Imaging of cell surface antigens on tumor lymph-node sections using fluorescence quantum dots

In this work we explored the potential of quantum dots for fluorescent detection of lymphoid surface antigens. To optimize detection with quantum dots, we upgraded a fluorescent microscope that allowed us obtaining multiple images from different quantum dots on a single section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under the mercury lamp during scores of minutes. Double staining of frozen sections with direct conjugates of quantum dots with primary mouse monoclonal antibodies demonstrated direct conjugate high specificity and sensitivity. High stability of quantum dots’ fluorescence allows their use in diagnostics to analyze antigen coexpression on lymphoid tissue sections. “Spillover” of fluorescent signals from quantum dots into adjacent fluorescent channels maximally separated by 40 nm did not exceed 8%, which renders spectral compensation unnecessary.     

d‐penicillamine capped cadmium telluride quantum dots as a novel fluorometric sensor of copper(II)

d ‐penicillamine‐capped cadmium telluride quantum dots (DPA‐capped CdTe QDs) were synthesized as the new fluorescent semiconductor nanocrystal in aqueous solution. Fourier transmission infrared spectroscopy, X‐ray diffraction, transmission electron microscopy, ultraviolet‐visible and photoluminescence spectroscopy were used for characterization of the QDs. Based on the quenching effect of Cu²⁺ ions on the fluorescence intensity of DPA‐capped CdTe QDs, a new fluorometric sensor for copper(II) detection was developed that showed good linearity over the concentration range 5 × 10^–9–3 × 10^–6 m with the detection limit 0.4 × 10^–9 m . Owing to the strong affinity of the DPA to copper(II), the sensor showed appropriate selectivity for copper(II) compared with conventional QDs. The DPA‐capped CdTe QDs was successfully applied for determination of Cu²⁺ concentration in river, well and tap waters with satisfactory results. Copyright © 2013 John Wiley & Sons, Ltd.     

Quinacrine fluorescence analysis of the chromosomes of Dipsacus fullonum L. (Dipsacaceae)

Abstract

Quinacrine fluorescence analysis of the chromosome complement of Dipsacus fullonum L. shows clear heterochromatic regions appearing as brightly fluorescent bands or clusters of fluorescent dots in most chromosomes. The resulting fluorescent pattern is chromosome specific.     

Long pepper (Piper longum) derived carbon dots as fluorescent sensing probe for sensitive detection of Sudan I

In this piece of work, microwave-assisted conversion of a natural precursor in to high-valued nano-scale material was carried out by a completely greener method. The fluorescent carbon dots prepared, designated as long pepper derived carbon dots (LPCDs), have been thoroughly characterized to explore the physical and chemical properties. The system exhibits excitation dependent emission behavior and from the optimal studies the excitation and emission wavelength of the system was found to be 330 nm and 455 nm respectively. On account of the superior fluorescent behavior of the LPCDs, it was successfully employed as a fluorescent sensing probe to detect Sudan I with good level of selectivity and sensitivity. This carcinogenic dye extensively used as food adulterant can impart several health issues. Food product safety is of high concern, therefore a simple facile and economical analytical method was proposed based on the fluorescence of LPCDs for this dye detection with satisfactory statistical parameters. A linear relationship was maintained in the range of 0 to 27.27 μM Sudan I with limit of detection of 0.92 μM. The quenching mechanism was studied and finally attributed to Förster resonance energy transfer (FRET) mechanism. In addition, the probe was effectively implemented for Sudan I detection in commercial chili powder samples with good level of recovery parameters.     

Multiplex targeting, tracking, and imaging of apoptosis by fluorescent surface enhanced Raman spectroscopic dots

We have developed multifunctional fluorescent surface enhanced Raman spectroscopic tagging material (F-SERS dots) composed of silver nanoparticle-embedded silica spheres with fluorescent organic dye and specific Raman labels for multiplex targeting, tracking, and imaging of cellular/molecular events in the living organism. In this study, F-SERS dots fabricated with specific target antibodies (BAX and BAD) were employed for the detection of apoptosis. The F-SERS dots did not show any particular toxicity in several cell lines. The F-SERS dots could monitor the apoptosis effectively and simultaneously through fluorescent images as well as Raman signals in both cells and tissues with high selectivity. Our results clearly demonstrate that F-SERS dots can be easily applicable to multiplex analysis of diverse cellular/molecular events important for maintaining cellular homeostasis.     

