Errors in quantitative backscattered electron analysis of bone standardized by energy-dispersive x-ray spectrometry

Backscattered electron (BSE) imaging has proven to be a useful method for analyzing the mineral distribution in microscopic regions of bone. However, an accepted method of standardization has not been developed, limiting the utility of BSE imaging for truly quantitative analysis. Previous work has suggested that BSE images can be standardized by energy-dispersive x-ray spectrometry (EDX). Unfortunately, EDX-standardized BSE images tend to underestimate the mineral content of bone when compared with traditional ash measurements. The goal of this study is to investigate the nature of the deficit between EDX-standardized BSE images and ash measurements. A series of analytical standards, ashed bone specimens, and unembedded bone specimens were investigated to determine the source of the deficit previously reported. The primary source of error was found to be inaccurate ZAF corrections to account for the organic phase of the bone matrix. Conductive coatings, methylmethacrylate embedding media, and minor elemental constituents in bone mineral introduced negligible errors. It is suggested that the errors would remain constant and an empirical correction could be used to account for the deficit. However, extensive preliminary testing of the analysis equipment is essential.     

Calibration of Soil Constitutive Models with Spatially Varying Parameters

Soil constitutive models are frequently calibrated from laboratory tests that utilize global boundary measurements, which necessarily relegate soil response to that of a homogenized equivalent medium. This paper demonstrates the applicability of advanced experimental technologies to enhance the state of model-based predictions in soil mechanics by taking into account the possibility of material heterogeneity during model calibration. By utilizing the full-field displacement measurement technique of three-dimensional digital image correlation, displacements of the surfaces of deforming triaxial sand specimens are measured throughout deformation. These displacements are assimilated into finite-element (FE) models of the test specimen through solution of an inverse problem. During optimization, in which the difference between measured and predicted displacements across the specimen surface form the basis for the objective function, model parameters are allowed to vary spatially throughout the specimen volume. FE models allowing three different levels of spatial variability are tested. Results indicate that accommodating consideration of material heterogeneity during calibration leads to more accurate predictions of global stress-strain behavior that are more faithful to observed full-field response.     

Visualization and Analysis of Microstructure in Three-Dimensional Discrete Numerical Models

A computer program has been developed to allow for the virtual slicing of irregularly spaced and irregularly shaped three-dimensional image data. The program was used to virtually slice three-dimensional particle assemblies from discrete element method (DEM) simulations, allowing, for the first time, direct comparison to two-dimensional slices extracted from solidified physical specimens. Based on slices obtained from the numerical specimens, it is possible to compare quantitatively numerical microstructure directly to its physical analog, which should lead to greatly improved calibrations of granular mechanics models, and could facilitate the calibration of models across all scales of interest rather than solely at specimen boundaries. Improved confidence in the ability of the DEM to realistically simulate the microstructure of granular assemblies (through improved multiscale calibration) should result in increased confidence in microstructural parameters measurable in numerical simulations but inaccessible in the laboratory. Algorithm development within the framework of the open-source Visualization Toolkit is described and performance of the algorithm is quantified for two platforms. Results from virtual slices of a test assembly with regular particle packing are verified against known analytical solutions. A slice of a more complex assembly comprised of nearly 40,000 spheres is quantified statistically and compared to an analogous slice from a physical specimen of uniform sand.     

A novel method for viewing heavy metal stained and embedded biological tissue by field emission scanning electron microscopy

Backscattered electron (BSE) imaging was used to display heavy metal stained biological structures of various embedded specimens. Samples were fixed, stained and embedded in resin blocks as with preparation for the transmission electron microscope (TEM). Blocks were trimmed to center the specimens in a trapezoidal face of up to 5 mm2 and their sides painted with conductive silver paint leaving the face uncovered. Blocks were sputter coated with 6-8 nm of silver, chromium or aluminum, with aluminum providing the best specimen contrast in BSE mode. Samples were examined in a field emission scanning electron microscope operated at a high emission current of 50 microA. Both the fixation protocol and microscope operating parameters were optimized to maximize the number of BSE available from the smallest probe. An accelerating voltage of 10 keV was found optimal for resolution and contrast. The technique allowed the direct visualization of embedded samples at resolutions beyond light microscopy with good contrast, without cutting sections, and avoiding grid bars obscuring areas of interest. The two dimensional images provided averaged information on the internal structures of the specimens in relation to the predicted emission depth of the BSE. The technique could be used for rapid diagnostics in pathological examinations, or for routine preselection of areas of interest within a sample face before final trimming for ultrathin sectioning for higher resolution TEM study.     

