Journal of Electron Microscopy - recent issues

Heat- and electron-beam-induced transport of gold particles into silicon oxide and silicon studied by in situ high-resolution transmission electron microscopy

Biskupek, J., Kaiser, U., Falk, F. — 2008-05-26

In this study, we describe the transport of gold (Au) nanoparticles from the surface into crystalline silicon (Si) covered by silicon oxide (SiO₂) as revealed by in situ high-resolution transmission electron microscopy. Complete crystalline Au nanoparticles sink through the SiO₂ layer into the Si substrate when high-dose electron irradiation is applied and temperature is raised above 150°C. Above temperatures of 250°C, the Au nanoparticles finally dissolve into fragments accompanied by crystallization of the amorphized Si substrate around these fragments. The transport process is explained by a wetting process followed by Stokes motion. Modelling this process yields boundaries for the interface energies involved.

Preparation of TEM samples of ferritic alloys

Yao, Z., Xu, S., Jenkins, M. L., Kirk, M. A. — 2008-05-26

We describe techniques for electropolishing irradiated ferritic specimens for examination under the TEM in situations where the foil quality is of utmost importance. First, we describe some modifications to the standard technique for making plan-view specimens aimed at optimizing the foil quality. Second, we describe a technique for making plan-view specimens from a region of buried damage in a specimen irradiated with 2 MeV Fe⁺ ions.

Surface replicas of normal and vitrified leaves of Datura insignis, Barb Rodr

Miguens, F. C. — 2008-05-26

Vitrification is a morphological and physiological disorder affecting plants during their in vitro vegetative propagation. Vitrified plants have a poor survival rate when transferred from in vitro to greenhouse conditions, a fact mainly due to water loss and dissecation. It has been shown that normal and vitreous leaves of Datura insignis differ in the frequency of normal and abnormal stomata. The purpose of this work was to compare the surface of normal and vitreous leaves of D. insignis, using a modification of the platinum/carbon replica method. Adaxial and abaxial leaf surfaces of normal plantlets have a smooth and homogenous cuticle. A granular aspect, probably due to leaf age, rarely occurs at the periphery of the epidermal cells. Both adaxial and abaxial leaf surfaces of vitrified plantlets show discontinuities in the cuticle, occurring at several regions of the outer periclinal cell walls. However, such discontinuities are most noticeable in the region between adjacent epidermal cells. Fibrils 20–30 nm thick show a random arrangement or an oriented pattern in cuticular discontinuities. In D. insignis vitrified plantlets, adaxial and abaxial leaf cuticle has discontinuities or gaps which may cause an increase in cuticular transpiration contributing to the low survival rate of vitrified plantlets.

Evaluation of a 2k CCD camera with an epitaxially grown CsI scintillator for recording energy-filtered electron cryo-micrographs

Yasunaga, T., Wakabayashi, T. — 2008-05-26

Zero-loss imaging of frozen-hydrated specimens requires a detector with high sensitivity, a low noise level and high spatial resolution, because more electrons are scattered inelastically than elastically by cryo-specimens and the number of electrons detected is ~1/4 of incident electrons after energy filtering. Cameras using charge-coupled devices (CCDs) are good candidates due to their high sensitivity. They have been used mainly to record electron diffraction patterns for electron crystallography due to their limited spatial resolution but recently used for acquiring direct images due to their convenience. The spatial resolution has been limited by the characteristics of a phosphor that is necessary to convert high-energy electrons to photons and the coupling. We adopted a CsI scintillator with good modulation transfer function (MTF), which was epitaxially grown from each of optical fibres. The stripes of carbon graphite with 3.4 Å spacing and 1.4 Å stripes of gold thin crystals could be recorded with a magnification of 240 000x and 560 000x at 200 kV, respectively. A computed Fourier transform of an image of a frozen-hydrated crystal of catalase containing about 1000 units showed diffraction spots at spatial frequencies of 1/9.6 Å^–1 up to 1/8 Å^–1 without background subtraction, when the image was recorded at 140 000x. These results show that the resolution of the developed camera was good enough to record images. Our used test method for MTF determination may be useful for others.

