Standard Transmission Electron Microscopy Research Articles

Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites

This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined using standard transmission electron microscopy (EM). Pre-embedding EM is a common method where the antibodies are introduced before embedding and sectioning. This method avoids the problem that antigens are often difficult to detect on EM-sections after embedding. In the method described here antigens in the parasite-infected host cell are detected. Entry of the antibody is made possible through permeabilisation of the host cell with tetanolysin. In principle this method could also be used to detect antigens within the parasite if the sample is appropriately fixed and permeabilised before addition of the relevant antibody. While access of the antibody will avoid the detection problems often seen with post-embedding methods, this procedure will produce comparably poorer morphology.

Environmentally Compatible Synthesis of Superparamagnetic Magnetite (Fe3O4) Nanoparticles with Prehydrolysate from Corn Stover

An environmentally compatible and size-controlled method has been employed for synthesis of superparamagnetic magnetite nanoparticles with prehydrolysate from corn stover. Various characterizations involving X-ray diffraction (XRD), standard and high-resolution transmission electron microscopy (TEM and HRTEM), selected area electron diffraction (SAED), and thermogravimetric analysis (TGA) have integrally confirmed the formation of magnetite nanoparticles with homogeneous morphology and the formation mechanism of magnetite only from ferric precursor. Organic materials in the prehydrolysate act as a bifunctional agent: (1) a reducing agent to reduce ferric ions to prepare magnetite with the coexistence of ferric and ferrous ions; and (2) a coating agent to prevent particle growth and agglomeration and to promote the formation of nanoscale and superparamagnetic magnetite. The size of the magnetite nanoparticles can be easily controlled by tailoring the reducing sugar concentration, reaction time, or hydrothermal temperature.

Solvothermal synthesis of N-doped TiO2 nanoparticles using different nitrogen sources, and their photocatalytic activity for degradation of benzene

Anatase-brookite mixed-phase N-doped TiO2 (N-TiO2) nanoparticles were synthesized through a solvothermal method using different nitrogen sources. The resulting samples were characterized by X-ray diffraction, specific surface area measurement, X-ray photoelectron spectroscopy, and standard and high-resolution transmission electron microscopy. The effects of the different nitrogen sources on phase composition, particle size, microstructure, and specific surface area are investigated. The photocatalytic activity of the TiO2 samples was evaluated through photocatalytic degradation of gaseous benzene under UV-light irradiation. N-TiO2 prepared using hydrazine hydrate achieved the highest photocatalytic performance in all the samples studied (including the commercial P25). Different intermediates during the photocatalytic degradation of benzene over HNT were identified by GC-MS analysis. A detailed reaction mechanism was proposed to explain their formation as intermediates in the reaction. Moreover, the photocatalytic activity of the nanoparticles remained almost unchanged after 15 gaseous-benzene degradation test cycles.

Development of a Transfer Function for a Personal, Thermophoretic Nanoparticle Sampler

Effective assessment of nanoparticle exposures requires accurate characterization of the aerosol. Of increasing concern is personal exposure to engineered nanoparticles that are specifically designed for use in the nanotechnology sector. This manuscript describes the operation and use of a personal sampler that utilizes thermophoretic force to collect nanoparticles onto a standard TEM (transmission electron microscope) grid. After collection, nanoparticles on the TEM grid are analyzed with an electron microscope, and the resultant data used to determine the characteristics of the nanoparticle aerosol sampled. Laboratory experiments were conducted to determine the inlet losses and collection efficiency of the thermophoretic sampler for particles between 20 and 600 nm in diameter. These results are used together with theory for thermophoretic velocity to form a transfer function that relates the properties of the collected particles to the properties of the sampled aerosol. The transfer function utilizes a ...

