Detection of aluminium by energy dispersive X-ray microanalysis at high accelerating voltages with semi-thin sections of biological sample

Aluminium (Al) was detected in semi-thin sections of three organs, the duodenum, liver and kidney, of ddY strain mouse by energy dispersive X-ray microanalysis at high accelerating voltages around 300 kV. Firstly, to determine the conditions best for detecting Al, several adult ddY mice were injected intraperitoneally with aluminium chloride (AlCl3) and the duodenums were fixed, embedded and sectioned at various thicknesses and subjected to energy dispersive X-ray microanalysis at various accelerating voltages from 100 to 400 kV. From the results obtained, 1.0 microm-thick sections observed at 300 kV resulted in the highest peak-counts/background ratios and were shown to be the most suitable for X-ray microanalysis. Secondly, ddY mice aged four weeks were administered orally with 2% AlCl3 at pH 2.5 for two weeks and the three organs (duodenum, liver and kidney) were subjected to X-ray microanalysis under the same condition found above. The results were compared with light microscopic Al staining of the same tissues. Aluminium was detected in lysosomes of the three kinds of tissues with higher sensitivity by energy dispersive X-ray microanalysis by light microscopic observation. From the results, it was suggested that Al dissolved in acidic water was absorbed in the duodenum and accumulated in the liver and kidney.     

A new method for detecting and localizing cell markers endocytosed by fibroblasts in epoxy resin semi-thin sections using scanning electron microscopy combined with energy dispersive X-ray microanalysis after ion-etching

Cell marking is widely used to examine cell development and differentiation in developmental biology. We developed a new method for localizing cell markers in a semi-thin epoxy section with scanning electron microscopy. Cultured fibroblasts ingesting carbon particles were autologously transplanted into a rabbit transparent ear chamber, 6 mm in diameter and 100 microm in depth. Eight days after the transplantation, tissues in the chamber were fixed and embedded in epoxy resin. Semi-thin sections were cut and stained with toluidine blue. Fibroblasts in connective tissues which contained black spots were observed with a light microscope. These sections were subsequently ion-etched with an ion-coater and coated with platinum. The same fibroblasts were then visualized by secondary electron imaging using a scanning electron microscope. A nucleus with nuclear envelope, nuclear pores, a nucleolus and heterochromatin, mitochondria with cristae and rough endoplasmic reticulum were observed in the fibroblasts. The black spots in the fibroblasts were identified as bright bodies with the scanning electron microscope. The bright bodies were found to be a lump of tiny particles less than 100 nm in diameter. In order to analyse such particles with energy dispersive X-ray microanalysis, ion-etched sections were coated with carbon. X-ray energy spectrometry clearly demonstrated that these were carbon particles, which had been endocytosed by the fibroblast. This suggests that scanning electron microscopy combined with energy dispersive X-ray microanalysis is useful for detecting carbon particles in the cytoplasm at an ultrastructural level in semi-thin epoxy sections subsequent to ion etching and that this method may be applicable to other cell markers, such as gold particles to track cells in the field of cell development and cell differentiation.     

Appearance of electron-dense segments: indication of possible conformational changes of pre-mineralizing collagen fibrils in the osteoid of rat bones

To elucidate precise mechanisms of appositional mineralization of bone, structural features of mineralizing collagen fibrils of the osteoid in normal and hypocalcaemic rats were examined in detail by transmission electron microscopy. Ultrathin sections of the osteoid of various types of bones of the rats fed with regular or normal calcium diet often displayed electrondense segments in the specific regions of the collagen fibrils located immediately adjacent to the mineralization front or to the mineralization nodules. Such dense segments appeared only after Ur-Pb staining and were more distinct in undecalcified specimens. Dense segments were undetectable in ultrathin sections picked up on ethylene glycol instead of water in the trough, even after Ur-Pb staining. Collagen fibrils in the widened osteoid of hypocalcaemic rats fed with calcium-free diet failed to show electron-dense segments. A careful comparison between the hydrously or anhydrously processed adjacent sections of a normal rat bone indicated a drastic dissolution of electron-dense material from the bone matrix near the mineralization front in hydrously processed sections and, thus, implicated the presence of labile mineral-matrix complexes in the recently mineralized bone matrix. Such labile sediments were readily dissociated within the ultrathin sections while the sections were floating on water and immediately adsorbed onto the pre-mineralizing collagen fibrils, where some conformational changes might have occurred. These data indicate that highly electron-dense segments appearing in the osteoidal collagen fibrils are a type of process-induced product, which indirectly represent possible structural alterations in the segmental portions of pre-mineralizing collagen fibrils in the osteoid of rat bones.     