Removal characteristics of metal ions using degreased coffee beans: adsorption equilibrium of cadmium(II)

The feasibility of using coffee beans after being dripped and degreased (DCB) as an adsorbent for base metals such as copper(II), zinc(II), lead(II), iron(III) and cadmium(II) were examined. The compositions of the DCB were characterized by Fourier transform infrared spectroscopy, scanning electronic micrograph and fluorescent X-ray. It was found that DCB contain sulfur and calcium from the analysis using fluorescent X-ray. The plant cell wall in DCB has the porous structure from the scanning electron microscopy (SEM) analysis, and the specific surface area was determined to be 1.2 m2/g using the specific surface area analyzer. Batch adsorption experiments on DCB were carried out at various pHs in order to elucidate the selectivity of metal ions. All metals were adsorbed at low pH region (3.0-5.0). Of particular interest was the adsorption characteristics of cadmium(II) on DCB. The adsorption isotherm for cadmium(II) at pH 8 fitted with a Langmuir equation to yield an adsorption equilibrium constant of 55.2 mmol dm(-3) and an adsorption capacity of 5.98 x 10(-2) mmol g(-1). The desorption of cadmium(II) was easily achieved over 90% by a single batchwise treatment with an aqueous solution of hydrochloric acid or nitric acid at more than 0.01 mol dm(-3). These results suggested that DCB behaves as a cation exchanger.     

Luminescence Enhancement of Carboxyl-Coated CdTe Quantum Dots by Silver Nanoparticles

Metallic nanoparticles (NPs) have been used to improve the sensibility of biosensors and bioassays either by enhancing radiative emission or inducing quenching process on fluorescent probes. The aim of this research was to study the interaction of silver and silver-pectin NPs with water-dispersed carboxyl-coated cadmium telluride (CdTe) quantum dots (QDs). Metallic NPs were observed to change the emission of these fluorophores through local field effects. In a solution-base platform, an increase of 82 % was observed for the CdTe emission due to the interaction of QDs and silver-pectin NPs. QDs interaction with silver NPs without pectin was also investigated and a smaller emission enhancement of 20 % was detected. We observed that the NPs’ nature and QDs’ surface charge and concentration are important parameters for NPs-QDs interaction. Moreover, the presence of the pectin polymer shows to be a key component to the observed fluorescence enhancement.     

Visualization of functional rotor proteins of the bacterial flagellar motor in the cell membrane

The bacterial flagellar motor is a rotary motor driven by the electrochemical potentials of specific ions across the cell membrane. Direct interactions between the rotor protein FliG and the stator protein MotA are thought to generate the rotational torque. Here, we used total internal reflection fluorescent microscopy to observe the localization of green fluorescent protein (GFP)-fused FliG in Escherichia coli cells. We identified three types of fluorescent punctate signals: immobile dots, mobile dots that exhibited simple diffusion, and mobile dots that exhibited restricted diffusion. When GFP-FliG was expressed in a DeltafliG background, most of the cells were not mobile. When the cells were tethered to a glass side, however, rotating cells were commonly observed and a single fluorescent dot was always observed at the rotational center of the tethered cell. These fluorescent dots were likely positions at which functional GFP-FliG had been incorporated into a flagellar motor. Our results suggest that flagellar basal bodies diffuse in the cytoplasmic membrane until the axial structure and/or other structures assemble.     

量子点在生物医学中的应用

半导体量子点是无机纳米结晶,构成于硒化镉核心和硫化锌外壳.这种荧光标记物的发射光强是常用有机荧光染料的20倍,稳定性是其100倍.量子点的发射波长取决于核心粒子的大小,而每一种单色量子点的发射波长窄而对称.这些光学特性使量子点在医学诊断、药物的高速筛选以及基因和蛋白质的高通量分析方面具有广泛的应用前景.基于量子点的稳定性和生物相容性,有可能通过标记不同颜色的量子点到不同的分子,观察它们在活细胞内的运动.     

