Journal of Electron Microscopy - Advance Access

Structural analysis of hydroxyapatite coating on magnetite nanoparticles using energy filter imaging and electron tomography

Fri, 06 Nov 2009 00:57:50 PST

Magnetic nanoparticle (MNP) composites with a magnetite (Fe₃O₄) core and a hydroxyapatite (HAp, Ca₁₀(PO₄)₆(OH)₂) coating were prepared using a precipitation method and a subsequent hydrothermal treatment. The hydrothermal treatment diminished the lepidocrocite layer on the magnetite, enhanced the crystal growth of HAp and dissolved the MNPs. The divalent iron ions dissolved into solvent were not substituted for the HAp lattice. The three-dimensional (3D) nanostructure, the crystal morphology of HAp covered with the MNPs and the interfacial nanostructure of magnetite/HAp were analyzed using an energy-filter transmission electron microscopy (EF-TEM) and visualized by computer tomography in transmission electron microscopy (TEM). EF-TEM and 3D reconstruction images using a tilted series of high-angle annular dark-field images showed that the needlelike HAp nanocrystals covered with a magnetite core and the crystal growth of HAp attached to the magnetite surface was inhibited as a result of the lower density of the nucleation site of the lepidocrocite layer. The dissolution of iron ion from MNPs and the interfacial interaction of HAp and magnetite could cause the needlelike morphology of HAp nanocrystals.

The spindle pole body of the pathogenic yeast Cryptococcus neoformans: variation in morphology and positional relationship with the nucleolus and the bud in interphase cells

Yamaguchi, M., Biswas, S. K., Kuwabara, Y., Ohkusu, M., Shimizu, M., Takeo, K. — Mon, 26 Oct 2009 20:57:23 PDT

The spindle pole body (SPB) in the interphase cell of the pathogenic yeast Cryptococcus neoformans was studied in detail by freeze-substitution and serial ultrathin sectioning electron microscopy. The SPB was located on the outer nuclear envelope and appeared either dumbbell- or bar shaped. The dumbbell-shaped SPBs were 228–365 nm long with amorphous spheres on each end, each sphere being 78–157 nm in diameter. The bar-shaped SPBs were 103–260 nm long and 32–113 nm thick. They consisted of filamentous materials. The dumbbell-shaped SPBs were more frequent (61%) than the bar-shaped SPBs. The bar-shaped SPBs may be regarded as dumbbell-shaped SPBs whose spherical parts became sufficiently small. There seemed to be no relationship between the SPB shape and the cell cycle stage of G1–G2, since both types of SPB appeared not only in unbudded cells but also in budded cells and their appearance seems to be random. It is not clear at present whether morphological changes between dumbbell- and bar shapes have any physiological function. The SPB tended to be localized away from the nucleolus (141° ± 44°), but localized randomly to the bud (97° ± 50°). The present study highlights the necessity of observing a large number of micrographs in three dimensions to describe accurately the ultrastructure of the SPB in yeast.

Fibre width measurement and quantification of filler size distribution in paper-based materials by SEM and image analysis

Bennis, H., Benslimane, R., Vicini, S., Mairani, A., Princi, E. — Sun, 25 Oct 2009 21:59:23 PDT

In this paper the development of image processing and analysis routines for acquiring information about the paper structure from images obtained by scanning electron microscopy (SEM) is described. A new method to measure the fibre width based on the processing of superficial SEM observation is herein proposed and successfully applied to some commercial paper-based materials. The quantification of fillers in the Z-direction of paper was based on paper cross-sectional SEM images. The results obtained by image processing demonstrated the excellent performance and suitability of the proposed method to quantify exactly information concerning fraction and size distribution of digital fillers. The fraction of digital fillers was correlated with the surface roughness of a given paper grade.

Morphological changes in murine skeletal muscle in response to exercise and mesterolone

Fri, 23 Oct 2009 09:08:24 PDT

Light and electron microscopy and quantitative morphometry were used to determine the effects of exercise and mesterolone on the soleus muscles of mice. Both exercise and mesterolone caused a significant hypertrophy of extrafusal muscle fibres. The hypertrophy of Type I fibres was greater than that of Type II fibres. There was no hyperplasia. Mitochondria were more numerous and larger than in the muscles of sedentary animals. Capillarity increased and small centrally nucleated muscle fibres appeared, usually in small clusters and most often in the muscles of animals exposed to mesterolone. A small proportion of satellite cells exhibited signs of activation but there were more in the muscles of mesterolone-treated animals than after exercise. Muscles from animals that had been both exercised and treated with mesterolone exhibited the largest changes: muscle mass and muscle fibre hypertrophy was greater than in all other groups of animals, capillarity was higher and >30% of all recognized satellite cells exhibited signs of activation. Groups of small centrally nucleated muscle fibres were commonly seen in these muscles. They appeared to be the result of splits in the form of sprouts from existing muscle fibres. With both exercise and mesterolone, alone or in combination, there was an increase in the proportion of Type I muscle fibres and a decrease in the proportion of Type II.

