Synchrotron-based X-ray fluorescence imaging of human cells labeled with CdSe quantum dots

Synchrotron-based X-ray fluorescence (S-XRF) is a powerful technique for imaging the distribution of many biologically relevant elements as well as of “artificial” elements deliberately introduced into tissues and cells, for example, through functionalized nanoparticles. In this study, we explored the potential of S-XRF for chemical nanoimaging (100 nm spatial resolution, nanoXRF) of human cells through the use of functionalized CdSe/ZnS quantum dots (QDs). We used a commercially available QD-secondary antibody conjugate to label the cancer marker HER2 (human epidermal growth factor receptor 2) on the surface of SKOV3 cancer cells and β-tubulin, a protein associated with cytoskeleton microtubules. We set up samples with epoxy inclusion and intracellular labeling as well as samples without epoxy inclusion and with surface labeling. Epoxy inclusion, also used in electron microscopy, has the advantage of preserving cell morphology and guaranteeing long-term stability. QDs proved to be suitable probes for nanoXRF due to the Se emission band, which is not in close proximity to any other emission band, and the signal specificity, which is preserved in both types of labeling. Therefore, nanoXRF using QD-based markers can be very effective at colocalizing specific intracellular targets with elements naturally present in the cell and may complement confocal fluorescence microscopy in a synergistic fashion.     

Quantum dot-based HIV capture and imaging in a microfluidic channel

Globally, over 33.2 million people who mostly live in developing countries with limited access to the appropriate medical care suffer from the human immunodeficiency virus (HIV) infection. We developed an on-chip HIV capture and imaging method using quantum dots (Qdots) from fingerprick volume (10 μl) of unprocessed HIV-infected patient whole blood in anti-gp120 antibody-immobilized microfluidic chip. Two-color Qdots (Qdot525 and Qdot655 streptavidin conjugates) were used to identify the captured HIV by simultaneous labeling the envelope gp120 glycoprotein and its high-mannose glycans. This dual-stain imaging technique using Qdots provides a new and effective tool for accurate identification of HIV particles from patient whole blood without any pre-processing. This on-chip HIV capture and imaging platform creates new avenues for point-of-care diagnostics and monitoring applications of infectious diseases.     

Intracellular imaging of targeted proteins labeled with quantum dots

We developed a new method for imaging the movement of targeted proteins in living cancer cells with photostable and bright quantum dots (QDs). QDs were conjugated with various molecules and proteins, such as phalloidin, anti-tubulin antibody and kinesin. These bioconjugated QDs were mixed with a transfection reagent and successfully internalized into living cells. The movements of individual QDs were tracked for long periods of time. Phalloidin conjugated QDs bound to actin filaments and showed almost no movement. In contrast, anti-tubulin antibody conjugated QDs bound to microtubules and revealed dynamic movement of microtubules. Kinesin showed an interesting behavior whereby kinesin came to be almost paused briefly for a few seconds and then moved once again. This is in direct contrast to the smoothly continuous movement of kinesin in an in vitro assay. The maximum velocity of kinesin in cells was faster than that in the in vitro assay. These results suggest that intracellular movement of kinesin is different from that in the in vitro assay. This newly described method will be a powerful tool for investigating the functions of proteins in living cells.     

Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.     

From analog to digital: exploring cell dynamics with single quantum dots

Semiconductor quantum dots (QDs) have emerged as new fluorescent probes for biology. When combined with ultrasensitive optical techniques, they allow motions of individual biomolecules to be tracked in live cells with high signal-to-noise and over unprecedented durations. Single QD imaging readily offers a powerful tool to investigate the organization in cell membranes. Altogether QDs will contribute to more advanced biological imaging and enable new studies on the dynamics of cellular processes.Robert Feulgen Lecture 2005 presented at the Joint Meeting of the Society for Histochemistry and The Histochemical Society in Noordwijkerhout, The Netherlands     