Imaging the interaction between dengue 2 virus and human blood platelets using atomic force and electron microscopy

Ghosh, K., Gangodkar, S., Jain, P., Shetty, S., Ramjee, S., Poddar, P., Basu, A. — 2008-05-26

Thrombocytopenia is frequently associated with dengue virus infection. Host factors such as anti-platelet immunopathogenic processes have been implicated in the origin of dengue-associated thrombocytopenia but the role of dengue virus in directly interacting with platelets and altering their hemostatic property remains incompletely understood. In the present study, we examined the effect of dengue 2 virus on the morphology and physiological activation profile of normal human platelets using atomic force microscopy, electron microscopy and flowcytometry. Platelets obtained from healthy donors were exposed to a cell culture-adapted 10⁴ LD₅₀ dengue 2 virus isolate in vitro and the subsequent effect on morphology and activation biology studied. Our results show that dengue 2 virus exposure at doses comparable to natural viremic states in human infections can activate platelets with an increase in P-selectin expression and fibrinogen-binding property. Atomic force, scanning and transmission electron microscopy also showed typical activation-related morphological changes such as altered platelet membrane architecture, degranulation, presence of filopodia and dilatation of the open canalicular system in the dengue 2 virus-exposed platelets but not in the controls. Importantly, Japanese encephalitis virus exposure at the same dose did not activate platelets or show any morphological changes. Our findings suggest that dengue 2 virus may directly interact with and activate platelets – an event that might be important in the origin of dengue-associated thrombocytopenia. Detailed molecular characterization of this effect might provide key knowledge toward better prophylaxis of the hemostatic complications of dengue disease.

The development and characteristics of a high-speed EELS mapping system for a dedicated STEM

Isakozawa, S., Kaji, K., Jarausch, K., Terada, S., Baba, N. — 2008-03-05

A new EELS (electron energy loss spectroscopy) real-time elemental mapping system has been developed for a dedicated scanning transmission electron microscope (STEM). The previous two-window-based jump-ratio system has been improved by a three-window-based system. It is shown here that the three-window imaging method has less artificial intensity in elemental maps than the two-window-based method. Using the new three-window system, the dependence of spatial resolution on the energy window width was studied experimentally and also compared with TEM-based EELS. Here it is shown experimentally that the spatial resolution of STEM-based EELS is independent of the energy window width in a range from 10 eV to 60 eV.

Back-etch method for plan view transmission electron microscopy sample preparation of optically opaque films

Yao, B., Coffey, K. R. — 2008-03-05

Back-etch methods have been widely used to prepare plan view transmission electron microscopy (TEM) samples of thin films on membranes by removal of the Si substrate below the membrane by backside etching. The conventional means to determine when to stop the etch process is to observe the color of the light transmitted through the sample, which is sensitive to the remaining Si thickness. However, most metallic films thicker than 75 nm are opaque, and there is no detectable color change prior to film perforation. In this paper, a back-etch method based on the observation of an abrupt change of optical reflection contrast is introduced as a means to determine the etch endpoint to prepare TEM samples for these films. As the acid etchant removes the Si substrate material a rough interface is generated. This interface becomes a relatively smooth and featureless region when the etchant reaches the membrane (film/SiO₂). This featureless region is caused by the mirror reflection of the film plane (film/SiO₂ interface) through the optically transparent SiO₂ layer. The lower etch rate of SiO₂ (compared with Si) gives the operator enough time to stop the etching without perforating the film. A clear view of the morphology and control of Si roughness during etching are critical to this method, which are discussed in detail. The procedures of mounting wax removal and sample rinsing are also described in detail, as during these steps damage to the membrane may easily occur without appropriate consideration. As examples, the preparation of 100-nm-thick Fe-based amorphous alloy thin film and 160-nm-thick Cu-thin film samples for TEM imaging is described.

Characterization of the charging effect in a ZrO2 sintered body by Ga ion beam irradiation

Kim, K. H., Kim, J. J., Suzuki, T., Shindo, D. — 2008-03-05

The charging effect in a ZrO₂ sintered body was investigated by using scanning ion microscope (SIM) images. In this study, we report interesting features caused by the charging effect in the ZrO₂ sintered body during the Ga ion beam irradiation: a bright contrast with a distorted net shape appears around the positively charged specimen. From this feature in the SIM image, it is clarified that the Ga ion beam is strongly deflected and the wide area of the internal parts of the focused ion beam machine is irradiated by the Ga ion beam, depending on the extent to which the specimen is charged. We discuss the mechanism of the characteristic charging effect through observing SIM images by varying the intensity of the Ga ion beam.

