Image analysis of mineralized and non-mineralized type I collagen fibrils

Turkey leg tendons at an early stage of mineralization have been thin sectioned and imaged by electron microscopy. At this stage collagen-associated mineral apatite was found to be present within both the gap and overlap zones. The earliest apatite occurs in a microcrystalline form which gives a rather generalized and characteristic density to both the gap and overlap zones; with subsequent development larger defined apatite crystals arise which span gap/overlap zones. Fourier transformation of such images revealed the major 67 nm axial repeat of the gap/overlap zone plus four other maxima corresponding to repeat spacings of 22, 16, 13, and 11 nm respectively. Computer imaging techniques were used to reconstruct images by using selected spatial frequencies from such transforms. In this manner the subperiodic distributions of mineral were visually enhanced. These subperiodicities are positioned in an asymmetric fashion over the entire D unit repeat aligning with the molecular orientation of the fibril. Analyses of both negatively stained collagen and computer-generated maps of collagen hydrophobicity were compared to the mineral distribution of collagen. Densitometric comparisons showed a positional correlation between the axial banding patterns of mineralized fibrils and those of negatively stained non-mineralized fibrils. Comparable spatial frequencies were also present in transforms between hydrophobic maps and mineral distribution of collagen. These results suggest that the lateral clusterings of hydrophobic residues which span the fibril at specific sites in both the gap and overlap zones serve to prohibit early mineral deposition. This observed hydrophobic influence in combination with the gap space appear as contributing factors in the observed axial distribution of mineral within collagen.     

The thickness determination of organic crystals under low dose conditions using electron energy loss spectroscopy.

In the 3-dimensional (3-D) reconstruction of protein crystals with variable thicknesses the electron images and diffraction patterns can only be merged if the crystal thickness is known. Measurement of the thickness using the ratio of the number of inelastically scattered electrons to the number of electrons in the zero loss peak can be accomplished with parallel electron energy loss spectrometry (PEELS). A theoretical analysis of the accuracy of the technique on paraffin crystals of different thicknesses is presented. Our experimental studies with paraffin crystals show the feasibility of measuring a single layer of 47A with good accuracy under low dose and low temperature conditions. A simple experimental apparatus is proposed to obtain thicknesses from small regions of unstained protein crystals prior to collecting the 3-D data sets from the unexposed area of the same crystal.     

Snow metamorphism as revealed by scanning electron microscopy

Current theories of snow metamorphism indicate that sublimating snow crystals have rounded shapes, while growing crystals have shapes that depend on growth rates. At slow growth rates, crystals are rounded. At moderate rates, they have flat faces with rounded edges. At fast growth rates, crystals have flat faces with sharp edges, and they have hollow faces at very fast growth rates. The main growth/sublimation mechanism is thought to be by the homogeneous nucleation of new layers at or near crystal edges. It was also suggested that the equilibrium shape of snow crystals would be temperature dependent: rounded above -10.5 degrees C, and faceted below. To test these paradigms, we have performed SEM investigations of snow samples having undergone metamorphism under natural conditions, and of snow samples subjected to isothermal metamorphism at -4 degrees and -15 degrees C in the laboratory. In general, current theories predicting crystal shapes as a function of growth rates, and of whether crystals are growing or sublimating, are verified. However, the transition in equilibrium shapes from rounded to faceted at -10.5 degrees C is not observed in our isothermal experiments that reveal a predominance of rounded shapes after more than a month of metamorphism at -4 and -15 degrees C. Some small crystals with flat faces that also have sharp angles at -15 degrees C, are observed in our isothermal experiments. These faces are newly formed, and contradict current theory. Several hypotheses are proposed to explain their occurrence. One is that they are due to sublimation at emerging dislocations.     