Detection of CD133 (prominin‐1) in a human hepatoblastoma cell line (HuH‐6 clone 5)

We examined CD133 distribution in a human hepatoblastoma cell line (HuH-6 clone 5). We directly observed the cultured cells on a pressure-resistant thin film (silicon nitride thin film) in a buffer solution by using the newly developed atmospheric scanning electron microscope (ASEM), which features an open sample dish with a silicon nitride thin film window at its base, through which the scanning electron microscope beam scans samples in solution, from below. The ASEM enabled observation of the ventral cell surface, which could not be observed using standard SEM. However, observation of the dorsal cell surface was difficult with the ASEM. Therefore, we developed a new method to observe the dorsal side of cells by using Aclar® plastic film. In this method, cells are cultured on Aclar plastic film and the dorsal side of cells is in contact with the thin silicon nitride film of the ASEM dish. A preliminary study using the ASEM showed that CD133 was mainly localized in membrane ruffles in the peripheral regions of the cell. Standard transmission electron microscopy and scanning electron microscopy revealed that CD133 was preferentially concentrated in a complex structure comprising filopodia and the leading edge of lamellipodia. We also observed co-localization of CD133 with F-actin. An antibody against CD133 decreased cell migration. Methyl-β-cyclodextrin treatment decreased cell adhesion as well as lamellipodium and filopodium formation. A decrease in the cholesterol level may perturb CD133 membrane localization. The results suggest that CD133 membrane localization plays a role in tumor cell adhesion and migration.

Technical Note: A novel rocket-based in situ collection technique for mesospheric and stratospheric aerosol particles

Abstract. A technique for collecting aerosol particles between altitudes of 17 and 85 km is described. Spin-stabilized collection probes are ejected from a sounding rocket allowing for multi-point measurements. Each probe is equipped with 110 collection samples that are 3 mm in diameter. The collection samples are one of three types: standard transmission electron microscopy carbon grids, glass fibre filter paper or silicone gel. Collection samples are exposed over a 50 m to 5 km height range with a total of 45 separate ranges. Post-flight electron microscopy will give size-resolved information on particle number, shape and elemental composition. Each collection probe is equipped with a suite of sensors to capture the probe's status during the fall. Parachute recovery systems along with GPS-based localization will ensure that each probe can be located and recovered for post-flight analysis.

Superb resolution and contrast of transmission electron microscopy images of unstained biological samples on graphene-coated grids

BackgroundIn standard transmission electron microscopy (TEM), biological samples are supported on carbon films of nanometer thickness. Due to the similar electron scattering of protein samples and graphite supports, high quality images with structural details are obtained primarily by staining with heavy metals. MethodsSingle-layered graphene is used to support the protein self-assemblies of different molecular weights for qualitative and quantitative characterizations. ResultsWe show unprecedented high resolution and contrast images of unstained samples on graphene on a low-end TEM. We show for the first time that the resolution and contrast of TEM images of unstained biological samples with high packing density in their native states supported on graphene can be comparable or superior to uranyl acetate-stained TEM images. ConclusionOur results demonstrate a novel technique for TEM structural characterization to circumvent the potential artifacts caused by staining agents without sacrificing image resolution or contrast, and eliminate the need for toxic metals. Moreover, this technique better preserves sample integrity for quantitative characterization by dark-field imaging with reduced beam damage. General significanceThis technique can be an effective alternative for bright-field qualitative characterization of biological samples with high packing density and those not amenable to the standard negative staining technique, in addition to providing high quality dark-field unstained images at reduced radiation damage to determine quantitative structural information of biological samples.

Efficient photocatalytic activity with carbon-doped SiO2 nanoparticles

Photocatalysis provides a 'green' approach to completely eliminate various kinds of contaminants that are fatal for current environmental and energy issues. Semiconductors are one of the most frequently used photocatalysts as they can absorb light over a wide spectral range. However, it is also well known that naked SiO2 is not an efficient photocatalyst due to its relatively large band gap, which could only absorb shortwave ultraviolet light. In this report, nanoscale particles of carbon-doped silicon dioxide (C-doped SiO2) for use in photocatalysis were successfully prepared by a facile one-pot thermal process using tetraethylorthosilicate (TEOS) as the source of both silicon and carbon. These particles were subsequently characterized by thermogravimetric analysis, X-ray diffraction, standard and high resolution transmission electron microscopy and X-ray photoelectron spectroscopy. The C-doped SiO2 displayed outstanding photocatalytic properties, as evidenced by its catalysis of Rhodamine B degradation under near-UV irradiation. We propose that carbon doping of the SiO2 lattice creates new energy states between the bottom of the conduction band and the top of the valence band, which narrows the band gap of the material. As a result, the C-doped SiO2 nanoparticles exhibit excellent photocatalytic activities in a neutral environment. The novel synthesis reported herein for this material is both energy efficient and environmentally friendly and as such shows promise as a technique for low-cost, readily scalable industrial production.