Comparing emergency department and psychiatric setting patients with panic disorder

A solid state magnetic resonance imaging technique is used to measure true three-dimensional mineral density of synthetic hydroxyapatite phantoms and specimens of bone ex vivo. The phosphorus-31 free induction decay at 2.0 T magnetic field strength is sampled following application of a short, hard radiofrequency excitation pulse in the presence of a fixed amplitude magnetic field gradient. Multiple gradient directions covering the unit sphere are used in an efficient spherical polar to Cartesian interpolation and Fourier transform projection reconstruction scheme to image the three-dimensional distribution of phosphorus within the specimen. Using 3-6 Gauss/cm magnetic field gradients, a spatial resolution of 0.2 cm over a field of view of 10 cm is achieved in an imaging time of 20-35 minutes. Comparison of solid state magnetic resonance imaging with dual energy X-ray absorptiometry (DXA), gravimetric analysis, and chemical analysis of calcium and phosphorus demonstrates good quantitative accuracy. Direct measurement of bone mineral by solid state magnetic resonance opens up the possibility of imaging variations in mineral composition as well as density. Advantages of the solid state magnetic resonance technique include avoidance of ionizing radiation; direct measurement of a constituent of the mineral without reliance on assumptions about, or models of, tissue composition; the absence of shielding, beam hardening, or multiple scattering artifacts; and its three-dimensional character. Disadvantages include longer measurement times and lower spatial resolution than DXA and computed tomography, and the inability to scan large areas of the body in a single measurement, although spatial resolution is sufficient to resolve cortical from trabecular bone for the purpose of measuring bone mineral density.     

The identification of response regulators of Branhamella catarrhalis using PCR

A variable-pressure scanning electron microscope (VP-SEM) equipped with a high-sensitive backscattered electron (BSE) detector of the YAG type was applied to studies of biological tissue samples. The rat kidney and trachea were fixed, dehydrated in ethanol, critical point-dried and examined in the VP-SEM under a specimen chamber pressure of 1 to 150 Pa. The high-resolution surface images of the non-coated specimens were obtained in a low-vacuum (1-20 Pa) environment at an accelerating voltage of 5 kV, while the images at 10-20 kV contained information beneath the surface of the specimens. The observation in the VP-SEM equipped with YAG detector in a low-vacuum (1-20 Pa) environment at low (3-5 kV) accelerating voltages is useful for the three-dimensional analysis of the surface morphology of biological non-coated samples.     

Lesion contrast enhancement in medical ultrasound imaging

Methods for improving the contrast-to-noise ratio (CNR) of low-contrast lesions in medical ultrasound imaging are described. Differences in the frequency spectra and amplitude distributions of the lesion and its surroundings can be used to increase the CNR of the lesion relative to the background. Automated graylevel mapping is used in combination with a contrast-weighted form of frequency-diversity speckle reduction. In clinical studies, the techniques have yielded mean CNR improvements of 3.2 dB above ordinary frequency-diversity imaging and 5.6 dB over sharper conventional images, with no post-processing graylevel mapping.     

(110) HREM of interfacial structures in semiconductor hetero-structures

We have established a (110) cross-sectional high-resolution transmission electron microscopy (HREM) method to observe atomic structures of semiconductor hetero-interfaces. We show theoretically that the semiconductors in a hetero-structure exhibit strong contrast for an EM specimen thickness near their extinction distances, allowing atomic-scale observations of the interfacial structures between them. Furthermore, to obtain a clear HREM image, an EM specimen preparation technique is developed in which chemical etching is used to remove ion milling artifacts. This HREM method allows edge-on imaging of interfaces formed along the (110) direction; observations of atomic steps at AlAs/GaAs interfaces and a chemically ordered structure at Si/Ge interfaces are demonstrated.     

Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging

Multiphoton excitation fluorescence imaging generates an optical section of sample by restricting fluorophore excitation to the plane of focus. High photon densities, achieved only in the focal volume of the objective, are sufficient to excite the fluorescent probe molecules by density-dependent, multiphoton excitation processes. We present comparisons of confocal with multiphoton excitation imaging of identical optical sections within a sample. These side-by-side comparisons of imaging modes demonstrate a significant advantage of multiphoton imaging; data can be obtained from deeper within biological specimens. Observations on a variety of biological samples showed that in all cases there was at least a twofold improvement in the imaging penetration depth obtained with multiphoton excitation relative to confocal imaging. The more pronounced degradation in image contrast deep within a confocally imaged sample is primarily due to scattered emission photons, which reduce the signal and increase the local background as measurements of point spread functions indicated that resolution does not significantly change with increasing depth for either mode of microscopy. Multiphoton imaging does not suffer from degradation of signal-to-background to nearly the same extent as confocal imaging because this method is insensitive to scatter of the emitted signal. Direct detection of emitted photons using an external photodetector mounted close to the objective (possible only in a multiphoton imaging system) improves system sensitivity and the utilization of scattered emission photons for imaging. We demonstrate that this technique provides yet further improvements in the capability of multiphoton excitation imaging to produce good quality images from deeper within tissue relative to confocal imaging.     

X射线荧光光谱微区分析在钨矿石鉴定中的应用

为解决类质同象矿物在显微镜下鉴定困难的问题,采用X射线荧光光谱仪微区分析功能对黑钨矿((Fe,Mn)WO₄)、白钨矿(CaWO₄)等钨矿石中主元素和主要伴生元素进行定性扫描和微区分布分析,获取矿物标本中存在的异常元素及其所处部位,然后在该部位确定重点测区进行定量分析以获取矿物组分的含量,最后计算各组分的配位数,并结合矿物组分含量的理论值给矿物定名。根据矿物标本的扫描图,重点对分析谱线、背景点、能量窗口、测量方式进行选择,有效规避了干扰。对谱线的重叠干扰、基体效应和矿物效应进行了仔细的研究,选择了合适的校正方式。各主要检测组分校准曲线的相关系数均大于0.999。对定量鉴定方法的精密度进行考察,各组分的相对标准偏差(RSD,n=12)均小于4%。将实验方法用于测定黑钨矿和白钨矿标本,定量鉴定结果与电子探针分析结果进行了比较,3个矿物标本各主要组分检测结果的相对偏差(RD)均小于4%,3组数据完全吻合。     

A new scanning electron microscopy approach to the quantification of bone mineral distribution: backscattered electron image grey-levels correlated to calcium K alpha-line intensities

The introduction of backscattered electron (BSE) imaging in scanning electron microscopy (SEM) has led to new possibilities for the evaluation of mineral distributions in bone on a microscopic level. The different grey-levels seen in the BSE-images can be used as a measure for the local mineral content of bone. In order to calibrate these BSE-grey-levels (BSE-GL) and correlate them to mineral contents, various attempts, using reference samples with known weighted mean atomic number and/or using simulated bone tissues with known hydroxyapatite concentrations, have been made. In contrast, a new approach is presented here based on measurements of the X-ray intensities of the calcium K alpha-line on selected areas of real bone samples; the measured intensities are then related to the corresponding BSE-GL. A linear positive correlation between weight percent (wt%) calcium and BSE-GL was found. When the BSE-mode is standardized using carbon and aluminum as references, the different mineral contents in bone samples can be recorded as BSE-GL, calibrated to wt% of calcium or hydroxyapatite (HA), respectively. The resulting mineral concentration histograms have a dynamic range from 0 to 89 wt% HA and have a binwidth resolution of 0.45 wt% HA. The presented modifications of the BSE method strongly enhance its feasibility in the field of bone research and its application as a special diagnostic tool for bone diseases.     

Functional brain networks in movement disorders

The measurement of bone mineral density (BMD) using X-rays is usually employed to monitor the mineral content in a given portion of bone. However, this method cannot differentiate between changes in bone volume or in degree of mineralization of the bone matrix. In contrast to BMD, bone mineral density distribution (BMDD), as measured on bone sections by quantitative backscattered electron imaging (qBEI), is able to distinguish differences in the degree of mineralization. For routine clinical research, we have validated the method of calibration and standardization of the backscattered electron (BE) signal. Carbon and aluminum were used as reference materials for BE gray levels and osteoid and apatite for calcium concentration. Experiments were performed to get knowledge about precision (intraassay variance-instrumental stability and interassay variance-reproducibility) and accuracy (standardization) of this method as well as the biological variance (intraindividual and interindividual) in human bone. On transiliac biopsies or necropsies from 20 individuals having had accidental death (13 females, 7 males, age 30-85 years) BMDD measurements were conducted. The patients' medical history as well as the histomorphology of these bones showed no evidence of metabolic bone disease. For instance, the standard deviations of the weighted mean calcium concentrations were <0.3%, <0.4%, <0.9%, and <2.6% of the mean for the intraassay, interassay, intraindividual, and interindividual variations, respectively. In addition, a mean BMDD histogram for transiliac bone specimens was calculated from the 20 aforementioned individuals. The method used allows detection of the degree of mineralization independently from the actual bone volume, a result that seems to be of special interest in the assessment of the effect of treatments for osteoporosis. The power of this technique is demonstrated by using bone from a patient with a metabolic bone disease. In this case of osteomalacia due to celiac disease, the mean calcium concentration in the bone matrix was reduced by 19.3% as compared with normal.     