The nacre protein perlucin nucleates growth of calcium carbonate crystals

Atomic force microscopy (AFM) in aqueous solution was used to investigate native nacre of the marine snail Haliotis laevigata on the microscopic scale and the interaction of purified nacre proteins with calcium carbonate crystals on the nanoscopic scale. These investigations were controlled by scanning electron microscopy (SEM), light microscopy (LM) and biochemical methods. For investigations with AFM and SEM, nacre was cleaved parallel to the aragonite tablets in this biogenic polymer/mineral composite. Multilamellar organic sheets consisting of a core of chitin with layers of proteins attached on both sides lay between the aragonite layers consisting of confluent aragonite tablets. Cleavage appeared to occur between the aragonite tablet layer and the protein layer. AFM images revealed a honeycomb‐like structure to the organic material with a diameter of the ‘honeycombs’ equalling that of the aragonite tablets. The walls of the structures consisted of filaments, which were suggested to be collagen. The flat regions of the honeycomb‐like structures exhibited a hole with a diameter of more than 100 nm. When incubated in saturated calcium carbonate solution, aragonite needles with perfect vertical orientation grew on the proteinacous surface. After treatment with proteinase K, no growth of orientated aragonite needles was detected. Direct AFM measurements on dissolving and growing calcite crystals revealed a surface structure with straight steps the number of which decreased with crystal growth. When the purified nacre protein perlucin was added to the growth solution (a super‐saturated calcium carbonate solution) new layers were nucleated and the number of steps increased. Anion exchange chromatography of the water‐soluble proteins revealed a mixture of about 10 different proteins. When this mixture was dialysed against saturated calcium carbonate solution and sodium chloride, calcium carbonate crystals precipitated together with perlucin leaving the other proteins in the supernatant. Thus perlucin was shown to be a protein able to nucleate calcium carbonate layers on calcite surfaces, and in the presence of sodium chloride, is incorporated as an intracrystalline protein into calcium carbonate crystals.     

Micro-Raman spectroscopy used to identify and grade human skin pilomatrixoma

Raman microspectroscopy was applied to analyze the changes in structural conformation and chemical composition of the mass of human skin pilomatrixoma (PMX). The normal skin dermis, collagen type I, and hydroxyapatite (HA) were used as control. The excised specimens from two patients diagnosed as a typical PMX were detected, in which one specimen was a soft mass, but the other was a hard mass with somewhat calcified deposits via histopathological examination. The Raman spectrum of normal skin dermis was found to be similar to the Raman spectrum of collagen type I, confirming that the collagen type I was a predominant component in normal skin dermis. The differences of Raman peak intensity between normal skin dermis and soft or hard PMX mass were obvious at 1,622-1,558, 1,400-1,230, 1,128, 1,000-850, 749, and 509 cm(-1). In particular, the peak at 1,665 cm(-1) assigned to amide I band shifted to 1,655 cm(-1) and the peak at 1,246 cm(-1) corresponding to amide III band was reduced in its intensity in hard PMX mass. The significant changes in collagen content and its structural conformation, the higher content of tryptophan, and disulfide formation in PMX masses were markedly evidenced. In addition, the shoulder and weak peak at 960 cm(-1) assigned to the stretching vibration of PO(4) (3-) of HA also appeared respectively in the Raman spectra of soft and hard PMX masses, suggesting the occurrence of calcification of HA in the PMX tissue, particularly in the hard PMX mass. The result indicates that the micro-Raman spectroscopy may provide a highly sensitive and specific method for identifying normal skin dermis and how it differs in chemical composition from different PMX tissues.     

Characterization of the calcification of cardiac valve bioprostheses by environmental scanning electron microscopy and vibrational spectroscopy

