A public software for energy filtering transmission electron tomography (EFTET-J): application to the study of granular inclusions in bacteria from Riftia pachyptila

Energy-filtering transmission electron microscopy (EFTEM) allows the determination of elemental distributions out of a sequence of energy filtered images. Combined with electron tomography, EFTEM is a powerful tool to obtain three-dimensional chemical maps from sub-cellular structures. However, there is no existing software in the public-domain for the computation and analysis of 3D-chemical maps. Here, we present a Java-based program to compute 3D-elemental distribution. This program is available as a set of plug-ins for the public-domain Java image processing program Image J inspired by NIH Image. Its implemented algorithms have been successfully applied to the three-dimensional localization of iron granules in semi thin (200 nm) epon sections from the vent worm Riftia pachyptalia.     

Quantum dots targeted to the assigned organelle in living cells

Fluorescent nanocrystal quantum dots (QDs) have the potential to be applied to bioimaging since QDs emit higher and far longer fluorescence than conventional organic probes. Here we show that QDs conjugated with signal peptide obey the order to transport the assigned organelle in living cells. We designed the supermolecule of luminescent QDs conjugated with nuclear- and mitochondria-targeting ligands. When QDs with nuclear-localizing signal peptides were added to the culture media, we can visualize the movements of the QDs being delivered into the nuclear compartment of the cells with 15 min incubation. In addition, mitochondrial signal peptide can also transport QDs to the mitochondria in living cells. In conclusion, these techniques have the possibility that QDs can reveal the transduction of proteins and peptides into specific subcellular compartments as a powerful tool for studying intracellular analysis in vitro and even in vivo.     

In situ localization of sulphur in the thioautotrophic symbiotic model Lucina pectinata (Gmelin, 1791) by cryo-EFTEM microanalysis

BACKGROUND INFORMATION: Lucina pectinata is a large tropical lucinid known to harbour sulphide-oxidizing bacteria in specialized gill cells. Conventional TEM (transmission electron microscopy) has shown that bacteriocytes also harbour visibly 'empty' vesicles whose chemical content remains, to date, only roughly determined. RESULTS: In the present study, L. pectinata gill tissues were cryo-fixed as fast as possible by performing high-pressure freezing before a freeze-substitution process and finally performing a cryo-embedding in Lowicryl. Ultrathin sections were then used for a cryo-EFTEM (where EFTEM stands for energy-filtered TEM) microanalysis. Results show that bacteriocytes within the gill tissues contain elemental sulphur in small vesicles produced by the host itself. In instances of sporadic depletion of sulphur in the environment, such structures may act as energy sources for bacterial endosymbionts. CONCLUSIONS: The cryo-EFTEM techniques represent (i) the only method used to date to locate and preserve sulphur at the cellular level and (ii) a powerful tool for sulphur metabolism analysis in thioautotrophic symbiont relationships.     

Bactericidal actions of a silver ion solution on Escherichia coli, studied by energy-filtering transmission electron microscopy and proteomic analysis

Bactericidal actions of the silver ion on Escherichia coli as a model microorganism were studied using energy-filtering transmission electron microscopy (EFTEM), two-dimensional electrophoresis (2-DE), and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). EFTEM observations demonstrated that the silver ion readily infiltrates the interior of E. coli, contrary to the early hypothesis that it resides initially in the cell membrane area. Furthermore, 2-DE and MALDI-TOF MS indicated that the expression of a ribosomal subunit protein as well as that of some other enzymes and proteins is affected by the silver ion. The present results demonstrate for the first time that one of the major bactericidal functions of the silver ion is its interaction with the ribosome and the ensuing inhibition in expression of the enzymes and proteins essential to ATP production.     

Bactericidal Actions of a Silver Ion Solution on Escherichia coli, Studied by Energy-Filtering Transmission Electron Microscopy and Proteomic Analysis

Bactericidal actions of the silver ion on Escherichia coli as a model microorganism were studied using energy-filtering transmission electron microscopy (EFTEM), two-dimensional electrophoresis (2-DE), and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). EFTEM observations demonstrated that the silver ion readily infiltrates the interior of E. coli, contrary to the early hypothesis that it resides initially in the cell membrane area. Furthermore, 2-DE and MALDI-TOF MS indicated that the expression of a ribosomal subunit protein as well as that of some other enzymes and proteins is affected by the silver ion. The present results demonstrate for the first time that one of the major bactericidal functions of the silver ion is its interaction with the ribosome and the ensuing inhibition in expression of the enzymes and proteins essential to ATP production.     

