Journal of Electron Microscopy - recent issues

Development of an analytical environmental TEM system and its application

Sun, 15 Nov 2009 01:00:01 PST

Many automotive materials, such as catalysts and fuel cell materials, undergo significant changes in structure or properties when subjected to temperature change or the addition of a gas. For this reason, in the development of these materials, it is important to study the behavior of the material under controlled temperatures and gaseous atmospheres. Recently, a new environmental transmission electron microscope (TEM) has been developed for observation with a high resolution at high temperatures and under gaseous atmospheres, thus making it possible to analyze reaction processes in details. Also, the new TEM provides a high degree of reproducibility of observation conditions, thus making it possible to compare and validate observation of various specimens under a given set of conditions. Furthermore, easiness of gas condition and temperature control can provide a powerful tool for the studying of the mechanism of material change, such as oxidation and reduction reactions.

Correction of higher order geometrical aberration by triple 3-fold astigmatism field

Sun, 15 Nov 2009 01:00:02 PST

A new concept of a spherical aberration correction system using three dodecapoles is proposed. The system compensates for higher order aberration of 6-fold astigmatism, which generally limits a uniform phase area for image forming and probe forming in an electron microscope with a conventional two-hexapole corrector. Triple 3-fold astigmatism field is used to correct the spherical aberration of the objective lens, and the total 3-fold astigmatism is eliminated by their combination. The optimum azimuth relationship among three dodecapoles is calculated to eliminate the 6-fold astigmatism. The principle of the method was verified using a mathematically complex representation. This new concept was experimentally tested with a scanning transmission electron microscope at 60 kV acceleration. The 6-fold astigmatism was certainly compensated and the coherent convergent angle became almost twice compared to a conventional double hexapole system.

HREM and EDS analysis of (La, M)TiO3 (M = Zn, Mn) prepared by M/Li ion exchange

Tsurui, T., Wataru, M., Watanabe, M., Katsumata, T., Inaguma, Y. — Sun, 15 Nov 2009 01:00:02 PST

High-resolution electron microscopy (HREM) and energy dispersive X-ray spectroscopy (EDS) studies of (La, M)TiO₃ (M = Zn, Mn) prepared by M/Li ion exchange were performed to clarify the microscopic mechanism of ion-exchange reactions. At a macroscopic level, EDS analysis revealed that Zn and Mn atoms were homogeneously dispersed in matrix grains of (La, Zn)TiO₃ and (La, Mn)TiO₃ samples, respectively. EDS analysis also revealed that no segregation of Zn and Mn atoms was observed even in the vicinity of grain boundary regions. HREM and EDS analysis demonstrated that the microscopic mechanism of ion-exchange reactions was different by the ion-exchanged metals.

STEM imaging of 47-pm-separated atomic columns by a spherical aberration-corrected electron microscope with a 300-kV cold field emission gun

Sun, 15 Nov 2009 01:00:02 PST

A spherical aberration-corrected electron microscope has been developed recently, which is equipped with a 300-kV cold field emission gun and an objective lens of a small chromatic aberration coefficient. A dumbbell image of 47 pm spacing, corresponding to a pair of atomic columns of germanium aligned along the [114] direction, is resolved in high-angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) with a 0.4-eV energy spread of the electron beam. The observed image was compared with a simulated image obtained by dynamical calculation.

Immunohistochemical localization of the STRO-1 antigen in developing rat teeth by light microscopy and electron microscopy

Kaneko, R., Akita, H., Shimauchi, H., Sasano, Y. — Sun, 15 Nov 2009 01:00:02 PST

STRO-1 is a cell-surface antigen. A cell population that is positive for the anti-STRO-1 antibody has been shown to contain mesenchymal stem cells. STRO-1-positive cells have been reported to reside in dental pulp and periodontal ligaments as well as in bone marrow. However, the tissue localization of STRO-1 in developing teeth is not clear. The present study was designed to investigate the spatiotemporal localization of STRO-1 in developing rat teeth by immunohistochemistry using light microscopy and electron microscopy. Wistar rats at ages 2, 3, 6 and 12 weeks postnatum and embryos at 18 days postcoitum were fixed with 4% paraformaldehyde. Mandibles and maxillae were resected and decalcified in 10% EDTA. The specimens were embedded in paraffin, and sections were cut and processed for immunohistochemistry using the anti-STRO-1 antibody. Selected specimens were frozen, sectioned and processed for immunoelectron microscopy. Immunoreactions for STRO-1 were identified in some bone marrow cells. Some odontoblasts and dental pulp cells showed positive immunoreactivity in developing rat tooth crowns and roots. Alveolar osteoblasts, cementoblasts and periodontal ligament cells were also immunoreactive. Electron microscopy localized the antigen in plasma membrane and some vesicles in dental pulp cells and odontoblasts. The study suggests that the STRO-1 antigen is involved in the differentiation of mesenchymal cell lineages and formation of the matrix in dental tissues.