Optical coding of mammalian cells using semiconductor quantum dots

Cell-based assays are widely used to screen compounds and study complex phenotypes. Few methods exist, however, for multiplexing cellular assays or labeling individual cells in a mixed cell population. We developed a generic encoding method for cells that is based on peptide-mediated delivery of quantum dots (QDs) into live cells. The QDs are nontoxic and photostable and can be imaged using conventional fluorescence microscopy or flow cytometry systems. We created unique fluorescent codes for a variety of mammalian cell types and show that our encoding method has the potential to create > 100 codes. We demonstrate that QD cell codes are compatible with most types of compound screening assays including immunostaining, competition binding, reporter gene, receptor internalization, and intracellular calcium release. A multiplexed calcium assay for G-protein-coupled receptors using QDs is demonstrated. The ability to spectrally encode individual cells with unique fluorescent barcodes should open new opportunities in multiplexed assay development and greatly facilitate the study of cell/cell interactions and other complex phenotypes in mixed cell populations.     

Lead sulfide near-infrared quantum dot bioconjugates for targeted molecular imaging

In this paper, we report the use of lead sulfide quantum dot (PbS QD) bioconjugates as near infrared (NIR) contrast agents for targeted molecular imaging with expanded emission wavelengths beyond 1000 nm. The red-shifted emission band, coupled with the small particle size, which will facilitate clearance, both afford PbS QDs unique properties for noninvasive, high resolution in vivo NIR imaging applications. We have performed imaging experiments at the molecular level using surface-modified PbS NIR QDs, together with our lab-built NIR imaging system. This novel instrumentation and fluorescent contrast agent have enabled us to study the relatively unexplored NIR biomedical imaging spectral region of 900-1200 nm. Preliminary experimental results indicate that PbS-QD/antibody bioconjugates are promising candidates for targeted NIR molecular imaging and future in vivo NIR tissue imaging applications.     

Mortalin imaging in normal and cancer cells with quantum dot immuno-conjugates

Quantum dots are the nanoparticles that are recently emerging as an alternative to organic fluorescence probes in cell biology and biomedicine, and have several predictive advantages. These include their ⑴broad absorption spectra allowing visualization with single light source, ⑵exceptional photo-stability allowing long term studies and ⑶narrow and symmetrical emission spectrum that is controlled by their size and material composition. These unique properties allow simultaneous excitation of different size of quantum dots with a single excitation light source, their simultaneous resolution and visualization as different colors. At present there are only a few studies that have tested quantum dots in cellular imaging. We describe here the use of quantum dots in mortalin imaging of normal and cancer cells. Mortalin staining pattern with quantum dots in both normal and cancer cells mimicked those obtained with organic florescence probes and were considerably stable.     

声光可调谐滤波器的细胞生物学研究应用

波长选择在荧光光谱仪和显微镜等光学应用中发挥了至关重要的作用。声光可调谐滤波器(AOTF)作为一种电光器件可实现多光源入射波长、功率的同时调制。在声光可调谐滤波器中,压电换能器结合于二氧化碲或石英晶体产生高频声波,改变晶体折射率形成周期性分布。该现象在晶体中生成衍射光栅,使以布拉格角正交入射的光束被高效衍射至一阶光束。当改变施加到晶体的信号频率时将改变折射率变化周期,因此,衍射光的波长随之改变。同时,衍射光强度由施加到晶体的信号振幅决定。本文从声光可调谐滤波器原理和特点出发,总结了声光可调谐滤波器在细胞生物学研究系统中的应用模型。得益于作用时间短、波长分辨率高、无振动部件等特性,声光可调谐滤波器提升了多波长光源功率调制能力,使细胞计数系统具备了细胞高光谱成像能力。所以不仅限于传统细胞生物学研究,包含声光可调谐滤波器件的系统还将在多参数高内涵成像分析、扫描荧光显微术、药物毒理研究等领域成为有力的研究工具。     