Water‐soluble mercaptoundecanoic acid (MUA)‐coated CdTe quantum dots: one‐step microwave synthesis,characterization and cancer cell imaging

ABSTRACT: In this study, a one‐step approach for aqueous synthesis of highly luminescent semiconductors, CdTe quantum dots (QDs), using long‐chain thiols‐mercaptoundecanoic acid (MUA) as surface ligand, was developed in a microwave irradiation system. The synthetic conditions were systematically investigated. The as‐prepared MUA‐coated QDs were characterized by various spectroscopy techniques, transmission electron microscopy (TEM) and X‐ray powder diffraction (XRD). The experimental results document that MUA‐coated CdTe QDs have small diameter, good stability, high luminescence and long lifetime. Particularly, it was confirmed, using fluorescence correlation spectroscopy (FCS) that, compared with other ligand, MUA formed a thicker ligand layer on the QD surfaces, which will help their stability and conjugation with biomolecules. Furthermore, MUA‐coated QDs were successfully used for HeLa cell imaging. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis and biological assay of GSH functionalized fluorescent quantum dots for staining Hydra vulgaris

Quantum dots (QDs) have been used extensively as fluorescent markers in several studies on living cells. Here, we report the synthesis of conjugates based on glutathione (GSH) and QDs (GSH-QDs) and we prove how these functionalized fluorescent probes can be used for staining a freshwater invertebrate called Hydra vulgaris. GSH is known to promote Hydra feeding response by inducing mouth opening. We demonstrate that GSH-QDs as well are able to elicit biological activity in such an animal, which results in the fluorescent staining of Hydra. GSH-QDs, once they reach the gastric region, are internalized by endodermal cells. The efficiency of GSH-QD internalization increases significantly when nanoparticles are coadministrated with free GSH. We also compared the behavior of bare QDs to that of GSH-QDs both in the presence and in the absence of free GSH. The conclusions from these series of experiments point to the presence of GSH binding proteins in the endodermal cell layer and uncover a novel role played by glutathione in this organism.     

Formulation, characterization, and in vitro evaluation of quantum dots loaded in poly(lactide)-vitamin E TPGS nanoparticles for cellular and molecular imaging

We developed a novel system of poly(lactide acid)-d-alpha-tocopheryl polyethylene glycol 1000 succinate (PLA-TPGS) nanoparticles (NPs) for quantum dots (QDs) formulation to improve imaging effects and reduce side effects as well as to promote a sustainable imaging. The QDs-loaded PLA-TPGS NPs were prepared by a modified solvent extraction/evaporation method, which were then characterized by laser light scattering (LLS) for size and size distribution; field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM) and transmission electron microscope (TEM) for surface morphology. Surface chemistry of the QDs-loaded PLA-TPGS NPs was analyzed by X-ray photoelectron microscopy (XPS) and Fourier transform infra-red spectroscopy (FTIR). Encapsulation efficiency of the QDs in the polymeric nanoparticles was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). The photostability of the QDs formulated in the PLA-TPGS nanoparticles was investigated as changes in the florescence intensity versus the irradiation time. Confocal laser scanning microscopy (CLSM) was used to image the cellular uptake of the QDs-loaded NPs by MCF-7 cells. Methylthiazolyldiphenyl-tetrazolium (MTT) assay was employed to assess the viability of MCF-7 cells incubated with the QDs formulated by the PLA-TPGS NPs versus the mercaptoacetic acid (MAA)-coated QDs. It was found that the QDs formulated in the PLA-TPGS NPs can result in higher fluorescence intensity and higher photostability than the bare QDs as well as lower cytotoxicity than the MAA-coated QDs.     

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.     

Scanning transmission X-ray,laser scanning,and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.     

Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.     