Using cadmium telluride quantum dots as a proton flux sensor and applying to detect H9 avian influenza virus

Semiconductor nanocrystals, often known as quantum dots, have been used extensively for a wide range of applications in bioimaging and biosensing. In this article, we report that the pH-sensitive cadmium telluride (CdTe) quantum dots (QDs) were used as a proton sensor to detect proton flux that was driven by ATP synthesis in chromatophores. To confirm that these QD-labeled chromatophores were responding to proton flux pumping driven by ATP synthesis, N,N'-dicyclohexylcarbodiimide (DCCD) was used as an inhibitor of ATPase activity. Furthermore, we applied the QD-labeled chromatophores as a virus detector to detect the H9 avian influenza virus based on antibody-antigen reaction. The results showed that this QD virus detector could be a new virus-detecting device.     

Self-Assembling Complexes of Quantum Dots and scFv Antibodies for Cancer Cell Targeting and Imaging

Semiconductor quantum dots represent a novel class of fluorophores with unique physical and chemical properties which could enable a remarkable broadening of the current applications of fluorescent imaging and optical diagnostics. Complexes of quantum dots and antibodies are promising visualising agents for fluorescent detection of selective biomarkers overexpressed in tumor tissues. Here we describe the construction of self-assembling fluorescent complexes of quantum dots and anti-HER1 or anti-HER2/neu scFv antibodies and their interactions with cultured tumor cells. A binding strategy based on a very specific non-covalent interaction between two proteins, barnase and barstar, was used to connect quantum dots and the targeting antibodies. Such a strategy allows combining the targeting and visualization functions simply by varying the corresponding modules of the fluorescent complex.     

Flow cytometric analysis of the cadmium-exposed green alga Chlamydomonas reinhardtii (Chlorophyceae)

A flow-cytometric method was developed and evaluated as a rapid ecotoxicological tool using cultures of the microalga Chlamydomonas reinhardtii (Chlorophyceae) under cadmium exposure. Three staining protocols were developed to assess the toxicological impact of this trace metal on algal physiology. Algal cells were exposed to total nominal cadmium concentrations of 5 and 100 µM. After 48 and 72 h exposure the fluorescent probes, fluorescein diacetate (FDA), dihydrorhodamine 123 (DHR123) and tetramethylrhodamine methyl ester (TMRM), were used to assess esterase activity, presence of reactive oxygen species and membrane potential, respectively. Results indicated that cell size, cell granularity and internal complexity were influenced by cadmium, confirming earlier findings on ultrastructural changes in microalgae exposed to trace metals. An increase was observed in the percentage of DHR123 positive cells as well as in their mean fluorescence intensity, on increasing cadmium concentration, confirming that this metal exerts its toxicity through the generation of reactive oxygen species. Furthermore, cadmium exposure resulted in an increase in esterase activity, as reflected in fluorescein fluorescence. We suggest this observation was linked to possible detoxification activity and defence mechanisms. Measurements of control samples during protocol optimization for TMRM proved not to be reproducible, leading us to defer any judgment on results of exposed samples and to conclude that TMRM does not seem suitable for flow cytometric use in algae. Our results demonstrate that although very rarely used in ecotoxicology, flow cytometry is a quick and convenient technique to assess toxic effects that can generate mechanistic information on the mode of action of contaminants.     

Synthesis of CdS nanoparticles from cadmium sulfate solutions using the extracellular polymeric substances of B. licheniformis as stabilizing agent

Mining and hydrometallurgical industries produce large amounts of hazardous metal sulfate solutions as a by-product which can be recycled and exploited to produce valuable and advanced materials. Here, for the first time, extracellular polymeric substances of Bacillus licheniformis were applied as biosurfactants to synthesize quantum dots of cadmium sulfide from pure artificial and impure industrial cadmium sulfate solutions. The bacterial biopolymers stabilized the generated crystalline nuclei as colloidal dots and prevented their further growth or agglomeration. In order to discover the composition and size distribution of the produced particles, characterization was performed by X-ray diffraction (XRD), and transmission electron microscopy (TEM). Results showed that the particles biosynthesized from the pure solution were nano-sized cubic crystals of CdS with the dimensions of 2–10 nm. The same product was also derived from the impure industrial solution. The outcomes of this study indicate the feasibility of cadmium or probably other metal recovery from industrial solutions and wastewaters in the form of valuable metal sulfide nanoparticles.     