High-resolution spin-polarized scanning electron microscopy (spin SEM)

Kohashi, T., Konoto, M., Koike, K. — Mon, 19 Oct 2009 01:22:54 PDT

We have developed spin-polarized scanning electron microscopy (spin SEM) with a 5-nm resolution. The secondary electron optics is very important, as it needs to transfer a sufficient number of secondary electrons to the spin polarimeter, due to the low efficiency of the polarimeter. The optics was designed using a three-dimensional (3D) simulation program of the secondary electron trajectories, and it achieves highly efficient collection and transport of the secondary electrons even though the distance between the sample and the objective lens exit of the electron gun remains short. Moreover, the designed optics enables us to obtain clear SEM images in the spin SEM measurement and to precisely adjust the probe beam shape. These functions lead to images with high spatial resolution and sufficient signal-to-noise (S/N) ratios. This optics has been installed in an ultra-high vacuum (UHV) spin SEM chamber with a Schottky-type electron gun for the probe electron beam. We observed recorded bits on a perpendicular magnetic recording medium and visualized small irregularities in the bit shapes around the track edges and bit boundaries. The high resolution of 5 nm was demonstrated by observing the smallest domain composed by a single grain in the recording medium.

Dimethylthiourea pretreatment inhibits endotoxin-induced compound exocytosis in goblet cells and plasma leakage of rat small intestine

Liu, S.-P., Chang, C.-Y., Huang, W.-H., Fu, Y.-S., Chao, D., Huang, H.-T. — Mon, 12 Oct 2009 23:26:17 PDT

Intravenous application of a high dose of endotoxin, also called lipopoly-saccharide (LPS), results in endotoxemia in animals, that induces production of cytokines and free radicals, systemic inflammation and mucin discharge from mucous tissues. The present study was to investigate (1) whether LPS application increased goblet cell secretion by compound exocytotic activity in mucosal villi and crypts of rat small intestine, and (2) whether hydroxyl radicals were involved in LPS-induced compound exocytosis in goblet cells and plasma leakage. Scanning electron microscopy showed that the numbers of goblet cells undergoing compound exocytosis (cavitated goblet cells) per mm² of ileal villus epithelium in rats 5 and 30 min after LPS (15 mg kg^–1) were 693 ± 196 (N = 6) and 547 ± 213 (N = 6), respectively, which were 5.1 and 8.4 times (P < 0.05) the number of saline control. The percentage of villus cavitated goblet cell numbers, in both duodenum and ileum 5 min after LPS and in the ileum 30 min after LPS, increased significantly (P < 0.05). Pretreatment with dimethylthiourea (DMTU), a hydroxyl radical scavenger, decreased the number of cavitated goblet cells to saline control (P > 0.05). Morphometric analysis showed that the percentage of crypt epithelial area in the duodenum and ileum occupied by goblet cell mucin stores in the duodenum and ileum 30 min after LPS were 3.8 ± 0.2% (N = 6) and 6.9 ± 0.5 (N = 6), respectively reducing to one half the amount of control (P < 0.01). When DMTU was given prior to LPS the crypt goblet cell mucin stores and the amount of plasma leakage returned to the level of control. It is concluded that hydroxyl radicals were involved in the LPS-induced increase in compound exocytotic activity of goblet cells and the increase in plasma leakage during acute phases of inflammatory response in rat small intestine.

Ultrastructural disorder of the secretory pathway in temperature-sensitive actin mutants of Saccharomyces cerevisiae

Yamaguchi, M., Kopecka, M. — Thu, 08 Oct 2009 06:38:38 PDT

Phenotypes of the two temperature-sensitive actin mutants of Saccharomyces cerevisiae act1-1 and act1-2 at permissive, restrictive and semi-restrictive temperatures were studied by freeze fracture and thin section electron microscopy, and fluorescent microscopy. In contrast to secretory mutants where accumulations of either secretory vesicles, Golgi apparatus, or endoplasmic reticulum were reported, act1-1 and act1-2 mutants revealed accumulation of all the three components, even at permissive temperature. However, more distinct accumulation of secretory organelles was evident during cultivation at the sub-restrictive temperature of 30°C. At the restrictive temperature of 37°C, many cells died, and their empty cell walls remained. Some of the few living cells showed features of apoptosis. From the present study, actin cables are concluded to be necessary for (i) correct spatial positioning and orientation of secretary pathway to the bud and septum, and (ii) vectorial movement of vesicles of the secretory pathway along the actin cables to the bud and septum.