Bioprosthetic heart valve tissue and associated calcification were studied in their natural state, using environmental scanning electron microscopy (ESEM). Energy dispersive X‐ray micro‐analysis, X‐ray diffraction, Fourier‐transform infrared and Raman spectroscopy were used to characterize the various calcific deposits observed with ESEM. The major elements present in calcified valves were also analyzed by inductively coupled plasma–optical emission spectroscopy. To better understand the precursor formation of the calcific deposits, results from the elemental analyses were statistically correlated. ESEM revealed the presence of four broad types of calcium phosphate crystal morphology. In addition, two main patterns of organization of calcific deposits were observed associated with the collagen fibres. Energy dispersive X‐ray micro‐analysis identified the crystals observed by ESEM as salts containing mainly calcium and phosphate with ratios from 1.340 (possibly octacalcium phosphate, which has a Ca/P ratio of 1.336) to 2.045 (possibly hydroxyapatite with incorporation of carbonate and metal ion contaminants, such as silicon and magnesium, in the crystal lattice). Raman and fourier‐transform infrared spectroscopy also identified the presence of carbonate and the analyses showed spectral features very similar to a crystalline hydroxyapatite spectrum, also refuting the presence of precursor phases such as β‐tricalcium phosphate, octacalcium phosphate and dicalcium phosphate dihydrate. The results of this study raised the possibility of the presence of precursor phases associated with the early stages of calcification.     

单颗磨粒刻划氧化镓晶体表面的裂纹成核位置及扩展方向研究

为了分析新一代光电子材料氧化镓晶体在超精密磨削、研磨加工过程中的裂纹成核位置及扩展方向,建立了单颗磨粒刻划氧化镓(010)晶面的弹性应力场模型,分析了氧化镓(010)晶面的脆塑性转变临界切削深度。通过MATLAB软件分析预测刻划氧化镓晶体过程中表面径向裂纹的成核位置及扩展方向,分析结果表明：当切削深度小于临界切削深度时,径向裂纹成核位置在磨粒的后方,裂纹扩展方向与切削方向之间的夹角在33°左右;当切削深度超过临界切削深度时,径向裂纹成核位置进一步向磨粒后方移动,裂纹生成方向与刻划方向之间的夹角在51°左右。为验证理论分析结果,对氧化镓晶体进行了纳米刻划试验,对比分析表明,氧化镓应力场的解析结果与试验数据高度一致。在线性加载条件下,Cube金刚石压头在氧化镓晶体(010)晶面上产生的径向裂纹偏转角在33.37°~51.45°之间。     

用FDTD法分析二维光子晶体带隙

阐述了光子晶体的概念,用时域有限差分法,通过数值计算,模拟分析了准一维光子晶体和二维光子晶体的禁带,讨论一种准一维和二维的混合光子晶体,并对其禁带进行研究。结果表明当一维光子晶体的周期性结构与二维光子晶体的周期性结构混合在一起能形成更宽的光子禁带。     

Papain‐gel degrades intact nonmineralized type I collagen fibrils

Papain‐gel has been utilized as a chemo‐mechanical material for caries removal due to its ability to preserve underlying sound dentin. However, little is known about the effect of the papain enzyme on intact type I collagen fibrils that compose the dentin matrix. Here we sought to define structural changes that occur in intact type I collagen fibrils after an enzymatic treatment with a papain‐gel. Intact and nonmineralized type I collagen fibrils from rat tail were obtained and treated with a papain‐gel (Papacarie) for 30 s, rinsed with water and imaged using an atomic force microscope (AFM). Additionally, polished healthy dentin specimens were also treated using the same protocol described above and had their elastic modulus (E) and hardness (H) measured by means of AFM‐based nanoindentation. AFM images showed that the papain‐gel induced partial degradation of the fibrils surface, yet no rupture of fibrils was noticed. The distinction between gap and overlap zones of fibrils vanished in most regions after treatment, and overlap zones appeared to be generally more affected. Mechanical data suggested a gradual decrease in E and H after treatments. A significant two‐fold drop from the values of normal dentin (E=20±1.9, H=0.8±0.08 GPa) was found after four applications (E=9.7±3.2, H=0.24±0.1 GPa) (P<0.001), which may be attributed to the degradation of proteoglycans of the matrix. In summary, this study provided novel evidence that intact nonmineralized type I collagen fibrils are partially degraded by a papain‐gel. SCANNING 31: 253–258, 2009. © 2010 Wiley Periodicals, Inc.     