Ultrastructural megakaryocyte modifications after vanadium inhalation in spleen and bone marrow

Sun, 15 Nov 2009 01:00:02 PST

Previous reports from our laboratory informed in mice an increase in platelets in blood, and megakaryocytes in spleen and bone marrow after vanadium inhalation. This element has become important in recent years because of its increased presence as an air pollutant. With this precedent, we evaluate the ultrastructural modifications in MKs from the spleen and bone marrow in our mouse experimental model. Mice inhaled 0.02 M V₂O₅ 1 h twice a week for 12 weeks. Tissues were processed for transmission electron microscopy. Results indicate an increase in the size and cytoplasmic granular content, as well as nuclear changes in MKs of exposed mice, changes which correlate with the time of exposure. Modifications in MKs described here suggest that inhaled vanadium induce megakaryocytic maturation, a raise in its granules content and demarcation membrane systems, which may lead to a rise in circulating platelet production and an increased risk for thromboembolic events.

FGF23 is mainly synthesized by osteocytes in the regularly distributed osteocytic lacunar canalicular system established after physiological bone remodeling

Sun, 15 Nov 2009 01:00:02 PST

This study aimed to evaluate whether the immunolocalization of fibroblast growth factor (FGF) 23 and dentin matrix protein 1 (DMP1) is associated with the spatial regularity of the osteocyte lacunar canalicular system(s) (OLCS). Femora of 12-weeks-old male ICR mice were fixed with 4% paraformaldehyde, decalcified with a 10% EDTA solution and then embedded in paraffin. We have devised a triple staining procedure that combines silver impregnation, alkaline phosphatase (ALPase) immunohistochemistry and tartrate-resistant acid phosphatase (TRAPase) enzyme histochemistry on a single paraffin section. This procedure permitted the visualization of ALPase-positive plump osteoblasts and several TRAPase-positive osteoclasts on those bone matrices featuring irregularly arranged OLCS, and of ALPase-positive bone lining cells on the bone matrix displaying the well-arranged OLCS. As observations proceeded from the metaphysis toward the diaphysis, the endosteal cortical bone displayed narrower bands of calcein labeling, accompanied by increased regularity of the OLCS. This implies that the speed of bone deposition during bone remodeling would affect the regularity of the OLCS. While DMP1 was evenly localized in all regions of the cortical bones, FGF23 was more abundantly localized in osteocytes of cortical bones with regularly arranged OLCS. In cortical bones, the endosteal area featuring regular OLCS exhibited more intense FGF23 immunoreaction when compared to the periosteal region, which tended to display irregular OLCS. In summary, FGF23 appears to be synthesized principally by osteocytes in the regularly distributed OLCS that have been established after bone remodeling.

Electron microscopy of octacalcium phosphate in the dental calculus

Kakei, M., Sakae, T., Yoshikawa, M. — Sun, 15 Nov 2009 01:00:02 PST

The purpose of this study was to morphologically demonstrate the presence of octacalcium phosphate in the dental calculus by judging from the crystal lattice image and its rapid transformation into apatite crystal, as part of our serial studies on biomineral products. We also aimed to confirm whether the physical properties of octacalcium phosphate are identical with those of the central dark lines observed in crystals of ordinary calcifying hard tissues. Electron micrographs showed that crystals of various sizes form in the dental calculus. The formation of each crystal seemed to be closely associated with the organic substance, possibly originating from degenerated microorganisms at the calcification front. Many crystals had an 8.2-Å lattice interval, similar to that of an apatite crystal. Furthermore, some crystals clearly revealed an 18.7-Å lattice interval and were vulnerable to electron bombardment. After electron beam exposure, this lattice interval was quickly altered to about half (i.e. 8.2 Å), indicating structural conversion. Consequently, a number of apatite crystals in the dental calculus are possibly created by a conversion mechanism involving an octacalcium phosphate intermediate. However, we also concluded that the calcification process in the dental calculus is not similar to that of ordinary calcifying hard tissues.