Autophagy in transition from hypertrophic cardiomyopathy to heart failure

Fidzianska, A., Bilinska, Z. T., Walczak, E., Witkowski, A., Chojnowska, L. — Wed, 30 Sep 2009 23:55:33 PDT

Endomyocardial biopsy of a patient in transition stage from hypertrophic cardiomyopathy to heart failure was investigated. The tissue showed hypertrophy, atrophy of myocytes and an increased amount of fibrosis. In addition, numerous cardiomyocytes revealed ubiquitin positive inclusions. Ultrastructural analysis indicated that cardiomyocytes contained typical autophagic vacuoles including mitochondria, glycogen granules, degraded remnants and myelin structures. The most obvious ultrastructural finding was the presence of amorphous plaques and tubulofilamentous inclusions. Such ultrastructural abnormalities allow us to conclude that degeneration of myocardial cells by autophagy mechanisms leads to cardiomyocyte death, loss and heart failure.

Quantitativeness of phase measurement by transport of intensity equation

Mitome, M., Ishizuka, K., Bando, Y. — Sat, 19 Sep 2009 02:52:56 PDT

Based on the transport of intensity equation (TIE), a phase map can be readily retrieved from only three images of a focal series. The retrieval procedure does not require an iterative calculation and it is performed very fast with the use of the fast Fourier transform. However, it must be noted that sensitivity depends on the spatial frequency components and difficulties are encountered when phase information is retrieved to be of the same quality for all frequency components. Since the retrieved phase is influenced by conditions in which the focal series was taken, it is important to select the appropriate conditions and parameters used in this process. Optimal conditions to preserve the quantitativeness of phase maps measured by the TIE method are discussed theoretically and they are demonstrated by measuring phase shifts in silver particles. It was found that the high quantitativeness can be obtained by selecting appropriately a defocus difference and a high pass filter constant according to the frequency components for the objects.

Examination of electron stains as a substitute for uranyl acetate for the ultrathin sections of bacterial cells

Yamaguchi, K., Suzuki, K.-i., Tanaka, K. — Sat, 19 Sep 2009 02:52:55 PDT

Electron staining reagents were examined to find a possible substitute for uranyl acetate (UA) in electron microscopy of bacterial ultrathin sections. Four kinds of stains, platinum blue (Pt-blue), oolong tea extract (OTE), potassium permanganate (KMnO₄) and phosphotungstic acid (PTA), were examined in comparison with UA either with or without post-staining with lead citrate (Pb). Electron microscopy was performed on sections from Spurr-embedded cells of a Gram-positive bacterium, Bacillus cereus NBRC 13597, and a Gram-negative bacterium, Escherichia coli NBRC 3301. Both Pt-blue and OTE showed staining similar to each other and to that of double staining with UA and Pb in B. cereus, while in E. coli the cytoplasmic membrane appeared less dense when compared with UA and Pb. KMnO₄ stained excessively to some extent, but showed images of the best contrast in the cytoplasmic membrane comparable with UA and Pb among the four reagents. PTA could stain the peptidoglycan layer but gave images of low quality for both bacteria. This study demonstrated that none of the reagents examined showed staining results of the same quality or better than the conventional method with UA and Pb. However, stains of Pt-blue, OTE and KMnO₄ could possibly be an alternative candidate for the UA according to the structure in question.

A specimen preparation technique for plane-view studies of surfaces using transmission electron microscopy

Foss, S., Tafto, J., Haakenaasen, R. — Sat, 12 Sep 2009 03:36:07 PDT

A method for preparing plane-view transmission electron microscope (TEM) samples is presented. With this inclined pseudo-plane-view technique, the undisturbed surface of the sample can be studied in plane view. Thus, nanostructures on the surface of a substrate can be studied with TEM in much the same way as with scanning electron microscopy (SEM), but in transmission at a much higher spatial resolution and with the opportunity of performing nanoscale diffraction. A glued sandwich with two surfaces facing each other was thinned at a low angle relative to the surfaces. The resultant construction contained thin wedges of the surfaces upon which it was possible to do TEM analysis. SEM analysis before and TEM analysis after such sample preparation was found to be consistent.

