Dots In Cells Research Articles

Second Harmonic Imaging of PEG‑Coated Silicon Quantum Dots in Cells

Second Harmonic Imaging of PEG‑Coated Silicon Quantum Dots in Cells

Fate of CdSe/ZnS quantum dots in cells: Endocytosis, translocation and exocytosis

Semiconductor quantum dots (QDs) have been extensively explored for extensive bioapplications, yet their cellular fate, especially exocytosis, has not been thoroughly investigated. Herein, we systematically investigated the whole cellular process from the endocytosis, intercellular trafficking, to the exocytosis of a typical QD, core/shell CdSe/ZnS QD. Using confocal laser scanning microscopy and flow cytometry, and after carefully eliminating the effect of cell division, we found that the QDs were internalized by HeLa cells with a time-, dose-, and serum-dependent manner. The cellular uptake was inhibited by serum, but eventually peaked after 4–6 h incubation with or without serum. The primary endocytosis pathway was clathrin-mediated, and actin- and microtubule-dependent in the medium with serum, while the caveolae-mediated endocytosis and macropinocytosis were more important for the QDs in the serum-free medium. Inside cells, most QDs distributed in lysosomes, and some entered mitochondria, endoplasmic reticulum, and Golgi apparatus. The translocation of the QDs from other organelles to Golgi apparatus was observed. The exocytosis of QDs was faster than the endocytosis, reaching the maximum in about one hour after cultured in fresh culture medium, with around 60% of the internalized QDs remained undischarged. The exocytosis process was energy- and actin-dependent, and the lysosome exocytosis and endoplasmic reticulum/Golgi pathway were the main routes. This study provides a full picture of behavior and fate of QDs in cells, which may facilitate the design of ideal QDs applied in biomedical and other fields.

Intracellular distribution of nontargeted quantum dots after natural uptake and microinjection

Background:The purpose of this study was to elucidate the mechanism of natural uptake of nonfunctionalized quantum dots in comparison with microinjected quantum dots by focusing on their time-dependent accumulation and intracellular localization in different cell lines.Methods:The accumulation dynamics of nontargeted CdSe/ZnS carboxyl-coated quantum dots (emission peak 625 nm) was analyzed in NIH3T3, MCF-7, and HepG2 cells by applying the methods of confocal and steady-state fluorescence spectroscopy. Intracellular colocalization of the quantum dots was investigated by staining with Lysotracker®.Results:The uptake of quantum dots into cells was dramatically reduced at a low temperature (4°C), indicating that the process is energy-dependent. The uptake kinetics and imaging of intracellular localization of quantum dots revealed three accumulation stages of carboxyl-coated quantum dots at 37°C, ie, a plateau stage, growth stage, and a saturation stage, which comprised four morphological phases: adherence to the cell membrane; formation of granulated clusters spread throughout the cytoplasm; localization of granulated clusters in the perinuclear region; and formation of multivesicular body-like structures and their redistribution in the cytoplasm. Diverse quantum dots containing intracellular vesicles in the range of approximately 0.5–8 μm in diameter were observed in the cytoplasm, but none were found in the nucleus. Vesicles containing quantum dots formed multivesicular body-like structures in NIH3T3 cells after 24 hours of incubation, which were Lysotracker-negative in serum-free medium and Lysotracker-positive in complete medium. The microinjected quantum dots remained uniformly distributed in the cytosol for at least 24 hours.Conclusion:Natural uptake of quantum dots in cells occurs through three accumulation stages via a mechanism requiring energy. The sharp contrast of the intracellular distribution after microinjection of quantum dots in comparison with incubation as well as the limited transfer of quantum dots from vesicles into the cytosol and vice versa support the endocytotic origin of the natural uptake of quantum dots. Quantum dots with proteins adsorbed from the culture medium had a different fate in the final stage of accumulation from that of the protein-free quantum dots, implying different internalization pathways.