An in situ heating TEM analysis method for an interface reaction

Tanigaki, T., Ito, K., Nagakubo, Y., Asakawa, T., Kanemura, T. — Thu, 17 Sep 2009 02:41:20 PDT

In order to analyze the thermal property of nano-sized materials, an in situ observation technique that allows highly sensitive energy dispersive x-ray spectroscopic (EDX) analyses and high-resolution in situ heating observation of precision specimens is required. A method for the in situ observation of the interface reaction using an analytical transmission electron microscopy (TEM) and a specimen-heating holder was developed. The specimen holder used in this study was a direct-heating type having a fine tungsten wire heater. For sensitive analyses including an EDX map of composition changes during the interface reaction, a space toward the EDX detector with a take-off angle of 20° was made in the specimen holder. Samples were prepared by attaching a micro-sample directly to the heater using the focused ion beam (FIB) micro-sampling technique. It was confirmed that the sensitive EDX map and electron diffraction analyses were possible during the reaction, and that the resolution of this technique was of the order of 0.223 nm at 550°C.

Nanostructural characterization and catalytic analysis of hybridized platinum/phthalocyanine nanocomposites

Thu, 17 Sep 2009 02:41:20 PDT

Organic crystals, such as phthalocyanine nanocrystal, were successfully hybridized with Pt nanoparticles using a nanohybridization technique. The presence of highly dispersed Pt nanoparticles on the surface of phthalocyanine was confirmed by the combination of transmission electron microscopy and three-dimensional electron tomography. Catalytic activities of hybridized samples with different degrees of dispersions were also examined as oxygen reduction reactivity (ORR) with a linear potential sweep method. It was found that oxygen reduction activity increased with increasing Pt dispersion, and reasonably high ORR was observed on Pt-dispersed phthalocyanine nanocrystal even at 2 wt% Pt loading.

Characterization of Mn-doped ZnO nanobelts by electron energy-loss spectroscopy

Zhang, J., Yu, C., Liao, Z., Zhang, X., You, L., Yu, D. — Thu, 17 Sep 2009 02:41:20 PDT

Mn-doped ZnO nanobelts have been synthesized via the vapor phase evaporation method, which exhibited ferromagnetism at room temperature. Electron energy-loss spectroscopy was used to investigate the chemical state of Mn dopants. It revealed that the Mn species had the chemical valence of +2. Meanwhile, the fine structures of the Mn L₂₃ edges indicated that the Mn dopant was located at the center of an oxygen octahedron but not an oxygen tetrahedron. This suggested that the Mn dopants did not substitute on the Zn sites as expected and sub-nanoscale MnO clusters had been formed in the synthesized ZnO nanobelts.

Quantitative electron holographic tomography for a spherical object

Fujita, T., Chen, M. — Thu, 17 Sep 2009 02:41:20 PDT

We demonstrate quantitative electron holographic tomography using a latex sphere as a simple example. Although a simple object is used, we believe that the observations made in this study will lead to further developments in electron holographic tomography. In particular, we make the quantitative interpretation of artifacts caused by incomplete angular information, i.e. a ‘missing wedge’.

Three-dimensional structure of the cytoskeleton in Trichomonas vaginalis revealed new features

Lee, K. E., Kim, J. H., Jung, M. K., Arii, T., Ryu, J.-S., Han, S. S. — Thu, 17 Sep 2009 02:41:20 PDT

The flagellated protozoan Trichomonas vaginalis has been widely studied owing to its medical significance and unique structure. The complicated three-dimensional (3D) structure of the cellular components of T. vaginalis was reconstructed from serial sections to enable observation of the spatial features of the whole cell. Electron tomography was used to examine the detailed structure of the cellular organelles. Tomographic reconstruction showed the mastigont system and the parabasal filament of T. vaginalis in detail. The last thin filament (Pf3) was located close to the adjacent filament, and the two filaments appeared to be vertically parallel in the cross-sectional view. It is likely that Pf3 cannot be distinguished from the adjacent filament in 2D images obtained from transmission electron microscopy. Our 3D reconstruction of T. vaginalis revealed the presence of an additional striated fiber, and 3D reconstruction by electron tomography showed twisting of the split parabasal filament.