Application of quantum dots as probes for correlative fluorescence, conventional, and energy-filtered transmission electron microscopy.

Luminescent semiconductor quantum dots (QDs) are a new class of fluorescent label with wide-ranging applications for cell imaging. The electron density and elemental composition of these materials permit the extension of their use as probes in conventional electron microscopy (TEM) and energy-filtered TEM (EFTEM). Here we illustrate the feasibility of using streptavidin-conjugated QDs as TEM tags by labeling a nuclear protein on cell sections and obtaining correlative fluorescence and TEM data. We also show that QD probes can be employed in conjunction with immunogold for co-localization of proteins at the ultrastructural level. Furthermore, by obtaining cadmium elemental maps of CdSe/ZnS QDs distributed on a nuclear structure, we demonstrate the potential of QDs for co-localization of multiple proteins when used in combination with EFTEM.     

Plasmonic Tuning of Photoluminescence from Semiconducting Quantum Dot Assemblies

We report tuning of photoluminescence enhancement and quenching from closed packed monolayers of cadmium selenide quantum dots doped with gold nanoparticles. Plasmon-mediated control of the emission intensity from the monolayers is achieved by varying the size and packing density of the quantum dots as well as the doping concentration of gold nanoparticles. We observe a unique packing density dependent crossover from enhancement to quenching and vice versa for fixed size of quantum dots and doping concentration of gold nanoparticles. We suggest that this behavior is indicative of a crossover from single particle to collective emission from quantum dots mediated by gold nanoparticles.     

Synthesis of (E)-9-(1-pyrenyl)-4-hydroxynon-2-enal, a fluorescent probe of the (E)-4-hydroxynon-2-enal with retained biological properties

(E)-9-(1-pyrenyl)-4-hydroxynon-2-enal (FHNE), a fluorescent probe of (E)-4-hydroxynon-2-enal (HNE) is synthesised in seven steps and in 35% overall yield, starting from commercially available 1-pyrencarboxyaldehyde. When incubated with cultured HeLa cells this fluorescent probe penetrates cells and particularly concentrates in the region surrounding the nucleus. As the parent compound, HNE it is able to induce the activation of heat shock factor (HSF) and it is able to induce the binding of HSF to heat shock element (HSE).     

A two-photon excitation fluorescence cross-correlation assay for a model ligand-receptor binding system using quantum dots

Two-photon excitation fluorescence cross-correlation spectroscopy (TPE-XCS) is a very suitable method for studying interactions of two distinctly labeled fluorescent molecules. As such, it lends itself nicely to the study of ligand-receptor interactions. By labeling the ligand with one color of fluorescent dye and the receptor with another, it is possible to directly monitor ligand binding rather than inferring binding by monitoring downstream effects. One challenge of the TPE-XCS approach is that of separating the signal due to the receptor from that of the ligand. Using standard organic fluorescent labels there is almost inevitably spectral cross talk between the detection channels, which must be accounted for in TPE-XCS data analysis. However, using quantum dots as labels for both ligand and receptor this limitation can be alleviated, because of the dot's narrower emission spectra. Using solely quantum dots as fluorescent labels is a novel approach to TPE-XCS, which may be generalizable to many pairs of interacting biomolecules after the proof of principle and the assessment of limitations presented here. Moreover, it is essential that relevant pharmacological parameters such as the equilibrium dissociation constant, K(d), can be easily extracted from the XCS data with minimal processing. Herein, we present a modified expression for fractional occupancy based on the auto- and cross-correlation decays obtained from a well-defined ligand-receptor system. Nanocrystalline semiconductor quantum dots functionalized with biotin (lambda(em) = 605 nm) and streptavidin (lambda(em) = 525 nm) were used for which an average K(d) value of 0.30 +/- 0.04 x 10(-9) M was obtained (cf. native system approximately 10(-15)). Additionally, the off-rate coefficient (k(off)) for dissociation of the two quantum dots was determined as 5 x 10(-5) s(-1). This off-rate is slightly larger than for native biotin-streptavidin (5 x 10(-6) s(-1)); the bulky nature of the quantum dots and restricted motion/orientation of functionalized dots in solution can account for differences in the streptavidin-biotin mediated dot-dot binding compared with those for native streptavidin-biotin.     