Improving Fabrication and Application of Zach Phase Plates for Phase-Contrast Transmission Electron Microscopy

Zach phase plates (PPs) are promising devices to enhance phase contrast in transmission electron microscopy. The Zach PP shifts the phase of the zero-order beam by a strongly localized inhomogeneous electrostatic potential in the back focal plane of the objective lens. We present substantial improvements of the Zach PP, which overcome previous limitations. The implementation of a microstructured heating device significantly reduces contamination and charging of the PP structure and extends its lifetime. An improved production process allows fabricating PPs with reduced dimensions resulting in lower cut-on frequencies as revealed by simulations of the electrostatic potential. Phase contrast with inversion of PbSe nanoparticles is demonstrated in a standard transmission electron microscope with LaB6 cathode by applying different voltages.

Virtual nanoscopy: generation of ultra-large high resolution electron microscopy maps.

A key obstacle in uncovering the orchestration between molecular and cellular events is the vastly different length scales on which they occur. We describe here a methodology for ultrastructurally mapping regions of cells and tissue as large as 1 mm(2) at nanometer resolution. Our approach employs standard transmission electron microscopy, rapid automated data collection, and stitching to create large virtual slides. It greatly facilitates correlative light-electron microscopy studies to relate structure and function and provides a genuine representation of ultrastructural events. The method is scalable as illustrated by slides up to 281 gigapixels in size. Here, we applied virtual nanoscopy in a correlative light-electron microscopy study to address the role of the endothelial glycocalyx in protein leakage over the glomerular filtration barrier, in an immunogold labeling study of internalization of oncolytic reovirus in human dendritic cells, in a cryo-electron microscopy study of intact vitrified mouse embryonic cells, and in an ultrastructural mapping of a complete zebrafish embryo slice.

Preparation of stable Pd nanocubes and their use in biological labeling

Stable Pd nanocubes (PdNC) with the average size ∼15nm were prepared by the controlled reduction of sodium tetrachloropalladate with ascorbic acid in water, in the presence of polyvinylpyrrolidone and potassium bromide. Morphology of the particles was characterized by transmission electron microscopy (TEM) and their stability in the colloidal solution was verified by dynamic light scattering (DLS). It has been demonstrated that the Pd nanocubes can be distinguished from commercial Au nanospheres in a standard TEM microscope by means of automated image analysis. In the next step, the PdNC were successfully conjugated to immunoglobulin proteins and used for the detection of a specific protein (nucleophosmin) on ultrathin sections of HeLa cells. Our experiments showed that PdNC can be used for multiple immunolabeling in combination with commercial Au nanospheres.

Study of damage to red blood cells exposed to different doses of γ-ray irradiation.

The aims of this research were to study alterations in the ultrastructure of red blood cells, the changes in concentrations of plasma electrolytes and the killing effect of lymphocytes in samples of blood exposed to different doses of γ-ray irradiation. Blood samples were treated with different doses of γ-ray irradiation and then preserved for different periods. Specimens were prepared for standard electron microscopy and transmission electron microscopy. At the same time, changes in the concentrations of Na(+), K(+) and Cl(-) and pH values in the plasma as well as Fas and FasL expression of lymphocytes before and after irradiation were determined. The proportions of reversibly and irreversibly transformed cells, for example, echinocytes, sphero-echinocytes, and degenerated forms, increased with increasing doses of irradiation and storage period, while the number of discocyte shaped red blood cells decreased. The change in K(+) concentration was greater than that of Na+ or Cl(-) after irradiation and was dosage-dependent. Plasma pH was influenced by different doses of radiation and storage time. After exposure to (137)Cs γ-irradiation, the expression of both Fas and FasL in lymphocytes differed significantly from that in the control group: the expression was positively correlated with irradiation dose (r=0.95, 0.96), but no significant difference in the Fas/FasL ratio was observed (P>0.05). We conclude that the ultrastructure of red blood cells is not changed obviously by irradiation with some doses of γ-rays and various periods of storage. However, irradiation does have some dose-dependent and time-dependent adverse effects on the erythrocytes.