Chemical mapping of DNA and counter-ion content inside phage by energy-filtered TEM

Double-stranded DNA in many bacterial viruses (phage) is strongly confined, which results in internal genome pressures of tens of atmospheres. This pressure is strongly dependent on local ion concentration and distribution within the viral capsid. Here, we have used electron energy loss spectroscopy (EELS), energy-filtered TEM (EFTEM) and X-ray energy dispersive spectroscopy to provide such chemical information from the capsid and the phage tail through which DNA is injected into the cell. To achieve this, we have developed a method to prepare thin monolayers of self-supporting virus/buffer films, suitable for EELS and EFTEM analysis. The method is based on entrapment of virus particles at air–liquid interfaces; thus, the commonly used method of staining by heavy metal salts can be avoided, eliminating the risk for chemical artifacts. We found that Mg^2 + concentration was approximately 2–4 times higher in the DNA-filled capsid than in the surrounding TM buffer (containing 10 mM Mg^2 +). Furthermore, we also analyzed the DNA content inside the phage tail by mapping phosphorus and magnesium.     

Co-localization of apoptosis-regulating proteins in mouse mammary epithelial HC11 cells exposed to TGF-beta1

TGF-beta1 is an apoptogenic agent for mammary epithelial cells (MEC). The molecular mechanism of the TGF-beta1-induced apoptosis remains, however, obscure. In the present study we used laser scanning cytometry, confocal microscopy and immunogold electron microscopy to analyze the expression, aggregation and co-localization of caspase-8, Bid, Bax and VDAC-1. These proteins are regarded as the most important factors involved in the regulatory phase of TGF-beta1-induced apoptosis. Apoptosis in HC11 mouse MEC manifested with a simultaneous increase in expression and subcellular aggregation of caspase-8, Bid, Bax and VDAC-1. Confocal microscopy revealed a strong pattern of co-localization of examined proteins during both early and late apoptosis. Experiments with double- and triple-staining immunoelectron microscopy showed a co-localization of Bax/Bid, caspase-8/Bax/Bid, and Bax/VDAC-1, on the membranes of mitochondria, Golgi apparatus, rough endoplasmic reticulum, nuclear envelope, nuclear pore, and within the nucleus. In conclusion, the observed pattern of changes in aggregation and subcellular localization of caspase-8, Bid, Bax and VDAC-1 during TGF-beta1-induced apoptosis in HC11 mouse MEC suggests an interaction between these proteins and formation of multimeric complexes on organellar membranes, thus controlling their permeability for intracellular mediators of apoptosis.     

Quantum dots do not affect the behaviour of mouse embryonic stem cells and kidney stem cells and are suitable for short-term tracking

Quantum dots (QDs) are small nanocrystals widely used for labelling cells in order to enable cell tracking in complex environments in vitro, ex vivo and in vivo. They present many advantages over traditional fluorescent markers as they are resistant to photobleaching and have narrow emission spectra. Although QDs have been used effectively in cell tracking applications, their suitability has been questioned by reports showing they can affect stem cell behaviour and can be transferred to neighbouring cells. Using a variety of cellular and molecular biology techniques, we have investigated the effect of QDs on the proliferation and differentiation potential of two stem cell types: mouse embryonic stem cells and tissue-specific stem cells derived from mouse kidney. We have also tested if QDs released from living or dead cells can be taken up by neighbouring cells, and we have determined if QDs affect the degree of cell-cell fusion; this information is critical in order to assess the suitability of QDs for stem cell tracking. We show here that QDs have no effect on the viability, proliferation or differentiation potential of the two stem cell types. Furthermore, we show that the extent of transfer of QDs to neighbouring cells is <4%, and that QDs do not increase the degree of cell-cell fusion. However, although the QDs have a high labelling efficiency (>85%), they are rapidly depleted from both stem cell populations. Taken together, our results suggest that QDs are effective cell labelling probes that are suitable for short-term stem cell tracking.     

Molecular profiling of single cancer cells and clinical tissue specimens with semiconductor quantum dots

Semiconductor quantum dots (QDs) are a new class of fluorescent labels with broad applications in biomedical imaging, disease diagnostics, and molecular and cell biology. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to multifunctional nanoparticle probes that are highly bright and stable under complex in vitro and in vivo conditions. New designs involve encapsulating luminescent QDs with amphiphilic block copolymers, and linking the polymer coating to tumor-targeting ligands and drug-delivery functionalities. These improved QDs have opened new possibilities for real-time imaging and tracking of molecular targets in living cells, for multiplexed analysis of biomolecular markers in clinical tissue specimens, and for ultrasensitive imaging of malignant tumors in living animal models. In this article, we briefly discuss recent developments in bioaffinity QD probes and their applications in molecular profiling of individual cancer cells and clinical tissue specimens.     