Irregular Shaped,Assumably Semi‐Crystalline Calciumphosphate Platelet Deposition at the Mineralization Front of Rabbit Femur Osteotomy: A HR‐TEM Study

Although bone minerals have been widely studied by various techniques in previous studies, crystal structures, morphology of bone minerals and its building pathway remained still controversy. In this work, the ultrastructure of the mineralization front of rabbit femur has been studied by conventional and high‐resolution (HR) transmission electron microscopy (TEM). In order to induce a healing and demineralization process the animals were subjected to a standardized osteotomy stabilized with titan screws and sonic pins. After 84 days follow‐up time the newly build bone was investigated. The mineralization front of rabbit femur osteotomy contains partly mineralized collagen fibrils with a pronounced striped pattern together with a large number of agglomerated apatite platelets. The striation is caused by mineralization in the hole zones of the collagen fibrils, corresponding to the early stage of mineralization. In the TEM micrographs, the mineralization zone appears denser and compact when compared with fully mineralized bone, although most of the collagen fibrils are completely mineralized in the latter (higher concentration of interfibrillar apatite platelets within the mineralization zone). In bone some partly mineralized collagen fibrils are also observed, revealing the same arrangement, regular shape, and size of apatite platelets as collagen fibrils in the mineralization zone. Apatite platelets with irregular shapes are observed at the vortex‐shaped outer boundary of the mineralization zone, i.e. at the interfaces with nonmineralized collagen or osteoblasts. HR TEM micrographs reveal that the platelets are assumably semicrystalline and that within the platelet nanocrystalline domains of apatite are embedded in an amorphous calciumphosphate matrix. SCANNING 35: 169‐182, 2013. © 2012 Wiley Periodicals, Inc.     

A nanostructured look of collagen apatite porosity into human mineralized collagen fibril

Bone tissue is a hierarchical material characterized at nanoscale by the mineralized collagen fibril, a recurring structure mainly composed of apatite minerals, collagen and water. Bone nanostructure has a fundamental role in determining the mechanical behavior of the tissue and its mass transport properties. Diffusion phenomenon allows to maintain an adequate supply of metabolites in the mechanisms of bone remodeling, adaptation and repair. Several analytical and computational models have been developed to analyze and predict bone tissue behavior. However, the fine replication of the natural tissue still represents a challenge. Insights on the structural organization at nanoscale and on the influence of apatite mineral crystals on the diffusion coefficient lead to outline the functional conditions for the development of biomimetic strategies for bone tissue engineering. Thorough understanding of bone nanostructure is essential to improve longevity of bioscaffolds and to decrease the risk of failure by controlling their mechanical and biological performance.     

Morphometrical study of plant vacuolar dynamics in single cells using three-dimensional reconstruction from optical sections

In higher plants, vacuoles increase their volumes in accordance with cell enlargement and occupy most of the cell volume. However, quantitative analyses of vacuolar contributions during changes in cell morphology have been hampered by the inadequacies and frequent artifacts associated with current three-dimensional (3-D) reconstruction methods of images derived from light microscopy. To overcome the limitations of quantifying 3-D structures, we have introduced 3-D morphometrics into light microscopy, adopting a contour-based approach for which we have developed an interpolation method. Using this software, named REANT, the morphological and morphometrical changes in protoplasts and vacuoles during plasmolysis could be investigated. We employed the tobacco (Nicotiana tabacum) BY-2 cell line No.7, expressing a GFP-AtVam3p fusion protein, BY-GV7, using GFP as a marker of vacuolar membranes (VMs). By vital staining of the plasma membrane (PM) of cells, we simultaneously obtained optical sections of both the PM and VM. We, therefore, reconstructed the 3-D structures of protoplasts and vacuoles before and after plasmolysis. We were able to identify the appearance of elliptical structures of VMs in the vacuolar lumen, and to determine that they were derived from cytoplasmic strands. From the 3-D structures, the volumes and surface areas were measured at the single cell level. The shrinkage of vacuoles accounted for most of the decrease in protoplast volume, while the surface area of the vacuoles remained mostly unchanged. These morphometrical analyses suggest that the elliptical structures are reservoirs for excess VMs that result from the response to rapid decreases in vacuolar and protoplast volumes.     