Morphological and ultrastructural changes in the cell structure of enterohaemorrhagic Escherichia coli O157:H7 following treatment with Quercus infectoria nut galls

Suwalak, S., Voravuthikunchai, S. P. — Thu, 17 Sep 2009 02:41:20 PDT

Some information is available on the oak (Quercus infectoria) nut gall as an effective medicinal plant against Shiga toxin-producing Escherichia coli (STEC) O157:H7. However, its antibacterial mechanisms have not yet been elucidated. In this study, some antibacterial actions against STEC O157:H7 were investigated by observing cell viability as well as morphological and ultrastructural changes. An ethanolic extract of Q. infectoria demonstrated inhibitory and bactericidal effects on all of the strains tested with minimal inhibition concentrations (MICs) at 0.78–1.56 mg ml^–1 and minimal bactericidal concentrations (MBCs) at 1.56–3.12 mg ml^–1. Cell numbers treated with 4MIC of the extract decreased at least two log-fold within 4 h and were completely killed within 12 h. Scanning electron microscopy illustrated a complete loss of surface appendages and pronounced morphological changes at MIC and 2MIC. The whole cell collapsed at 4MIC. Ultrastructural changes from corresponding transmission electron micrographs further verified that damages in the treated cells increased with the increase in the extract concentrations. At MIC (0.78 mg ml^–1), there was some evidence that the cytoplasmic membranes of the treated E. coli were bulging and/or ruptured, and the cells appeared to be discharging intracellular materials. At 2MIC, the outer membrane of the treated E. coli which was attached to the cell wall became separated from the wall. Disruption in the outer wall and cytoplasmic membranes, especially at the polar regions of the cells occurred and some vacuolization appeared. At 4MIC, the damage to E. coli cells was extensive, and there was loss of their cellular integrity.

Spontaneous [Ca2+]i oscillations in G1/S phase-synchronized cells

Russa, A. D., Maesawa, C., Satoh, Y.-i. — Thu, 17 Sep 2009 02:41:20 PDT

Ca²⁺ signaling controls a wide range of cellular functions such as division, fertilization, apoptosis and necrosis. Specifically, calcium signaling is thought to play a crucial role in driving cells through the different stages of the cell-division cycle. In most cells, however, this fact is far from being established. Few studies have examined this question from a different perspective: whether cells exhibit some characteristic cell cycle-dependent intracellular calcium-signaling patterns. This approach is effective in discerning the causal relationship between Ca²⁺ signaling and the cell cycle. Through synchronization of the cell cycle, flow cytometry and confocal scanning microscopic intracellular calcium ion concentration ([Ca²⁺]_i) imaging, the present study shows that the G1/S phase transition is uniquely characterized by spontaneous [Ca²⁺]_i oscillations that last for up to 40 min. Most likely, these oscillations emanate from the [Ca²⁺]_i signaling that accompanies DNA replication as the cell prepares for the next division cycle. These temporal signals further affirm the significance of Ca²⁺ in the cell cycle.

Development of a multifunctional TEM specimen holder equipped with a piezodriving probe and a laser irradiation port

Fri, 17 Jul 2009 01:01:15 PDT

The double-probe piezodriving specimen holder that was recently developed by some of the present authors is modified to introduce a laser irradiation port in one of its two arms. As a result, the new specimen holder consists of a piezodriving probe and a laser irradiation port, both of which can be three-dimensionally controlled by using piezoelectric elements and micrometers. While the piezodriving probe interacts with the specimen set in the holder in several ways, the laser beam causes photo-induced phenomena to occur. By performing electron holography using the new specimen holder, we demonstrate that it is possible to evaluate the change in the electric field resulting from the discharging effect of laser irradiation on organic photoconductors.