Regenerating axons and their growth cones observed by scanning electron microscopy

A predenervated sciatic nerve segment, which had been treated by repeated freezing and thawing to kill Schwann cells, was grafted to the original sciatic nerve in the rat. Three to five days later, the graft was chemically fixed and treated by KOH-collagenase digestion, a treatment which selectively removes almost all non-cellular elements including the collagen fibrils and basal laminae from the tissue, thus making it possible to observe regenerating axons by scanning electron microscopy. Debris of degraded Schwann cells and myelin sheaths remained in the form of "columns", and several thick (2-3 microns in diameter) and thin (less than 1 micron in diameter) axons ran singly or in bundles on such "cell debris columns." Thick axons have an almost straight contour, while there were various swellings at intervals along the thin axons. In most cases, the growing tips of regenerating axons were swollen as growth cones ranging from 2 microns to 5 microns in diameter. Growth cones exhibited fusiform to polygonal variations in structure and had only a few filopodial processes on the surface.     

Perichromatin granule-like granular structures in the nuclei of antral-follicular oocytes of the Chinese hamster

In the nuclei of antral-follicular oocytes of the Chinese hamster (Cricetulus griseus), numerous granular structures ranging from 60 nm to 250 nm in diameter were present in the nucleolar area, and a few were observed on the surface of heterochromatin materials and within the interchromatin space. When treated with bismuth staining en bloc after glutaraldehyde fixation (GA-Bi staining), these granular structures were shown to be composed of fine fibrils intensely contrasted with bismuth, indicating that these may be regarded as one type of perichromatin granule.     

High-resolution ultrastructure of amyloid fibrils in familial amyloid polyneuropathy

The ultrastructure of amyloid fibrils in familial amyloid polyneuropathy (FAP) was clearly demonstrated. Amyloid of three patients with FAP caused by the point mutation of the 30th amino acid of transthyretin (ATTR Val30Met) and one patient with FAP caused by two point mutations of the 30th and 104th amino acid of transthyretin (ATTR Val30Met/Arg104Cys) were partially isolated, stained negatively and examined with an electron microscope. Amyloid fibrils of both types were composed of two protofilaments and twisted at 180 degrees to the right and left alternately with a periodicity of 125-135 nm. This is the first report demonstrating such unique alternating twist structure of amyloid fibrils. There were no ultrastructural differences between the fibrils caused by the ATTR Val30Met and ATTR Val30Met/ Arg104His; therefore, it is suggested that the point mutation of the 30th amino acid of transthyretin might play an important role in the formation of amyloid fibrils. Further biochemical study on the mechanism of this alternating twist formation should be undertaken.     

基于负染－电镜的Aβ体外聚集表征测试要点分析

本文应用电镜负染色技术,对阿尔茨海默病（ Alzheimer′s disease, AD）中所涉及的β淀粉样肽（β?amyloid peptide,Aβ）的聚集规律进行了分析。通过对样品的浓度、pH值、温度和时间等设置系列梯度,以及调整负染试剂的种类、浓度和染色时间,优化了Aβ寡聚体样品制备以及电镜观察的最佳条件,获得了清晰的Aβ纤维图像并用以估算纤维长度。实验结果表明：经过六氟异丙醇给予单体化处理,并溶于无水二甲基亚砜（ DMSO）的Aβ（100μmol·L－1）,在pH7?2的PBS中4℃孵育24 h,Aβ寡聚体形态清晰,且Aβ42寡聚体比Aβ40寡聚体聚集趋势更明显。在pH 8?0的硼酸缓冲液中,Aβ42（40μmol·L－1）在37℃孵育72 h后,纤维形成明显;而Aβ纤维样品经过煮沸后再制备负染样品,所得电镜图像更为清晰,便于对纤维长度和结构进一步分析。因此电镜负染色技术,可作为一种快捷,直观的Aβ体外聚集形貌表征的质控方法。     

Discontinuous capillary segments in the extensor digitorum longus muscle of aged BUF/Mna rats