In vivo observation of a nuclear channel-like system: evidence for a distinct interchromosomal domain compartment in interphase cells

We have investigated the interchromosomal domain compartment in living cells by transfecting cDNA coding for Xenopus vimentin, engineered to contain a nuclear localization signal (NLS), coupled to the green fluorescent protein. In human vimentin-free SW13 cells, this chimeric protein was deposited in body-like "dots" both at 37 degrees C, the nonpermissive temperature for assembly of the amphibian vimentin, and 28 degrees C, the optimal temperature for Xenopus vimentin assembly, indicating that the chimeric protein was assembly incompetent. However, when transfected into a subclone stably expressing Xenopus NLS-vimentin (SW13-SC), the chimeric protein incorporated, as a fluorescent tracer, into the structures formed by NLS-vimentin and allowed us to visualize the outgrowth of the vimentin fibers after a temperature shift to 28 degrees C in living cells. In particular, we followed the time-dependent outgrowth of fibers from nuclear dots, first connecting two dots each and with time three and more, eventually generating a spatially restricted fiber system consisting of few loop-like arrays traversing the nucleus. Virtually identical results were obtained when the temperature was lowered only to 30 and 32 degrees C, respectively. An engineered human NLS-vimentin, without need for temperature shift, formed seemingly identical patterns of nuclear fibrils at 37 degrees C in three additionally transfected human cell lines: MCF-7, PLC, and HeLa. When the epithelial cytokeratin pair 8 and 18 was expressed in the nucleus via an engineered NLS in the cytokeratin 18 gene, more network-like, extended filament arrays were generated. Notably, in cotransfection experiments with Xenopus NLS-vimentin, we observed that the formation of these cytokeratin networks at 37 degrees C initiated from dots that nearly entirely colocalized with the aggregated amphibian NLS-vimentin. After a shift to 28 degrees C, extending Xenopus NLS-vimentin and cytokeratin filaments frequently followed the same path through the nucleus. These data indicate that interphase cells contain a seemingly equivalent, accessible interchromosomal space.     

Imaging of surface cell antigens on the tumor sections of lymph nodes using fluorescence quantum dots

The usefulness of quantum dots for the immunofluorescent detection of surface antigens on the lymphoid cells has been studied. To optimize quantum dots detection we have upgraded fluorescent microscope that allows obtaining multiple images from different quantum dots from one section. Specimens stained with quantum dots remained stable over two weeks and practically did not bleach under mercury lamp illumination during tens of minutes. Direct conjugates of primary mouse monoclonal antibodies with quantum dots demonstrated high specificity and sufficient sensitivity in the case of double staining on the frozen sections. Because of the high stability of quantum dots' fluorescence, this method allows to analyze antigen coexpression on the lymphoid tissue sections for diagnostic purposes. The spillover of fluorescent signals from quantum dots into adjacent fluorescent channels, with maxima differing by 40 nm, did not exceed 8%, which makes the spectral compensation is practically unnecessary.     

The fluorescent probe Bodipy-FL-verapamil is a substrate for both P-glycoprotein and multidrug resistance-related protein (MRP)-1.

Several fluorescent probes have been used in functional studies to analyze drug transport in multidrug-resistant cells by fluorescent microscopy. Because many of these molecules have some drawbacks, such as toxicity, nonspecific background, or accumulation in mitochondria, new fluorescent compounds have been proposed as more useful tools. Among these substances, Bodipy-FL-Verapamil, a fluorescent conjugate of the drug efflux blocker verapamil, has been used to study P-glycoprotein activity in different cell types. In this study we tested by fluorescent microscopy the accumulation of Bodipy-FL-Verapamil in cell lines that overexpress either P-glycoprotein (P-gp) or multidrug resistance-related protein 1 (MRP1). Expression of P-gp and MRP1 was evaluated at the mRNA level by RT-PCR technique and at the protein level by flow cytometric analysis using C219 and MRP-m6 monoclonal antibodies. Results indicate that Bodipy-FL-Verapamil is actually a substrate for both proteins. As a consequence, any conclusion about P-gp activity obtained by the use of Bodipy-FL-Verapamil as fluorescent tracer should be interpreted with caution.