Biodistribution study of carbogenic dots in cells and in vivo for optical imaging

Blue fluorescent carbon dots (C-dots) were synthesized and evaluated for their cytotoxicity and also for their optical imaging performance. The results showed that the C-dots could enter into the Hela cells in 15 min incubation and the uptake increased rapidly from 15 min to 2 h. In cytotoxicity study, C-dots were biocompatible and nontoxic to three human cells including two cancer cells (Hela and SMCC-7721) and one normal cell (HEK 293) in concentrations up to 500 μg/mL. Since the endocytic interference factors, including NaN3, MβCD, sucrose, and low temperature, could not play an inhibitory effect on C-dots entering into cells, the direct nonendocytic pathway for C-dots was speculated. The C-dots showed encouraging cell-imaging applications in vitro and in vivo. They entered into cells without any further functionalization, and the fluorescence property of these particles can be used for fluorescence-based cell-imaging applications.

Dimethyl sulfoxide reduces hepatocellular lipid accumulation through autophagy induction

Induction of autophagy is known not only to regulate cellular homeostasis but also to decrease triglyceride accumulation in hepatocytes. The aim of this study is to investigate whether DMSO (dimethyl sulfoxide) has a beneficial role in free fatty acid-induced hepatic fat accumulation. In HepG2 cells, treatment with 0.5 mM palmitate for six hours significantly increased lipid and triglyceride (TG) accumulation, assessed by Oil-red O staining and TG quantification assay. Treatment with 0.01% DMSO for 16 h statistically reduced palmitate-induced TG contents. Pretreatment of 10 mM 3-methyladenine (3MA) for 2 h restored hepatocellular lipid contents, which were attenuated by treatment with DMSO. DMSO increased LC3-II conversion and decreased SQSTM1/p62 expression in a time and dose-dependent manner. In addition, the number of autophagosomes and autolysosomes, as seen under an electron microscopy, as well as the percentage of RFP-LAMP1 colocalized with GFP-LC3 dots in cells transfected with both GFP-LC3 and RFP-LAMP1, as seen under a fluorescent microscopy, also increased in DMSO-treated HepG2 cells. DMSO also suppressed p-eIF2α/p-EIF2S1, ATF4, p-AKT1, p-MTOR and p-p70s6k/p-RPS6KB2 expression as assessed by western blotting. Knockdown of ATF4 expression using siRNA suppressed ATF4 expression and phosphorylation of AKT1, MTOR and RPS6KB2, but increased LC3-II conversion. DMSO reduced not only soluble but also insoluble mtHTT (mutant huntingtin aggregates) expressions, which were masked in the presence of autophagy inhibitor. DMSO, a kind of chemical chaperone, activated autophagy by suppressing ATF4 expression and might play a protective role in the development of fatty acid-induced hepatosteatosis.

Sendai virus-based liposomes enable targeted cytosolic delivery of nanoparticles in brain tumor-derived cells

BackgroundNanotechnology-based bioassays that detect the presence and/or absence of a combination of cell markers are increasingly used to identify stem or progenitor cells, assess cell heterogeneity, and evaluate tumor malignancy and/or chemoresistance. Delivery methods that enable nanoparticles to rapidly detect emerging, intracellular markers within cell clusters of biopsies will greatly aid in tumor characterization, analysis of functional state and development of treatment regimens.ResultsExperiments utilized the Sendai virus to achieve in vitro, cytosolic delivery of Quantum dots in cells cultured from Human brain tumors. Using fluorescence microscopy and Transmission Electron Microscopy, in vitro experiments illustrated that these virus-based liposomes decreased the amount of non-specifically endocytosed nanoparticles by 50% in the Human glioblastoma and medulloblastoma samples studied. Significantly, virus-based liposome delivery also facilitated targeted binding of Quantum dots to cytosolic Epidermal Growth Factor Receptor within cultured cells, focal to the early detection and characterization of malignant brain tumors.ConclusionsThese findings are the first to utilize the Sendai virus to achieve cytosolic, targeted intracellular binding of Qdots within Human brain tumor cells. The results are significant to the continued applicability of nanoparticles used for the molecular labeling of cancer cells to determine tumor heterogeneity, grade, and chemotherapeutic resistivity.