Quantum dot-based assay for Cu quantification in bacterial cell culture

A simple and sensitive method for quantification of nanomolar copper with a detection limit of 1.2 × 10⁻¹⁰ M and a linear range from 10⁻⁹ to 10⁻⁸ M is reported. For the most useful analytical concentration of quantum dots, 1160 μg/ml, a 1/K_sv value of 11 μM Cu²⁺ was determined. The method is based on the interaction of Cu²⁺ with glutathione-capped CdTe quantum dots (CdTe–GSH QDs) synthesized by a simple and economic biomimetic method. Green CdTe–GSH QDs displayed the best performance in copper quantification when QDs of different sizes/colors were tested. Cu²⁺ quantification is highly selective given that no significant interference of QDs with 19 ions was observed. No significant effects on Cu²⁺ quantification were determined when different reaction matrices such as distilled water, tap water, and different bacterial growth media were tested. The method was used to determine copper uptake kinetics on Escherichia coli cultures. QD-based quantification of copper on bacterial supernatants was compared with atomic absorption spectroscopy as a means of confirming the accuracy of the reported method. The mechanism of Cu²⁺-mediated QD fluorescence quenching was associated with nanoparticle decomposition.     

Indication of intracellular physiological pH changes by l-cysteine-coated CdTe quantum dots with an acute alteration in emission color

A novel quantum dots (QDs) based biosensor was developed to monitor physiological pH changes in both fixed and living cells by means of pH-dependent emission color of the QDs. In our system, the nominally single-sized colloidal solution samples of the l-cysteine-capped CdTe QDs with intrinsically broadened size distributions were prepared by employing aqueous synthesis technique. The quench of fluorescence intensities of the QDs with a 16 nm red shift of the emission maximum and a color change from green to yellow was observed with a slight pH decrease (from 7.0 to 6.8) in the system. This pH-dependent emission could be attributed to the efficient exciton energy transfer from smaller QDs to larger ones, which was controlled by electrostatic-tuned aggregation/disaggregation (low/high pH values) processes of the QDs. In addition to high stability, the emission shift of the QDs was reversible for at least one cycle under optimal conditions. Our pH biosensor may find potential application for monitoring the intracellular pH changes in both physiological and pathological conditions.     

Microwave‐assisted aqueous synthesis of new quaternary‐alloyed CdSeTeS quantum dots; and their bioapplications in targeted imaging of cancer cells

In this study, we report for the first time a one‐pot approach for the synthesis of new CdSeTeS quaternary‐alloyed quantum dots (QDs) in aqueous phase by microwave irradiation. CdCl₂ was used as a Cd precursor during synthesis, NaHTe and NaHSe were used as Te and Se precursors and mercaptopropionic acid (MPA) was used as a stabilizer and source of sulfur. A series of quaternary‐alloyed QDs of different sizes were prepared. CdSeTeS QDs exhibited a wide emission range from 549 to 709 nm and high quantum yield (QY) up to 57.7 %. Most importantly, the quaternary‐alloyed QDs possessed significantly long fluorescence lifetimes > 100 ns as well as excellent photostability. Results of high‐resolution transmission electron microscopy (HRTEM), energy dispersive X‐ray spectroscopy (EDX) and powder X‐ray diffraction (XRD) spectroscopy showed that the nanocrystals possessed a quaternary alloy structure with good crystallinity. Fluorescence correlation spectroscopy (FCS) showed that QDs possessed good water solubility and monodispersity in aqueous solution. Furthermore, CdSeTeS QDs were modified with alpha‐thio‐omega‐carboxy poly(ethylene glycol) (HS‐PEG‐COOH) and the modified QDs were linked to anti‐epidermal growth factor receptor (EGFR) antibodies. QDs with the EGFR antibodies as labeling probes were successfully applied to targeted imaging for EGFR on the surface of SiHa cervical cancer cells. We believe that CdSeTeS QDs can become useful probes for in vivo targeted imaging and clinical diagnosis. Copyright © 2012 John Wiley & Sons, Ltd.     