We examined the structural changes of capillaries in the extensor digitorum longus muscle of aged (25 months) male BUF/Mna rats, which caused severe muscle weakness of hind legs during aging. The aged muscle mostly consisted of bundles of muscle fibres 15-35 microm in diameter (type 1). In some small areas, however, muscle bundles contained small muscle fibres mainly 15-25 microm in diameter (type 2), possibly indicating that these small fibres are in the course of regeneration after necrosis. Examination of serial ultrathin sections revealed some remarkable changes of capillaries in the type 2 muscle bundle. In some capillaries, the vascular lumen became discontinuous by several close contacts of opposed endothelial cells in their course, forming plural closed vascular segments. Moreover, a solitary endothelial cell was often observed within a scaffold of basal lamina, which remained after destruction of endothelial cells. The segmentation of capillaries and the occurrence of the scaffolds of basal laminae are considered to indicate the degenerative process of capillaries. In some instances, on the other hand, endothelial cells closely apposed each other with no vascular lumen for distances of up to 100 microm, and some capillaries had a narrow vascular lumen (1-3 microm diameter) for a long distance (approximately 300 microm), probably indicating that these structures are in the course of regenerating capillaries to remodel the capillary networks around the muscle fibres. Pericytic processes circularly arranged outside the endothelium at the slit-like and narrow vascular lumen, like hoops, possibly preventing the rupture of the newly-formed vascular lumen from the increased blood flow and/or blood pressure. In addition, the occasional occurrence of capillaries with fenestrations may participate to increase the supply of nutrients and oxygen to the regenerating muscle fibres. The present findings suggest that the capillary networks in the extensor digitorum longus muscle of aged BUF/Mna rats are remodelled following the regeneration of muscle fibres after necrosis, and that on occasion, neighbouring endothelial cells may closely contact with each other both in the degenerative and regenerative processes of capillaries.     

利用热退火法从非晶硅薄膜中生长纳米硅粒

报道了一种从氢化非晶硅薄膜中生长纳米硅粒的方法.氢化非晶硅薄膜经过不同条件的热退火处理后,用拉曼散射和X射线衍射技术对样品进行了分析.实验结果表明:在快速升温条件下所形成的nc-Si在薄膜中的分布是随机均匀的,直径在1.6～15 nm范围内,硅粒大小随退火过程中升温快慢而变化.     

磷钨酸乙醇染色法在嗜铬颗粒电镜X射线显微分析中的应用

通过比较常规透射电镜制样法、快速冷冻固定－冷冻超薄切片法及磷钨酸乙醇（EPTA）染色法在嗜铬颗粒透射电镜X射线显微分析中的应用，发现磷钨酸乙醇染色法能使嗜铬颗粒电子着色，从而较好地显示嗜铬颗粒的超微结构。同时磷钨酸乙醇染色法也以在一定程度上原位保留生物样品中元素，可以应用于检测样品元素含量的变化或比较样品元素含量的组间差别，提示磷钨酸乙醇染色法是一种可应用于透射电镜X射线显微分析的经济简便的生物样品制备方法。     

Abnormal formation of the glucan network from regenerating protoplasts in Schizosaccharomyces pombe cps8 actin point mutant

To study the close relationship between the actin cytoskeleton and cell wall formation, the process of cell wall formation in reverting protoplasts of the fission yeast, Schizosaccharomyces pombe, cps8 actin point mutant was investigated by ultra-high-resolution low-voltage scanning electron microscopy (UHR-LVSEM) and transmission electron microscopy (TEM). The protoplast of the cps8 mutant began to form a glucan network in a unipolar manner and to secrete alpha-galactomannan. The site of cell wall formation grew in a cylindrical shape in the wild-type protoplast. The alpha-galactomannan did not fill in the intrafibrillar spaces completely, however, and the fibrils were exposed on the cell surface. UHR-LVSEM images indicated that the glucan fibrils were thin and rope-shaped, forming a looser network than the wild-type. TEM images indicated the finest fibrils were approximately 1.5 nm in diameter, the same diameter as the wild-type. These results suggest that the cps8 mutant was insufficient in developing cross-linkage with the glucan fibrils up to the wide ribbon shape as found in the wild-type [Osumi M et al. (1989) J. Electron Microsc. 38: 457-468; Osumi M (1998) Micron 29: 207-233]. These findings appear to indicate that the actin cytoskeleton controls formation of the glucan network and secretion of beta-1,6-glucan, and confirm the close relationship of the actin cytoskeleton and glucan formation.     