Microcalorimeter-type energy dispersive X-ray spectrometer for a transmission electron microscope

Thu, 27 Aug 2009 22:15:46 PDT

A new energy dispersive X-ray spectrometer (EDS) with a microcalorimeter detector equipped with a transmission electron microscope (TEM) has been developed for high- accuracy compositional analysis in the nanoscale. A superconducting transition-edge-sensor-type microcalorimeter is applied as the detector. A cryogen-free cooling system, which consists of a mechanical and a dilution refrigerator, is selected to achieve long-term temperature stability. In order to mount these detector and refrigerators on a TEM, the cooling system is specially designed such that these two refrigerators are separated. Also, the detector position and arrangement are carefully designed to avoid adverse affects between the superconductor detector and the TEM lens system. Using the developed EDS system, at present, an energy resolution of 21.92 eV full-width-at-half maximum has been achieved at the Cr K line. This value is about seven times better than that of the current typical commercial Si(Li) detector, which is usually around 140 eV. The developed microcalorimeter EDS system can measure a wide energy range, 1–20 keV, at one time with this high energy resolution that can resolve peaks from most of the elements. Although several further developments will be needed to enable practical use, highly accurate compositional analysis with high energy resolution will be realized by this microcalorimeter EDS system.

Morphological changes in the tongue as a consequence of manganese inhalation in a murine experimental model: Light and scanning electron microscopic analysis

Thu, 27 Aug 2009 22:15:44 PDT

Air pollution by suspended particles has become a worldwide health problem. The main sources of these particles are fossils and additives combustion. Mn enters the body through inhalation, but part of the particles accesses contact with tongue's posterior surface where lingual tonsils and lingual papillae are placed. We decided to explore in a mouse model, the impact that the deposit of inhaled Mn has on the tongue's surface. Atrophy of the lingual tonsil, filiform papillae, as well as the swelling of taste buds in fungiform papillae, were the predominant changes. Ferropenic anemia is associated with the changes described and could be related to the interference of Mn in iron metabolism and riboflavin absorption. More research should be done to explore the participation of suspended particles trapped in the oral cavity in toxicology of Mn or other inhaled pollutants.

Atomic force microscopy observation of the enamel roughness and depth profile after phosphoric acid etching

Tue, 18 Aug 2009 22:46:24 PDT

The aim was to compare the enamel surface roughness (ESR) and absolute depth profile (ADP) (mean peak-to-valley height) by atomic force microscopy (AFM) before and after using four different phosphoric acids. A total of 160 enamel samples from 40 upper premolars were prepared. The inclusion criterion was that the teeth have healthy enamel. Exclusion criteria included any of the following conditions: facial restorations, caries lesions, enamel hypoplasia and dental fluorosis. Evaluations of the ESR and ADP were carried out by AFM. The Mann–Whitney U-test was used to compare continuous variables and the Wilcoxon test was used to analyze the differences between before and after etching. There were statistically significant differences (P ≤ 0.05) among mean surface roughness and absolute depth before and after using four different phosphoric acids in healthy enamel; Etch-37 and Scotchbond Etching Gel showed higher profiles after etching (P ≤ 0.05). There were statistically significant differences (P ≤ 0.05) among roughness and ADP before and after using four different phosphoric acids in healthy enamel. However, consistently Etch-37 and Scotchbond Etching Gel showed the highest increase regarding the ESR and ADP after etching healthy enamel. AFM was a useful tool to study site-specific structural topography changes in enamel after phosphoric acid etching.

Cyanoacrylate glue as an alternative mounting medium for resin-embedded semithin sections

Sat, 08 Aug 2009 07:10:47 PDT

Commercially available generic Superglue (cyanoacrylate glue) can be used as an alternative mounting medium for stained resin-embedded semithin sections. It is colourless and contains a volatile, quick-setting solvent that produces permanent mounts of semithin sections for immediate inspection under the light microscope. Here, we compare the use of cyanoacrylate glue for mounting semithin sections with classical dibutyl phthalate xylene (DPX) in terms of practical usefulness, effectiveness and the quality of the final microscopic image.