Immunofluorescent labeling of cancer cells with quantum dots synthesized in aqueous solution

Thioglycolic-acid-stabilized CdTe quantum dots, synthesized directly in aqueous solution, are successfully conjugated with biotin and polyethylene glycol. Using these conjugates, we report the development of this kind of water-soluble quantum dot for immunofluorescent labeling of cancer cells. The results show that these conjugates have very low nonspecific binding and good stability against photobleaching, enabling them to be applied in many biological fields, such as cellular labeling, intracellular tracking, and other imaging applications.     

Simple and sensitive progesterone detection in human serum using a CdSe/ZnS quantum dot-based direct binding assay

In this study, we developed a CdSe/ZnS quantum dot (QD)-based immunoassay for use in determining the presence of progesterone (P₄) in human serum. Hydrophilic QDs were conjugated to anti-progesterone antibody (P₄Ab) via ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling reagents. After purification, the P₄Ab–QD conjugates were immobilized onto the wells of a 96-well microtiter plate, and a direct-binding immunoassay based on the binding of P₄ to immobilized P₄Ab–QD conjugates had a detection limit of 0.21 ng/ml and a sensitivity of 1.37 ng/ml, with a linear range of 0.385 to 4.55 ng/ml. The proposed immunoassay was successfully used to determine the P₄ concentration in real human serum, and the results showed a good correlation with the accredited radioimmunoassay (RIA).     

The preparation of ethylenediamine‐modified fluorescent carbon dots and their use in imaging of cells

In this work, fluorescent carbon dots (CDs) were synthesized using a hydrothermal method with glucose as the carbon source and were surface‐modified with ethylenediamine. The properties of as‐prepared CDs were analyzed by transmission electron microscopy (TEM), Fourier transform infrared (FTIR), ultraviolet–visible light (UV/vis) absorption and fluorescent spectra. Furthermore, CDs conjugated with mouse anti‐(human carcinoembryonic antigen) (CEA) monoclonal antibody were successful employed in the biolabeling and fluorescent imaging of human gastric carcinoma cells. In addition, the cytotoxicity of CDs was also tested using human gastric carcinoma cells. There was no apparent cytotoxicity on human gastric carcinoma cells. These results suggest the potential application of the as‐prepared CDs in bioimaging and related fields. Copyright © 2015 John Wiley & Sons, Ltd.     

Modeling bidirectional transport of quantum dot nanoparticles in membrane nanotubes

This paper develops a model of transport of quantum dot (QD) nanoparticles in membrane nanotubes (MNTs). It is assumed that QDs are transported inside intracellular organelles (called here nanoparticle-loaded vesicles, NLVs) that are propelled by either kinesin or dynein molecular motors while moving on microtubules (MTs). A vesicle may have both types of motors attached to it, but the motors are assumed to work in a cooperative fashion, meaning that at a given time the vesicle is moved by either kinesin or dynein motors. The motors are assumed not to work against each other, when one type of motors is pulling the vesicle, the other type is inactive. From time to time the motors may switch their roles: passive motors can become active motors and vice versa, resulting in the change of the vesicle’s direction of motion. It is further assumed that QDs can escape NLVs and become free QDs, which are then transported by diffusion. Free QDs can be internalized by NLVs. The effects of two possible types of MT orientation in MNTs are investigated: when all MTs have a uniform polarity orientation, with their plus-ends directed toward one of the cells connected by an MNT, and when MTs have a mixed polarity orientation, with half of MTs having their plus-ends directed toward one of the cells and the other half having their plus-ends directed toward the other cell. Computational results are presented for three cases. The first case is when organelles are as likely to be transported by kinesin motors as by dynein motors. The second case is when organelles are more likely to be transported by kinesin motors than by dynein motors, and the third case is when NLVs do not associate with dynein motors at all.     