Immunohistochemical detection of haem oxygenase in AA amyloidogenesis

Haem oxygenase (HO)-1, a rate-limiting enzyme in the degradation of haem, is increased in Alzheimer's disease and in inflammations such as AA amyloidosis. However, the specific association of HO-1 is poorly understood in AA amyloidosis. In this study, we designed the experiment to reveal the contribution and association of HO-1 in the spleen during experimental murine AA amyloidosis. Experimental murine AA amyloidosis was induced with injection of an emulsion consisting of Freund's complete adjuvant and Mycobacterium butyricum. The serum amyloid A level was highest on day 3. The distribution of cells containing iron, indicating an increase of HO-1 in the red pulp, was detected with Berlin blue staining. AA amyloid formation was immunohistochemically detected as a marker by chondroitin sulphate proteoglycan (CSPG), one of the components of AA amyloid fibrils. Immunolocalizations of HO-1 and CSPG indicated a conspicuous increase and scattering of positive cells in the red pulp of the spleen. Double positive cells were not detected. On day 7, amyloid deposition was detected with Congo red staining in the extracellular spaces in the marginal zone of the white pulp in the spleen and HO-1-positive cells accumulated near the amyloid deposition area. CSPG was detected within the cells and also localized in the amyloid deposition area. CSPG was still not localized in the HO-1-positive cells. Double positive cells of HO-1 and CSPG were localized in the red pulp and in the amyloid deposition area on day 14. X-ray microanalysis indicated the existence of iron in the electron-dense bodies of fibroblasts in the amyloid deposition areas. The fibroblasts extended amyloid fibrils into the extracellular spaces of the marginal zone. These results suggest that HO-1-positive fibroblasts, but not HO-1-positive macrophages, are associated with the late stage of amyloid fibril formation.     

Photoluminescence and composition of amorphous As<Subscript>2</Subscript>Se<Subscript>3</Subscript> films modified with Er(<Emphasis Type="Italic">thd</Emphasis>)<Subscript>3</Subscript> complex compound

The photoluminescence and composition of amorphous As₂Se₃ films modified with an Er(thd)₃ complex compound have been studied. A band centered at 1.54 μm, characteristic of photoluminescence from Er embedded in amorphous matrices, has been revealed at room temperature. The composition of thin amorphous As₂Se₃ films modified with an Er(thd)₃ complex compound has been examined by methods of nuclear microanalysis: Rutherford backscattering and nuclear resonant reactions. Dependences of the concentrations of Er ions, oxygen, and carbon on the growth conditions of the films are obtained. It is shown that the Er concentration in a thin film varies nonlinearly as the relative concentration of the starting complex compound increases. In addition, the increase in the Er content of a film is accompanied by a simultaneous rise in the content of such light elements as oxygen and carbon. Comparative analysis of the nuclear microanalysis data and IR spectra demonstrates that, in modification of As₂Se₃ with the Er(thd)₃ complex compound by the given method, the nearest environment of Er in the complex compound is partly preserved.     

Application of the environmental SEM in human dentin bleached with hydrogen peroxide in vitro.

The environmental SEM (E-SEM) can be used unfixed biological samples under a low vacuum and wet condition. In this study, the fractured dentin of unfixed human teeth was treated with a 30% hydrogen peroxide solution (H2O2) for the examination of tooth-bleaching prior to the E-SEM and a conventional SEM. The peritubular matrix (PM) always showed a few cracks along the long axis of a dentinal tubule, and the ends of fine fibrils rose to the smoothly changed surface of the intertubular matrix (IM). The E-SEM with non-fixation and the conventional SEM following fixation indicated that the hydrogen peroxide solution easily permeated the PM and dissolved the non-fibrillar substance including the cracks of the PM by the constriction. In the IM, the solution may partially dissolve the organic parts within mineralized fibrils as well as non-fibrillar substance between the fibrils, although these remnants might precipitate again there.     

Defective bone remodelling in osteoprotegerin-deficient mice

Previous studies have reported enhanced osteoclastogenesis, increased bone resorption and osteoporosis in osteoprotegerin (OPG)-deficient mice. In the present study, we show that the tibial epiphyses contain abundant, thin trabeculae lined with numerous osteoclasts and cuboidal osteoblasts. The increase in osteoblasts and osteoclasts was associated with a dramatic increase in calcein labelling of the mineralization fronts and replacement of much of the intertrabecular marrow with numerous alkaline phosphatase-positive preosteoblasts. Furthermore, the discrete, linear cement lines seen in wild-type mice were replaced by a randomly oriented meshwork of cement lines that were stained intensely for tartrate-resistant acid phosphatase and osteopontin in the OPG-/- mice. These indices of accelerated bone remodelling in mutant bone were associated with irregular trabecular surfaces, a disorganized collagen matrix interspersed with amorphous ground substance and numerous fissures between old and new bone. In total, these observations indicate that enhanced osteoclastic activity in OPG-/- epiphyses led to a coupled increase in osteoblast differentiation and activity and an increase in bone remodelling. The high bone turnover, disorganized matrix and impaired attachment of new to old bone in the cement lines in OPG-/- mice appear to cause bone fragility.     