Generalization of the paraxial trajectory method for the analysis of non-paraxial rays: simulation program G-optk for electron gun characterization

Fujita, S., Takebe, M., Ushio, W., Shimoyama, H. — Tue, 04 Aug 2009 05:05:31 PDT

The paraxial trajectory method has been generalized for the application to the cathode rays inside electron guns. The generalized method can handle rays that initially make a large angle with the optical axis with a satisfactory accuracy. The key to success of the generalization is the adoption of the trigonometric function sine for the trajectory slope specification, instead of the conventional use of the tangent. Formulas have been derived to relate the ray conditions (position and slope of the ray at reference planes) on the cathode to those at the crossover plane using third-order polynomial functions. Some of the polynomial coefficients can be used as the optical parameters in the characterization of electron sources; the electron gun focal length gives a quantitative estimate of both the crossover size and the angular current intensity. An electron gun simulation program G-optk has been developed based on the mathematical formulations presented in the article. The program calculates the principal paraxial trajectories and the relevant optical parameters from axial potentials and fields. It gives the electron-optical-column designers a clear physical picture of the electron gun in a much more faster way than the conventional ray-tracing methods.

Old age is associated with ultrastructural changes in isolated rat liver sinusoidal endothelial cells

O'Reilly, J. N., Cogger, V. C., Le Couteur, D. G. — Sat, 01 Aug 2009 04:43:28 PDT

Old age is associated with ultrastructural changes in the hepatic sinusoid called pseudocapillarization, which include defenestration and thickening of the sinusoidal endothelium. We investigated whether such changes also occur in isolated and cultured liver sinusoidal endothelial cells. Liver sinusoidal endothelial cells were isolated from young (6–10 months, n = 4) and old (24–26 months, n = 4) F344 rats and fenestrations evaluated using scanning electron microscopy. Fenestration diameter was reduced in old age from 194 ± 1 nm to 185 ± 1 nm (P < 0.001) and there was an age-related increase in the number fused fenestrations and large gaps. Age-related changes in the diameter of fenestrations in the liver sinusoidal endothelial cell are maintained following isolation and culture. This suggests that this age-related change may be intrinsic to the liver endothelial cell and/or irreversible.

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. — Sat, 01 Aug 2009 04:43:27 PDT

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.

A further observation of muscle spindles in the extensor digitorum longus muscle of the aged rat

Desaki, J., Nishida, N. — Fri, 31 Jul 2009 07:50:19 PDT

We observed three novel muscle spindles in the extensor digitorum longus muscle of the aged (20 months) rat. Two muscle spindles of the three contained thin muscle fibers lacking sensory innervation between the layers of the spindle capsule and within the periaxial space, respectively. The other one contained sensory-innervated thin muscle fibers with an indistinct equatorial nucleation between the layers of the spindle capsule. These findings suggest that the occurrence of thin muscle fibers may be intimately related to the degeneration and regeneration of extrafusal muscle fibers during aging and that these newly formed thin muscle fibers may often fail to receive sensory innervation.

Evaluation of imaging plates as recording medium for images of negatively stained single particles and electron diffraction patterns of two-dimensional crystals

Li, Z., Hite, R. K., Cheng, Y., Walz, T. — Thu, 30 Jul 2009 02:45:57 PDT

We evaluated imaging plates (IPs) and the DITABIS Micron scanner for their use in recording images of negatively stained single-particle specimens and electron diffraction patterns of two-dimensional crystals. We first established the optimal imaging and read-out conditions for images of negatively stained single-particle specimens using the signal-to-noise ratio of the images as the evaluation criterion. We found that images were best recorded on IPs at a magnification of 67 000x, read out with a gain setting of 20 000 and a laser power setting of 30% with subsequent binning over 2 x 2 pixels. Our results show that for images of negatively stained specimens, for which the resolution is limited to ~20 Å, IPs are a good alternative to EM film. We also compared IPs with a 2K x 2K Gatan charge-coupled device (CCD) camera for their use in recording electron diffraction patterns of sugar-embedded two-dimensional crystals. Diffraction patterns of aquaporin-0 recorded on IPs and with the CCD camera showed reflections beyond 3 Å and had similar R_Friedel as well as R_merge values. IPs can thus be used to collect diffraction patterns, but CCD cameras are more convenient and remain the best option for recording electron diffraction patterns.

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

Wed, 08 Jul 2009 07:35:23 PDT

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.

Ultrastructural megakaryocyte modifications after vanadium inhalation in spleen and bone marrow

Tue, 30 Jun 2009 05:24:27 PDT

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.

Electron microscopy of octacalcium phosphate in the dental calculus

Kakei, M., Sakae, T., Yoshikawa, M. — Fri, 26 Jun 2009 07:58:54 PDT

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.

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

Tue, 23 Jun 2009 15:01:48 PDT

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.

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

Mon, 22 Jun 2009 04:04:37 PDT

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. — Fri, 12 Jun 2009 09:05:30 PDT

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.

Development of an analytical environmental TEM system and its application

Thu, 11 Jun 2009 05:32:04 PDT

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.