Studies on quantum dots synthesized in aqueous solution for biological labeling via electrostatic interaction

3-Mercaptopropyl acid-stabilized CdTe nanoparticles synthesized in aqueous solution are effectively bound to a biomacromolecule, papain, via electrostatic interaction. The conjugation between the nanoparticles and the papain is demonstrated by UV-Vis absorption, photoluminescence spectroscopy, transmission electron microscopy, and fluorescence micrographs. The biological activity of papain is maintained after the conjugation. The effects of the quantity of papain and the size of nanoparticles on the fluorescence characteristics of the CdTe-papain bioconjugates were studied.     

Development of magnetic separation and quantum dots labeled immunoassay for the detection of mercury in biological samples

A rapid and sensitive immunoassays of mercury (Hg) in biological samples was developed using quantum dots (QDs) and magnetic beads (MBs) as fluorescent and separated probes, respectively. A monoclonal antibody (mAb) that recognizes an Hg detection antigen (BSA-DTPA-Hg) complex was produced by the injection of BALB/c mice with an Hg immunizing antigen (KLH-DTPA-Hg). Then the ascites monoclonal antibodies were purified. The Hg monoclonal antibody (Hg-mAb) is conjugated with MBs to separate Hg from biological samples, and the other antibody, which is associated with QDs, is used to detect the fluorescence. The Hg in biological samples can be quantified using the relationship between the QDs fluorescence intensity and the concentration of Hg in biological samples following magnetic separation. In this method, the detection linear range is 1–1000 ng/mL, and the minimum detection limit is 1 ng/mL. The standard addition recovery rate was 94.70–101.18%. The relative standard deviation values were 2.76–7.56%. Furthermore, the Hg concentration can be detected in less than 30 min, the significant interference of other heavy metals can be avoided, and the simultaneous testing of 96 samples can be performed. These results indicate that the method could be used for rapid monitoring Hg in the body.     

Four-dimensional imaging of filter-grown polarized epithelial cells

Understanding how epithelial cells generate and maintain polarity and function requires live cell imaging. In order for cells to become fully polarized, it is necessary to grow them on a permeable membrane filter; however, the translucent filter obstructs the microscope light path required for quantitative live cell imaging. Alternatively, the membrane filter may be excised but this eliminates selective access to apical and basolateral surfaces. Conversely, epithelial cells cultured directly on glass exhibit different phenotypes and functions from filter grown cells. Here, we describe a new method for culturing polarized epithelial cells on a Transwell filter insert that allows superior live cell imaging with spatial and temporal image resolution previously unachievable using conventional methods. Cells were cultured on the underside of a filter support. Epithelial cells grown in this inverted configuration exhibit a fully polarized architecture, including the presence of functional tight junctions. This new culturing system permits four-dimensional (three spatial dimension over time) imaging of endosome and Golgi apparatus dynamics, and permits selective manipulation of the apical and basolateral surfaces. This new technique has wide applicability for visualization and manipulation of polarized epithelial cells.     

Ultrasensitive detection of ferritin in human serum by Western blotting based on quantum dots-labeled avidin-biotin system

Various methods have been applied for serum ferritin detection, however, these methods still have some limitations. Over the last few years, quantum dots (QDs) have become very attractive for immunoassays because of their enormous potentials in ultrasensitive analysis. In this study, a Western blotting method combined with QDs‐labeled avidin–biotin system for detecting human serum ferritin was described. Meanwhile, the traditional diaminobenzidine (DAB)–horseradish peroxidase (HRP) method had been compared with the method. The linearity of this QDs‐based Western blotting method was from 0.27 to 1.1 ng, and the quantification limit was 0.27 ng, the sensitivity was up to pictogram values. Real serum samples such as hepatoma, thalassemia patient and normal individual sera were analyzed, the analysis results demonstrated that there was significant difference in the concentrations of ferritin between patients and normal individual serum. Furthermore, the recovery of ferritin from the serum samples of patients ranged from 98.15 to 119.67%, and the RSD (relative standard deviation) ranged from 8.73 to 11.61%, the repeatabilities were well within the acceptable range, which revealed that this method is a stable and reproducible method for detecting serum ferritin and have potential application prospect in clinical laboratory of serum ferritin detection.