视场外回波引起条纹场扫描成像重构混叠现象研究

为了消除成像视场外回波对条纹场扫描成像(又称为傅立叶望远镜或者相干场成像)的可能影响,本文从理论上分析回波引起重构混叠的主要原因,并针对不同的应用场合提出对应的最佳解决方案。本文通过计算机仿真手段详细研究不同照明区域回波对成像的影响,仔细分析增加短基线方案对重构模糊的改善效果。仿真结果表明:三光束(同时发射的最多光束)重叠区域的回波对重构结果有最严重影响;增加短基线可以消除图像混叠,单臂增加一个短基线则能使图像质量明显改善,对应的斯托里尔比(Strehl ratio)从0.49上升到0.59;且单臂增加基线数越多,则重构图像细节就越丰富。本文所提出的消除成像视场外回波影响的几种方案对于条纹场扫描成像系统在不同领域的应用均具有重要的借鉴价值。     

Three-dimensional reconstruction and modeling of microstructures and microchemistry in geological materials

Computer-aided three-dimensional (3-D) reconstruction of minerals and rock fabrics enables quantification, spatial analysis, and state of the art communication of the complex spatial relations typically encountered in geological materials. An efficient and versatile combination of sample preparation, data acquisition, and processing methods has been established which renders the 3-D reconstruction of microstructures in geological materials applicable on a routine basis: rock samples are subject to a cycle of precision serial lapping and subsequent image acquisition by an electron probe microanalyzer. The yielded backscattered electron images and x-ray maps depict mineral phase distribution and chemical variation; backscattered electron images are stacked in a voxel array to be interpolated by binary processing. X-ray map data are interpolated by geostatistical methods, and the results are forwarded to one voxel array per chemical element of interest. Merging the voxel arrays by boolean and image algebraic methods enables the integrated analysis and visualization of mineral morphology and associated chemical variation in geological materials.     

Scanning and transmission electron microscopy for evaluation of order/disorder in bone structure

A comparative characterization of the structure of normal and abnormal (osteoporotic) human lumbar and thoracic vertebrae samples was carried out to reveal the type of possible disorder. Samples from the bone fragments extracted during the surgery due to vertebra fractures were examined by scanning electron microscopy (SEM), conventional and high resolution transmission electron microscopy (TEM and HRTEM), and X-ray energy dispersive spectroscopy (EDS). Contrary to what might be expected in accordance with possible processes of dissolution, formation and remineralization of hard tissues, no changes in phase composition of mineral part, crystal sizes (length, width, and thickness), and arrangement of crystals on collagen fibers were detected in abnormal bones compared to the normal ones. The following sizes were determined by HRTEM for all bone samples: 相似文献   

Three-dimensional finite element analysis for estimation of the weld residual stress in the dissimilar butt weld piping

Numerous dissimilar metal welds are used to connect carbon steel and stainless steel in nuclear power plants. Recently, some cracks have occurred in the dissimilar metal welds, and welding residual stress is considered as a contributing factor to the cracks. In this study, welding residual stresses in dissimilar butt weld piping were evaluated by the 3-dimensional (3-D) finite element method. Welding residual stresses along the circumference of heat affected zones as well as weld regions were obtained through the analysis, which could not be obtainable with 2-dimensional (2-D) analysis. The differences between 2-D analysis and 3-D analysis are presented in this paper.     