Regeneration of muscle fibers in the extensor digitorum longus muscle of the aged rat

Desaki, J. — 2008-03-05

Regeneration of muscle fibers was observed in the extensor digitorum longus (EDL) muscle of aged (24 and 27 months) Wistar rats. The aged muscles consisted almost exclusively of medium-sized muscle fibers. In addition to degenerating and/or atrophied muscle fibers, very small muscle fibers <10 µm in diameter were observed in some muscle bundles which sporadically distributed in the muscle. In the degenerating muscle fibers, satellite cells mostly appeared to be normal, possibly surviving within the scaffold of basal lamina to form new (regenerating) muscle fibers. However, some of the satellite cells were degenerated and destroyed, suggesting the decrease in number of muscle fibers. On the other hand, very small muscle fibers existed between small and/or medium-sized muscle fibers or in the wide interstitial spaces between them solitarily or in small groups. In addition, immature muscle cells having a centrally located nucleus and sporadically distributed myofilaments were observed among the small and/or medium-sized muscle fibers and partially lacked a layer of basal lamina. These immature muscle cells were often closely apposed to fibroblasts with some slender cytoplasmic processes and/or to each other without an interposing basal lamina. These findings suggest that in addition to satellite cells within the basal lamina tubes, some of the regenerating muscle fibers in the aged EDL muscle may be originated from mesenchymal cells such as fibroblasts in the interstitial spaces.

Effect of Hydrofiber(R) wound dressings on bacterial ultrastructure

Hobot, J., Walker, M., Newman, G., Bowler, P. — 2008-03-05

Ionic silver has well-proven bactericidal properties, and silver-containing wound dressings are now widely used to aid in the creation of an antimicrobial environment in wounds. The effect of silver ions on bacterial ultrastructure can best be studied by viewing bacterial cells under a transmission electron microscope (TEM). Bacterial cells of Pseudomonas aeruginosa were incubated within a control dressing (e.g. a non-antimicrobial Hydrofiber® dressing) (Hydrofiber® is a registered trademark of E.R. Squibb and Sons, L.L.C.) and a silver-containing Hydrofiber® dressing, followed by processing for TEM. Liquid cultures, with and without silver, were prepared for comparison. The addition of silver to growing bacterial cultures stopped growth of the cells very quickly. Ultrastructurally, the presence of silver was found to affect both the shape of the bacterial nucleoid and the organization of bacterial DNA. X-ray microanalysis of bacteria from liquid cultures showed the presence of silver within silver-treated cells and the absence of calcium. It is suggested that the presence of available silver ions within the Hydrofiber® dressing could lead to the loss of cellular ions, vital for maintaining the structural integrity of the nuclear area.

Novel muscle spindles containing muscle fibers devoid of sensory innervation in the extensor digitorum longus muscle of aged rats

Desaki, J., Nishida, N. — 2008-03-05

We examined the structural features of muscle spindles at the equatorial and juxtaequatorial regions in the extensor digitorum longus muscle of adult (12 months) and aged (25 months) rats. In aged muscle spindles, the lamellated layers of the spindle capsule were a little increased in number compared to those in the adult ones. Two novel muscle spindles were observed in the aged muscle. In one muscle spindle, the spindle capsule contained four thin intrafusal muscle fibers invested by the inner capsule and two muscle fibers between the layers of the spindle capsule. Serial semithin sections revealed that the latter lacked the investment of the spindle capsule at the polar region. The other muscle spindle contained four intrafusal muscle fibers: two thin sensory-innervated muscle fibers invested by the inner capsule and two thick muscle fibers similar in structural features to neighboring extrafusal muscle fibers and lacking sensory innervation within the wide periaxial space. These findings suggest that two muscle fibers between the layers of the spindle capsule may be invested by the newly formed capsular cells during aging, while two thick fibers within the periaxial space may fail to receive the sensory innervation during the early development and follow the course of extrafusal fiber differentiation.

Sample Preparation of GaN-Based Materials on a Sapphire Substrate for STEM Analysis

Okuno, H., Takeguchi, M., Mitsuishi, K., Guo, X. J., Furuya, K. — 2008-01-29

In this work, a detailed TEM sample preparation recipe based on a wedge polishing technique for GaN-based materials is presented. The obtained samples have atomically flat surfaces without any obvious surface damages such as the formation of amorphous layers. A composition estimation of Al_xGa_1–_xN from Z-contrast STEM imaging is carried out using these samples. The results are in good accord with the nominal composition.

Polarity Determination for the CdxZn1-xS Nanocombs by EELS

Zhang, X. Z., Zhai, T. Y., Ma, Y., Yao, J. N., Yu, D. P. — 2008-01-29

Comb-like Cd_xZn_1-xS nanostructures have been synthesized by one-step metallo-organic chemical vapor deposition (MOCVD). The microstructure and composition were characterized by analytical transmission electron microscopy (TEM). The ribbon of the comb-like structure is along [2-1-10] (the a-axis), and the nanotips are along [0001] (the c-axis). The atomic location by channeling-enhanced microanalysis (ALCHEMI) technique was used to determine the polarity of the crystal via electron energy-loss spectroscopy (EELS). The thickness dependence, delocalization and beam convergent effect are discussed here. The investigations reveal that the growth mechanism is consistent with the asymmetric growth.