Some observations on the subfibrillar structure of collagen fibrils as noted during treatment with NKISK and cathepsin G with mechanical agitation

We observed the structure of collagen fibrils in rat tail tendons after treatment with NKISK and cathepsin G. NKISK is a pentapeptide that has been previously shown to bind fibronectin, while cathepsin G is a serine protease that cleaves fibronectin but not type I collagen. In tendons treated with NKISK, fibrils were seen to extensively dissociate into smaller-diameter subfibrils. These subfibrils were homogeneous in diameter with an average diameter of 26.3?±?5.8?nm. Similar, although less extensive, dissociation into subfibrils was found in tendons treated with cathepsin G. The average diameter of these subfibrils was 24.8?±?4.9?nm. The ability of NKISK and cathepsin G to release subfibrils at physiological pH without harsh denaturants may enhance the study of the subfibrillar structure of collagen fibrils.     

Penetration and stainability of modified Sato's lead staining solution.

The penetration and stainability of modified Sato's lead staining solution containing calcined lead citrate were studied. Modified Sato's lead solution was preserved for 1 week and for 2 years, each at room temperature and at 4 degrees C. Specimens were stained with these solutions to measure the stainability. After 2-min staining, specimens were stained to the depth of 1.0-1.2 microns even when there had been 2-year preservation of the staining solution. This modified solution could be preserved for a long time and good penetration and stainability could still be obtained. This solution is also suitable for the observation of semithin sections.     

Orientational Ordering within Semiconducting Polymer Fibrils

Due to a general paucity of suitable characterization methods, the internal orientational ordering of polymer fibrils has rarely been measured despite its importance particularly for semi-conducting polymers. An emerging tool with sensitivity to bond orientation is polarized resonant soft X-ray scattering (P-RSoXS). Here, P-RSoXS reveals the molecular arrangement within fibrils (if type I or type II fibrils), the extent of orientation in the fibril crystal, and an explicit crystal-amorphous interphase. Neat films as well as binary blends with a fullerene derivative are characterized for three different polymers, that are prototypical materials widely used in organic electronics applications. Anisotropic P-RSoXS patterns reveal two different fibril types. Analysis of the q-dependence of the anisotropy from simulated and experimental scattering patterns reveal that neat polymer fibrillar systems likely comprise more than two phases, with the third phase in addition to crystal and amorphous likely being an interphase with distinct density and orientation. Intriguingly, the fibril type correlates to the H- or J-aggregation signature in ultraviolet-visible (UV–vis) spectroscopy, revealing insight into the fibril formation. Together, the results will open the door to develop more sophisticated structure-function relationships between chemical design, fibril type, formation pathways and kinetics, interfacial ordering, and eventually device functions.     

表面层外全反射角X射线能谱微分析

电子探针微区分析(EPMA,XRMA)由于X射线激发深度较大而对薄层分析产生困难,无法准确确定分析结果是样品表面的成分还是样品体相的成分。本工作在通常的X射线微区分析设备上,采用外全反射角X射线能谱微分析方法,通过对硅村底上不同膜厚的铝膜和铜膜的测定,探索出一种区分膜成分和体相成分的新方法。结果表明：与常规的方法相比,该法有较高的表面灵敏度,可很好地解决薄层分析的困难。     

基于多孔氧化铝模板的一维ZnO纳米线的制备

多孔氧化铝由于具有纳米级的孔径、尺寸可调等独特的优点,成为合成纳米材料的一种常用模板.以多孔氧化铝为模板,制备出了纳米量级的纤维、纳米棒、金属管、半导体等新型材料.制备出了优良的多孔氧化铝有序孔洞阵列;以其为模板,采用直流电化学沉积的方法,在其规则排列的孔中沉积得到锌的纳米线;然后将其在高温下氧化,得到氧化锌的纳米线.利用X射线衍射谱、扫描电子显微镜等手段研究了它们的微结构性质,X射线衍射谱表明,用电化学沉积方法得到的锌和氧化锌纳米线均为多晶结构.