Stability of quantum dots in live cells

Quantum dots (QDs) are highly fluorescent and photostable, making them excellent tools for imaging. When using these QDs in cells and animals, however, intracellular biothiols (e.g., glutathione and cysteine) can degrade the QD monolayer compromising function. Here, we describe a label-free method to quantify the intracellular stability of monolayers on QD surfaces that couples laser desorption/ionization mass spectrometry (LDI-MS) with inductively coupled plasma mass spectrometry (ICP-MS). Using this new approach we have demonstrated that QD monolayer stability is correlated with both QD particle size and monolayer structure, with proper choice of both particle size and ligand structure required for intracellular stability.

Comparison of Ki67 and modified AgNOR in dysplastic epithelial lesion, primary squamous cell carcinoma and metastatic squamous cell carcinoma of head and neck

ABSTRACTThe variations in nucleolar organizer regions (AgNOR) and the increase in Ki67 expression and correlation with histological findings in 64 specimen squamous epithelium from normal epithelium to overt carcinoma were evaluated. Mean AgNOR counts (mAgNOR), proliferative index (pAgNOR), variation in size and dispersion of AgNOR dots in cells were graded and compared in normal, dysplastic, primary and metastatic squamous cell carcinoma (SCC). The Ki67 percentage is significantly increased from normal squamous to SCC group, and the reactivity of staining were related to histological differentiation. The mAgNOR counts were high in all 44 cases of primary and metastatic SCC and low in normal squamous tissue and increased in dysplastic lesions. The mAgNOR count in SCC increased from well differentiated to poorly differentiated. PAgNOR counts were gradually increased in different grade of SCC, 1.4% in well differentiated, 6% in moderately differentiated, 19% in poorly differentiated. There was only significant difference between well differentiated versus poorly differentiated cases. Only significant correlation between Ki67 percentage and mAgNOR and between Ki67 percentage and pAgNOR in primary SCC group was noticed. Therefore, Ki67 is gold standard method for evaluation of proliferation activity. AgNOR quantity is proportional to the proliferative activity of the cell but does not necessarily indicate malignancy.

Salvianolic acid B inhibits autophagy and protects starving cardiac myocytes

To investigate the protective or lethal role of autophagy and the effects of Salvianolic acid B (Sal B) on autophagy in starving myocytes. Cardiac myocytes were incubated under starvation conditions (GD) for 0, 1, 2, 3, and 6 h. Autophagic flux in starving cells was measured via chloroquine (3 μmol/L). After myocytes were treated with Sal B (50 μmol/L) in the presence or absence of chloroquine (3 μmol/L) under GD 3 h, the amount of LC3-II, the abundance of LC3-positive fluorescent dots in cells, cell viability and cellular ATP levels were determined using immunoblotting, immunofluorescence microscopy, MTT assay and luminometer, respectively. Moreover, electron microscopy (EM) and immunofluorescent duel labeling of LC3 and Caspase-8 were used to examine the characteristics of autophagy and apoptosis. Immunoblot analysis showed that the amount of LC3-II in starving cells increased in a time-dependent manner accompanied by increased LC3-positive fluorescence and decreased cell viability and ATP content. Sal B (50 μmol/L) inhibited the increase in LC3-II, reduced the abundance of LC3 immunofluorescence and intensity of Caspase-8 fluorescence, and enhanced cellular viability and ATP levels in myocytes under GD 3 h, regardless of whether chloroquine was present. Autophagy induced by starvation for 3 h led to cell injury. Sal B protected starving cells by blocking the early stage of autophagic flux and inhibiting apoptosis that occurred during autophagy.