Reduction of time-varying nanotesla magnetic fields from electric power lines by twisting

Been, A. J., Folkertsma, G. A., Verputten, H. H.J., Bolhuis, T., Abelmann, L. — Fri, 17 Jul 2009 01:01:15 PDT

Time-varying magnetic fields generated by electrical power lines in the laboratory can disturb electron microscope imaging. Modern microscopes require these fields to be below 10 nT [2]. We calculated and measured magnetic fields from straight and twisted current-carrying wires, and show that without twisting, this value cannot be reached.

Exposure of the gill epithelial cells of larval lampreys to an ion-deficient environment: a stereological study

Bartels, H., Schmiedl, A., Rosenbruch, J., Potter, I. C. — Fri, 17 Jul 2009 01:01:15 PDT

Three kinds of epithelial cells comprise the surfaces of the gill filaments and lamellae of larval lampreys (ammocoetes): ammocoete mitochondria-rich cells (AMRCs), intercalated mitochondria-rich cells (IMRCs) and pavement cells. Selected characteristics of these cell types in ammocoetes of Geotria australis held in distilled water and in 10% sea water were compared using an ultrastructural stereological approach to determine which of those cell type(s) respond to exposure to an ion-deficient environment in a manner that indicates that they are involved in ion uptake. Particular focus was placed on the enigmatic AMRC, which comprises ca 60% of the cells and contains numerous mitochondria. The mean percentage contributions of both AMRCs and pavement cells to the total number of the three cell types in the two experimental groups were not significantly different, whereas that of IMRCs was >7% in distilled water and <1% in 10% sea water (P < 0.001). Furthermore, the mean apical surface areas of neither AMRCs nor pavement cells differed significantly between the two experimental groups, whereas that of IMRCs was nearly 3-fold greater in distilled water than in 10% sea water. The volume densities and size of mitochondria in AMRCs did not differ between the two exposure regimes. The above comparisons provide no indications that the uptake of Na⁺ and Cl^– in the gill epithelium of ammocoetes involves either the AMRC or pavement cell but, when considered in conjunction with data on ion-transporting cells in other vertebrates, they are consistent with the conclusion that the IMRC plays a crucial role in this process.

Smart specimen preparation for freeze substitution and serial ultrathin sectioning of yeast cells

Yamaguchi, M., Okada, H., Namiki, Y. — Fri, 17 Jul 2009 01:01:15 PDT

A smart and efficient method for freeze substitution and serial sectioning of yeast cells is described. Yeast cells were placed in a single layer between two copper disks, rapidly frozen, freeze substituted and embedded in an epoxy resin. The cell layer was re-embedded by the same resin, the surface trimmed leaving 1 µm above the cell layer, and serially sectioned. The sections were collected on the two-slit grids and placed on a Formvar film mounted to cover the holes of an aluminum supporting rack. The grids were removed from the rack, stained together using a silicon tube and observed in a transmission electron microscope. The images of yeast cells observed were clear and natural, and would be useful for a detailed 3D structural analysis such as structome.

Establishment of a standardized post-embedding method for immunoelectron microscopy by applying heat-induced antigen retrieval

Yamashita, S., Katsumata, O., Okada, Y. — Fri, 17 Jul 2009 01:01:15 PDT

We have developed a new standardized method for the post-embedding immunoelectron microscopy using the same fixation, antigen retrieval and image contrasting procedures. Tissues were fixed with 4% formaldehyde containing 2.5 mM CaCl₂, 1.25 mM MgCl₂ in a 0.1 M 4-(2-hydroxyethyl)-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4) for 2 h and then with the same fixative composition in 0.1 M HEPES buffer (pH 8.5) overnight at room temperature. Vehicle osmolarity of fixatives was adjusted to 300–330 mOsm by adding glucose. The specimens were dehydrated with dimethylformamide on ice and embedded in LR-White resin. Ultrathin sections were heated in a 20 mM Tris–HCl buffer (pH 9.0) for 1–2 h at 95°C. After immuno-gold labeling, the sections were treated with 2% glutaraldehyde containing 0.05% tannic acid in a 0.1 M phosphate buffer (pH 5.5) for 5 min and with a 1% OsO₄/0.1 M phosphate buffer (pH 7.4) for 5 min, and then they were double stained with uranyl acetate and lead citrate. The standardized method yielded strong and reproducible immunoreactions for soluble, membrane-bound and filamentous proteins showing an excellent image contrast without destruction of the fine structures.