was used to visualize naked QDs, liposome controls, nontargeted QD-conjugated liposomes (QD-Ls), and QD-ILs. QD-ILs prepared by insertion of anti-HER2 scFv exhibited efficient receptor-mediated endocytosis in HER2-overexpressing SK-BR-3 and MCF-7/HER2 cells but not in control MCF-7 cells as analyzed by flow cytometry and confocal microscopy. In contrast, nontargeted QD-Ls showed minimal binding and uptake in these cells. Doxorubicin-loaded QD-ILs showed efficient anticancer activity, while no cytotoxicity was observed for QD-ILs without chemotherapeutic payload. In athymic mice, QD-ILs significantly prolonged circulation of QDs, exhibiting a plasma terminal half-life ( t 1/2) of approximately 2.9 h as compared to free QDs with t 1/2 < 10 min. In MCF-7/HER2 xenograft models, localization of QD-ILs at tumor sites was confirmed by in vivo fluorescence imaging.