The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture

BackgroundNanotechnology has the potential to provide agriculture with new tools that may be used in the rapid detection and molecular treatment of diseases and enhancement of plant ability to absorb nutrients, among others. Data on nanoparticle toxicity in plants is largely heterogeneous with a diversity of physicochemical parameters reported, which difficult generalizations. Here a cell biology approach was used to evaluate the impact of Quantum Dots (QDs) nanocrystals on plant cells, including their effect on cell growth, cell viability, oxidative stress and ROS accumulation, besides their cytomobility.ResultsA plant cell suspension culture of Medicago sativa was settled for the assessment of the impact of the addition of mercaptopropanoic acid coated CdSe/ZnS QDs. Cell growth was significantly reduced when 100 mM of mercaptopropanoic acid -QDs was added during the exponential growth phase, with less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs addition. They were up taken by Medicago sativa cells and accumulated in the cytoplasm and nucleus as revealed by optical thin confocal imaging. As part of the cellular response to internalization, Medicago sativa cells were found to increase the production of Reactive Oxygen Species (ROS) in a dose and time dependent manner. Using the fluorescent dye H2DCFDA it was observable that mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive and linear increase of ROS accumulation.ConclusionsOur results showed that the extent of mercaptopropanoic acid coated CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors including QDs properties, dose and the environmental conditions of administration and that, for Medicago sativa cells, a safe range of 1-5 nM should not be exceeded for biological applications.

Accurate detection and complete tracking of large populations of features in three dimensions

Localization and tracking of colloidal particles in microscopy images generates the raw data necessary to understand both the dynamics and the mechanical properties of colloidal model systems. Yet, despite the obvious importance of analyzing particle movement in three dimensions (3D), accurate sub-pixel localization of the particles in 3D has received little attention so far. Tracking has been limited by the choice of whether to track all particles in a low-density system, or whether to neglect the most mobile fraction of particles in a dense system. Moreover, assertions are frequently made on the accuracies of methods for locating particles in colloid physics and in biology, and the field of particle locating and tracking can be well-served by quantitative comparison of relative performances. We show that by iterating sub-pixel localization in three dimensions, the centers of particles can be more accurately located in three-dimensions (3D) than with all previous methods by at least half an order of magnitude. In addition, we show that implementing a multi-pass deflation approach, greater fidelity can be achieved in reconstruction of trajectories, once particle positions are known. In general, all future work must defend the accuracy of the particle tracks to be considered reliable. Specifically, other researchers must use the methods presented here (or an alternative whose accuracy can be substantianted) in order for the entire investigation to be considered legitimate, if the basis of the physical argument (in colloids, biology, or any other application) depends on quantitative accuracy of particle positions. We compare our algorithms to other recent and related advances in location/tracking in colloids and in biology, and discuss the relative strengths and weaknesses of all the algorithms in various situations. We carry out performance tests directly comparing the accuracy of our and other 3D methods with simulated data for both location and tracking, and in providing relative performance data, we assess just how accurately software can locate particles. We discuss how our methods, now applied to colloids, could improve the location and tracking of features such as quantum dots in cells.

Accumulation of O‐GlcNAc‐Displaying CdTe Quantum Dots in Cells in the Presence of ATP

Where the dots go. GlcNAc-, galactose-, glucose-, and mannose-displaying CdTe quantum dots have been synthesized. We found that GlcNAc-displaying quantum dots were specifically accumulated in digitonin-permeabilized HeLa cells only in the presence of ATP. This finding supports the hypothesis that the HSP70 family in the endoplasmic reticulum has an affinity for the GlcNAc moiety.

Nanometric three-dimensional tracking of individual quantum dots in cells

Wide-field single-molecule fluorescence microscopy has become an established tool for the study of dynamic biological processes which occur in the plane of a cellular membrane. In the current study we have extended this technique to the three-dimensional analysis of molecular mobility. Introduction of a cylindrical lens into the emission path of a microscope produced some astigmatism which was used to obtain the full three-dimensional position information. The localization accuracy of fluorescent objects was calculated theoretically and subsequently confirmed by simulations and by experiments. For further validation individual quantum dots were followed when passively diffusing and actively transported within life cells.