3D Reconstruction of the Glycocalyx Structure in Mammalian Capillaries using Electron Tomography

Visualising the molecular strands making up the glycocalyx in the lumen of small blood vessels has proved to be difficult using conventional transmission electron microscopy techniques. Images obtained from tissue stained in a variety of ways have revealed a regularity in the organisation of the proteoglycan components of the glycocalyx layer (fundamental spacing about 20 nm), but require a large sample number. Attempts to visualise the glycocalyx face-on (i.e. in a direction perpendicular to the endothelial cell layer in the lumen and directly applicable for permeability modelling) has had limited success (e.g. freeze fracture). A new approach is therefore needed. Here we demonstrate the effectiveness of using the relatively novel electron microscopy technique of 3D electron tomography on two differently stained glycocalyx preparations. A tannic acid staining method and a novel staining technique using Lanthanum Dysprosium Glycosamino Glycan adhesion (the LaDy GAGa method). 3D electron tomography reveals details of the architecture of the glycocalyx just above the endothelial cell layer. The LaDy GAGa method visually appears to show more complete coverage and more depth than the Tannic Acid staining method. The tomographic reconstructions show a potentially significant improvement in determining glycocalyx structure over standard transmission electron microscopy.

Amphiphilic Diblock Copolymer and Polycaprolactone Blends to Produce New Vesicular Nanocarriers

New Melatonin-loaded vesicular nanocarriers were prepared by interfacial deposition using a blend of an amphiphilic diblock copolymer, poly(methyl methacrylate)-block-poly(2-(dimethylamino)ethyl methacrylate), PMMA-b-PDMAEMA, with poly(epsilon-caprolactone), PCL. Particle size and morphology of the nanocarriers was evaluated. Dynamic light scattering shows that the nanocarriers have hydrodynamic radii between 100 and 180 nm, with unimodal particle size distribution for each formulation. Shape and structure were visualized by transmission electron microscopy (TEM), cryogenic TEM and scanning electron microscopy. Standard TEM for nanocapsules showed an oily core surrounded by a thin layer composed by PCL/PMMA-b-PDMAEMA. Cryo-TEM also indicated the presence of spherical nano-objects with a diffuse polymer corona. Encapsulation efficiencies were determined assaying the nanoparticles by HPLC and higher values of ca. 25% are shown by the nanocapsules. We could successfully incorporate platinum nanoparticles into the nanocarrier as evidenced by TEM, which opens up the possibility for promising applications like monitoring the encapsulated drug in the body.

High Temperature Deformation of Superalloy Inconel 718

Experimental results on hot deformation and dynamic structural processes of nickel based alloy Inconel 718 are reviewed. The focus is the analysis of dynamic precipitation processes which operate during hot deformation of these materials at elevated temperatures. Hot compression tests were performed on the solution treated precipitation hardenable nickel based superalloy Inconel 718 at 720-1150°C with a constant true strain rates of 10-4 and 4x10-4s-1. True stress - true strain curves and microstructure analysis of the deformed nickel based superalloy is presented. The properties and dynamic behaviour are explained through observation of the microstructure using standard optical, scanning and transmission electron microscopy. Structural observations of solution treated Inconel 718 deformed at high temperatures, reveal non uniform deformation effects. The distribution of niobium-rich carbides were affected by localized flow within the strain range investigated at relatively low deformation temperatures 720 - 850°C.

Probing the Structure of Colloidal Core/Shell Quantum Dots Formed by Cation Exchange

Cation-exchange reactions have greatly expanded the types of nanoparticle compositions and structures that can be prepared. For instance, cation-exchange reactions can be utilized for preparation of core/shell quantum dots with improved (photo)stability and photoluminescence quantum yield. Understanding the structure of these nanomaterials is imperative for explaining their observed properties and for their further development. Core/shell quantum dots formed by cation exchange are particularly challenging to characterize because shell growth does not lead to an increase in overall particle size that can easily be characterized by standard transmission electron microscopy (TEM). Here, we report on the direct observation of the PbSe/CdSe core/shell structure (formed by cation exchange) using high-angle annular dark field (HAADF) imaging and energy-filtered TEM (EF-TEM). These results are further confirmed by energy-dependent X-ray photoemission spectroscopy (XPS) data that show increasing Pb/Cd signal with ...