Electron holography study for two-dimensional dopant profile measurement with specimens prepared by backside ion milling

2008-01-29

The visualization of two-dimensional dopant profiles and the quantitative analysis of the built-in potential across the p–n junction, V_p-n, by electron holography were carried out with specimens prepared from the backside ion milling method combined with the focused ion beam technique. It was possible to obtain dopant profiling of the large field of view with low surface damage and gradually changed thickness. From the quantitative analysis using the phase information of electron holography and the thickness information of electron energy-loss spectroscopy, V_p-n was estimated to be about 0.78 V assuming that the thickness of the dead layer on both surfaces is 50 nm, which is to show the difference of within 12% from the calculated value. It demonstrates that the backside ion milling method is a very promising specimen preparation technique for the reliable and quantitative analysis of dopant profiling with electron holography.

Ultrastructural Alterations During Embryonic Rats' Lung Development Caused by Ozone

Lopez, I., Sanchez, I., Bizarro, P., Acevedo, S., Ustarroz, M., Fortoul, T. — 2008-01-29

Ozone (O₃) is an oxidizing agent that acts on phospholipids, proteins and sugars of cellular membranes producing free radicals, which cause oxidative damages. The O₃ exposure has been used as a model to study oxidative stress, in which the respiratory airways represent the entrance to the organism. In this study, ultrastructural alterations were identified at the bronchiolar level during the intra-uterine lung development, using an O₃ exposure model in pregnant rats during 18, 20 and 21 days of gestation. Twelve pregnant Wistar rats, six controls and six exposed to 1 ppm O₃ inhalation during 12 h per day, were used. The rats were sacrificed at gestational days 18, 20 and 21; the fetuses were obtained and their lungs dissected. The ultrastructural analysis evidenced swollen mitochondria, cytoplasmic vacuolization of the epithelial cells and structural disorder caused by the oxidative stress. At gestation day 20, flake-off epithelial cells and laminar bodies in the bronchiolar lumen were observed. In the 21-gestation-day group, the mitochondria were edematous and their cristae were disrupted by the damage caused in mitochondrial membranes.

Vogt-Koyanagi-Harada disease and vitiligo: Where does the illness begin?

Prignano, F., Betts, C. M., Lotti, T. — 2008-01-29

Vogt–Koyanagi–Harada (VKH) disease is a disorder affecting melanocytes of the skin, ocular, auditory and central nervous system. The pathogenesis is thought to be related to an aberrant T cell-mediated immune response directed against self-antigens present in melanocytes. Vitiligo is characterized by leukoderma arising at any age but usually before 30 years of age. The pathogenesis of vitiligo still remains puzzling; many hypotheses have been proposed, such as autoimmune, genetic, autocytotoxic, neural and each may contribute having its own important pathogenetic role. The expressions of the most representative melanocytic markers as HMB-45, tyrosinase, S-100 protein were investigated on the lesional, perilesional and healthy skin of a patient affected by VKH and his young daughter with vitiligo. An electronmicroscopy (EM) study was performed on the same clinical specimens. Immunohistochemical data for melanocytic cells using HMB-45 and tyrosinase were negative in the VKH patient, while the expression of both HMB-45 and tyrosinase was detected in the perilesional and lesional skin of the vitiligo patient. By EM, it was possible to show many Langerhans cells (LC) in many differentiative phases, most of which with irregular cristae or matrix swelling in both the ultraviolet (UV)-exposed and non-UV-exposed skin of VKH lesions. In both vitiligo and VKH lesions, there is a disappearance of melanocytes and an alteration of LC distribution and mitochondrial morphology which may impair the antigen-presenting functions. The vitiligo lesions in the patient's young daughter question the VKH inheritability.

Ultrastructural mitochondrial pathology in human astrocytic tumors: potentials implications pro-therapeutics strategies

Arismendi-Morillo, G. J., Castellano-Ramirez, A. V. — 2008-01-29

This study was realized to illustrate and analyze the ultrastructural mitochondrial pathology in human astrocytic tumors. Tumoral biopsies of 10 patients with pathological diagnosis of astrocytic tumors by means of transmission electron microscopy were examined. Mitochondria exhibits heterogeneous morphology in all the cases. Mitochondrial swelling with partial or total cristolysis was the most constant alteration observed. Mitochondrial fusion–fission phenomena have been demonstrated. These findings suggest that the majority of astrocytoma cells are incompetent to produce adequate amount of energy by means of oxidative phosphorylation. Ultrastructural mitochondrial pathology indicates that possibly both glycolytic inhibition and inhibition or down-regulation of mitochondrial respiration would be a potential tool for future therapeutic strategies in cases of human astrocytic tumors.