Modified method of AgNOR staining for tissue and interpretation in histopathology

This study was conducted in the department of Pathology King Edward Medical University, from June to December 2002 to introduce the new method of AgNOR staining and its interpretation to increase its reliability. A total of 60 brain specimens were stained with modified AgNOR technique. The diagnosis of malignancy was made on H & E staining. AgNOR counts, variation in size and dispersion of AgNOR dots in cells were graded and compared in malignant and non-malignant lesions. Modified method of AgNOR staining and interpretation was an easy, reliable and reproducible alternative to traditional AgNOR techniques for evaluating proliferation activity of cells in malignant and benign brain lesions. mAgNOR counts of different grades of astrocytoma (2.97+/-0.96, 3.97+/-0.43, 6.01+/-2.74 and 8.01+/-3.56) were significantly (P<0.01) greater when compared with counts of normal brain (0.40+/-0.01), and reactive gliosis (0.60+/-0.01). AgNOR size and dispersion were of higher grade in a significantly greater proportion of malignancy when compared with benign conditions (P<0.05). The AgNOR dots were brighter and more clear with modified staining when compared with previous studies. We conclude that modified AgNOR staining technique is simple, quick and reliable to evaluate cell proliferation by detecting AgNORs size and dispersion. In future, AgNOR size and dispersion should be considered rather than the count only. We recommend the use of morphometry for AgNOR size in future. We also recommend the use of modified AgNOR staining for obtaining sound and confidant results in routine paraffin sections.

OxfordGrid: a web interface for pairwise comparative map views

OxfordGrid is a web application and database schema for storing and interactively displaying genetic map data in a comparative, dot-plot, fashion. Its display is composed of a matrix of cells, each representing a pairwise comparison of mapped probe data for two linkage groups or chromosomes. These are arranged along the axes with one forming grid columns and the other grid rows with the degree and pattern of synteny/colinearity between the two linkage groups manifested in the cell's dot density and structure. A mouse click over the selected grid cell launches an image map-based display for the selected cell. Both individual and linear groups of mapped probes can be selected and displayed. Also, configurable links can be used to access other web resources for mapped probe information. OxfordGrid is implemented in C#/ASP.NET and the package, including MySQL schema creation scripts, is available at ftp://cggc.agtec.uga.edu/OxfordGrid/.

Photoactivation of Quantum Dot Fluorescence Following Endocytosis

We studied the fluorescence of quantum dots in cells. Coating quantum dots with cationic peptides caused them to be endocytosed and transported to lysosomes. After overnight incubation, their fluorescence apparently dimmed but became markedly "photoactivatable", increasing more than 3-fold within minutes on exposure to bright light, and decaying over hours in the dark. Photoactivation was greater in the presence of water than ethanol, and UV illumination compensated for lack of water during photoactivation. Dimming and photoactivation could affect the use of quantum dots as quantitative probes in vivo and lead to new uses, such as tracking molecular movement.

Dentatorubral-pallidoluysian atrophy protein interacts through a proline-rich region near polyglutamine with the SH3 domain of an insulin receptor tyrosine kinase substrate.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neuro degrees enerative disorder associated with CAG/glutamine repeat expansion. While the DRPLA gene is ubiquitously expressed, neuron death occurs in specific anatomical areas of the brain. This predicts that the DRPLA protein interacts with other proteins and that these interactions may play a role in pathogenesis. Here, we describe a protein that binds to the DRPLA product. One of the clones isolated with a yeast two-hybrid system was identified as a human homolog of the insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53). The gene produced two mRNA forms by differential splicing and encoded 552 and 521 amino acids, respectively. The longer form was mainly expressed in the brain and the shorter one in other tissues. The products were phosphorylated upon stimulation of cultured cells with insulin or insulin-like growth factor 1. Binding of the DRPLA protein to IRSp53 was ascertained by co-immunoprecipitation with antibodies and also by co-localization in perinuclear oval dots in cells expressing engineered constructs. A proline-rich region near the polyglutamine tract of the DRPLA protein and the SH3 domain of IRSp53 were involved in the binding. An extended polyglutamine tract significantly reduced binding ability in yeast cells, but not in in vitro binding assays. The identification of IRSp53 and other proteins detected by the yeast hybrid system predicts that DRPLA functions in a signal transduction pathway coupled with insulin/IGF-1.