Hydrothermal Stability of {001} Faceted Anatase TiO2

Due to its great importance in fundamental research and practical applications, tailored synthesis of anatase TiO2 dominated with highly energetic {001} facets has been extensively studied during the past few years. However, clean (001) surface of anatase TiO2 has been evidenced to be unstable and usually tends to reconstruct under ultrahigh-vacuum conditions. Thus, the stability of surface structure under other ambient conditions might be one of the most critical issues for anatase TiO2 with exposed high-reactive {001} facets. In this study, the hydrothermal stability of {001} faceted anatase TiO2 was systematically investigated by using single-crystalline anatase TiO2 nanosheets with 80% {001} facets as a model starting material. Under hydrothermal conditions (200 °C in deionized water), anatase TiO2 nanosheets can grow into larger single crystals with a truncated bipyramidal shape through an oriented attachment process along the [001] crystallographic direction, driven by the minimization of surface energy. Furthermore, the coarsening behavior and growth mechanism were discussed with the assistance of standard and high-resolution transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and inductively coupled plasma optical emission spectrometry, as well as theoretical calculations. A modified kinetic model was also developed to elucidate the asymptotic growth of anatase TiO2 nanosheets via oriented attachment mechanism. In addition, pH value and the solvents adopted during the treatments were revealed to have significant influence on the oriented attachment-based crystal growth due to suppression of hydrolysis of Ti–F groups on the surface. For example, the anatase TiO2 nanosheets remained their original morphology unchanged when ethanol, propanol, butanol, or 1.5 M HCl aqueous solution was used as reaction medium.

HIV-Gag VLPs presenting trimeric HIV-1 gp140 spikes constitutively expressed in stable double transfected insect cell line

We have previously described the establishment and characterization of a stably transfected insect cell line for the constitutive and efficient expression of Pr55 HIV Gag proteins, which auto-assemble into enveloped Virus-Like Particles (VLPs) released into the cell culture supernatant. Such HIV-Gag VLPs have been shown to elicit a specific systemic humoral response in vivo, proving the appropriate antigenic presentation of the HIV Gag protein to the immune system. Here we describe the establishment of a stable double transfected insect cell line for the constitutive and reproducible production of Pr55Gag-VLPs expressing on their surface trimeric forms of HIV-1 envelope glycoproteins. The persistence of HIV coding genes has been verified in clonal resistant insect cells, the protein expression and conformation has been verified by Western blot analysis. The resulting HIV-VLPs have been visualized by standard transmission electron microscopy and their immunogenicity has been evaluated in vivo. This represents, to our knowledge, the first example of stable double transfected insect cell line for the constitutive production of enveloped HIV-Gag VLPs presenting trimeric HIV-gp140 on their surface.

Spectroscopic and Microscopic Investigation of Gold Nanoparticle Formation: Ligand and Temperature Effects on Rate and Particle Size

We report a spectroscopic and microscopic investigation of the synthesis of gold nanoparticles (AuNPs) with average sizes of less than 5 nm. The slow reduction and AuNP formation processes that occur by using 9-borabicyclo[3.3.1]nonane (9-BBN) as a reducing agent enabled a time-dependent investigation based on standard UV-vis spectroscopy and transmission electron microscopy (TEM) analyses. This is in contrast to other borohydride-based syntheses of thiolate monolayer protected AuNPs which form particles very rapidly. We investigated the formation of 1-octadecanethiol (ODT) protected AuNPs with average diameters of 1.5-4.3 nm. By studying the progression of nanoparticle formation over time, we find that the nucleation rate and the growth time, which are interlinked with the amount of ODT and the temperature, influence the size and the size dispersion of the AuNPs. High-resolution TEM (HRTEM) analyses also suggest that the nanoparticles are highly single crystalline throughout the synthesis and appear to be formed by a diffusion-controlled Ostwald-ripening growth mechanism.

Imaging internal features of whole, unfixed bacteria

Wet scanning-transmission electron microscopy (STEM) is a technique that allows high-resolution transmission imaging of biological samples in a hydrated state, with minimal sample preparation. However, it has barely been used for the study of bacterial cells. In this study, we present an analysis of the advantages and disadvantages of wet STEM compared with standard transmission electron microscopy (TEM). To investigate the potential applications of wet STEM, we studied the growth of polyhydroxyalkanoate and triacylglycerol carbon storage inclusions. These were easily visible inside cells, even in the early stages of accumulation. Although TEM produces higher resolution images, wet STEM is useful when preservation of the sample is important or when studying the relative sizes of different features, since samples do not need to be sectioned. Furthermore, under carefully selected conditions, it may be possible to maintain cell viability, enabling new types of experiments to be carried out. To our knowledge, internal features of bacterial cells have not been imaged previously by this technique.