Direct and indirect immunolabelling of HeLa cells with quantum dots

With excellent optical properties, quantum dots (QDs) have been made as attractive molecular probes for labelling cells in biological research. In this study high‐quality CdSe QDs prepared in a paraffin–oleic acid system were used as fluorescent labels in direct and indirect detection of carcinoembryonic antigen (CEA), a cancer marker expressed on the surface of HeLa cells. The primary antibody (Ab) (rabbit anti‐CEA8) and secondary Ab (goat anti‐rabbit IgG) were covalently linked to carboxyl‐functioned CdSe QDs, and both the QDs–antibody and QDs–IgG probes were successfully used to label HeLa cells. The present study demonstrates the practicability of CdSe QDs as an attractive type of fluorescent labels for biological applications such as protein probes and cell imaging. Copyright © 2008 John Wiley & Sons, Ltd.     

Applications of T-lymphoma labeled with fluorescent quantum dots to cell tracing markers in mouse body

Photoluminescent semiconductor quantum dots (QDs) are novel nanometer-size probes that have found bioimaging. Here we imaged a cell line of mouse lymphocytes. QDs were actively taken into the target cells by endocytotic pathways. The fluorescence of QDs held in the endosomes could be studied for more than a week and remained stable luminescence against cell activation induced by concanavalin A, phytohemagglutinin, phorbol myristate acetate, and calcium ionophore A23187. These results suggested that QD-labeling was stable and did not affect either cell activation or cell function. When QD-labeled cells were intravenously injected into mouse, they remained in the peripheral blood in a concentration of approximately 10% up to 5 days after injection using both fluorescence microscopy and flow cytometry. In addition, approximately 20% of QDs were detected in the kidneys, liver, lung, and spleen and could still be observed 7 days after injection. These results suggested that fluorescent probes of QDs might be useful as bioimaging tools for tracing target cells over the period of a week in vivo.     

Quantum dots‐based fluorescence resonance energy transfer biosensor for monitoring cell apoptosis

The development of advanced methods for accurately monitoring cell apoptosis has extensive significance in the diagnostic and pharmaceutical fields. In this study, we developed a rapid, sensitive and selective approach for the detection of cell apoptosis by combining the site‐specific recognition and cleavage of the DEVD–peptide with quantum dots (QDs)‐based fluorescence resonance energy transfer (FRET). Firstly, biotin‐peptide was conjugated on the surface of AuNPs to form AuNPs‐pep through the formation of an Au‐S bond. Then, AuNPs–pep–QDs nanoprobe was obtained through the connection between AuNPs–pep and QDs. FRET is on and the fluorescence of QDs is quenched at this point. The evidence of UV–vis spectra, transmission electron microscopy (TEM), and Fourier transform infrared (FT‐IR) spectroscopy revealed that the connection was successful. Upon the addition of apoptosis cell lysis solution, peptide was cleaved by caspase‐3, and AuNPs was dissociated from the QDs. At this time, FRET is off, and thus the QDs fluorescence was recovered. The experimental conditions were optimized in terms of ratio of peptide to AuNPs, buffer solution, and the temperature of conjugation and enzyme reaction. The biosensor was successfully applied to distinguishing apoptosis cells and normal cells within 2 h. This study demonstrated that the biosensor could be utilized to evaluate anticancer drugs.     

Energy filtering transmission electron microscopy immunocytochemistry and antigen retrieval of surface layer proteins from Tannerella forsythensis using microwave or autoclave heating with citraconic anhydride

Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3′-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.     

Colistin-functionalised CdSe/ZnS quantum dots as fluorescent probe for the rapid detection of Escherichia coli

Intensely fluorescent, colistin-functionalised CdSe/ZnS QDs (Colis-QDs) nanoparticles, are synthesized and used as sensitive probes for the detection of Escherichia coli, a Gram-negative bacteria. Colistin molecules are attached to the terminal carboxyl of the mercaptoacetic acid-capped QDs in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as amide bond promoters. The TEM analysis of bacteria treated with Colis-QDs conjugates showed the accumulation of Colis-QDs in the cell wall of E. coli. Under the recommended working conditions, the method provides a detection limit as few as 28 E. coli cells per mL, which is competitive which more elaborate detection systems. The simplicity of the method together with short analysis time (< 15 min, without including preparation and photoactivation of the Colis-QDs conjugate) make the proposed approach useful as quick bacteria screening system.