Determination of mask opening size in creating a fluid hole on brittle material by double-side sand blasting

In creating a hole on brittle materials by double-side sand blasting, the rebounding sand particle flux during the process may result in underetching at the edge of the mask opening, and leads to a larger sized fluid hole than the desired one. Determination of the correct mask opening size was made mainly by trial and error in the past. In this paper, relationships between the mask opening size and desired size of a hole on both the front and the back sides of the substrate are derived. For the front side, by taking into account the underetching effect, an equation is derived based on the kinetic energy theory. For the back side, there is negligible rebounding sand particles, and the mask opening size is set to be equal to the desired size of the hole. Experiments were conducted to verify the derived relationships. It is found that the measured sizes of the eroded holes on both the front and the back sides of the wafer substrate are distributed normally. The desired hole sizes deviate slightly from the median of a normal distribution curve, and the maximum predicted errors are 2.4% and 3.0% for front side and the back side sand blasting, respectively. The very satisfactory result together with ANOVA and test of homogeneity of variance of the predicted errors for various hole size shows that the derived relationships is applicable for determination of the mask opening size in the sand blasting process.     

Determination of mask opening size in creating a fluid hole on brittle material by double-side sand blasting

In creating a hole on brittle materials by double-side sand blasting, the rebounding sand particle flux during the process may result in underetching at the edge of the mask opening, and leads to a larger sized fluid hole than the desired one. Determination of the correct mask opening size was made mainly by trial and error in the past. In this paper, relationships between the mask opening size and desired size of a hole on both the front and the back sides of the substrate are derived. For the front side, by taking into account the underetching effect, an equation is derived based on the kinetic energy theory. For the back side, there is negligible rebounding sand particles, and the mask opening size is set to be equal to the desired size of the hole. Experiments were conducted to verify the derived relationships. It is found that the measured sizes of the eroded holes on both the front and the back sides of the wafer substrate are distributed normally. The desired hole sizes deviate slightly from the median of a normal distribution curve, and the maximum predicted errors are 2.4% and 3.0% for front side and the back side sand blasting, respectively. The very satisfactory result together with ANOVA and test of homogeneity of variance of the predicted errors for various hole size shows that the derived relationships is applicable for determination of the mask opening size in the sand blasting process.     

Differential interference contrast and confocal reflectance imaging of collagen organization in three-dimensional matrices

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the process of 3-D extracellular matrix (ECM) remodeling at the cellular and subcellular level. In this study, we directly compare two imaging modalities for both quantitative and qualitative imaging of 3-D collagen organization in vitro: differential interference contrast (DIC) and confocal reflectance imaging. The results demonstrate that two-dimensional (2-D) DIC images allow visualization of the same population of collagen fibrils as observed in 2-D confocal reflectance images. Thus, DIC can be used for qualitative assessment of fibril organization, as well as tracking of fibril movement in sequential time-lapse 2-D images. However, we also found that quantitative techniques that can be applied to confocal reflectance images, such as Fourier transform analysis, give different results when applied to DIC images. Furthermore, common techniques used for 3-D visualization and reconstruction of confocal reflectance datasets are not generally applicable to DIC. Overall, obtaining a complete understanding of cell-matrix mechanical interactions will likely require a combination of both wide-field DIC imaging to study rapid changes in ECM deformation which can occur within minutes, and confocal reflectance imaging to assess more gradual changes in cell-induced compaction and alignment of ECM which occur over a longer time course.     

Diffractive-refractive optics: X-ray collimator

Diffractive-refractive optics are x-ray focusing monochromators based on the diffraction on profiled crystal surface. Diffraction on longitudinal parabolic groove machined in crystal surface forms a sagittaly focused synchrotron radiation beam. Such kind of monochromator may be realized as a crystal with parabolic hole, where the beam is diffracted on the inner wall of the hole. Two such asymmetrically cut crystals set into antiparallel position, creating a dispersive (+,-,-,+) arrangement, form a sagittaly focusing x-ray monochromator which should be practically aberration-free. The focusing properties of such kind of monochromator are discussed in detail and it is shown for the first time that it can be used not only for focusing but also for creating highly parallel monochromatic beam in the broad region of the Bragg angles. This device with parabolic hole has not been tested experimentally yet.