Palliation of large-bowel obstruction due to recurrent rectosigmoid tumor using self-expandable endoprostheses

STUDY DESIGN: This study assessed the variability of segmental bone mineral density in the lower cervical spine (C4 through C7). A mean segmental bone mineral density value at each level was determined for all specimens, and a mean coefficient of variation among the 17 specimens was calculated. OBJECTIVES: To quantify the degree of intersegmental bone mineral density variations within cadaveric lower cervical spine segments. SUMMARY OF BACKGROUND DATA: Bone mineral density studies in the thoracic and lumbar spine have shown a high degree of variability between spinal segments; however, the extent of segmental bone mineral density variability in the cervical spine is unknown. METHODS: Seventeen human cadaveric cervical spine specimens (C4 through C7) were scanned in a water bath using dual energy x-ray absorptiometry in a lateral direction. Segmental bone mineral density of the vertebral bodies of all specimens were analyzed with respect to differences between segments within each specimen. RESULTS: The mean coefficient of segmental bone mineral density variations within each specimen for all spines was 14.8% (range, 5.8%-22.9%). Bone mineral density mean values and ranges at each level were as follows: C4, 0.720 g/cm2 (range, 0.367-1.161 g/cm2); C5, 0.784 g/cm2 (range, 0.348-1.268 g/cm2); C6, 0.735 g/cm2 (range 0.367-1.450 g/cm2); C7, 0.590 g/cm2 (range, 0.340-1.040 g/cm2). Paired analysis of difference between all levels for 16 specimens demonstrated the bone mineral density at the C7 level to be significantly lower than at all other levels (P < 0.05). CONCLUSION: Our data show that significant interlevel bone mineral density variability exists in the lower cervical spine, and suggests that random single segment bone mineral density sampling or mean specimen bone mineral density values may not be relevant.     

二次电子成分衬度成像方法在异质结半导体材料和器件的微结构研究中的应用

阐述了高分辨二次电子成分衬度像的成像原理、成像条件和实验方法，介绍了一种新的试样制备方法，讨论了各种制样方法的特点。以多层Ｐ＋Ｓｉ１－ｘＧｅｘ／ｐＳｉ异质结内光发射红外探测器为例，介绍了二次电子成分衬度像观察技术在异质结半导体材料和器件微结构研究中的应用。将这种技术与通常的透射电子衍射衬度方法进行了比较，讨论了它在异质结半导体材料和器件中的应用前景。     

Automation and system integration of scanning tomographic acoustic microscope

The Scanning Tomographic Acoustic Microscope (STAM) is an instrument capable of performing subsurface imaging of microscopic specimens. Designed around the Scanning Laser Acoustic Microscope (SLAM), the STAM incorporates numerous hardware and software advances which allow automated imaging of the acoustic properties of thick specimens at high resolution. With these modifications the STAM is shown to be capable of image formation for tissue characterization and nondestructive evaluation (NDE) of microscopic specimens. This paper describes the design and operation of the STAM hardware, and software algorithms developed to permit high resolution imaging. Examples are provided to demonstrate the capability of multiple angle and multiple frequency tomographic reconstructions.     

The histopathology of nonsteroidal anti-inflammatory drug-associated colitis

We combined three techniques--mechanical testing, three-dimensional imaging, and finite-element modeling--to distinguish between the contributions of architecture and tissue modulus to mechanical function in human trabecular bone. The objectives of this study were 2-fold. The first was to assess the accuracy of micromechanical modeling of trabecular bone using high-contrast x-ray images of the trabecular architecture. The second was to combine finite-element calculations with mechanical testing to infer an average tissue modulus for the specimen. Specimens from five human L1 vertebrae were mechanically tested along the three anatomic axes. The specimens were then imaged by synchrotron x-ray tomography, and the elastic moduli of each specimen were calculated from the tomographic image by finite-element modeling. We found that 23-microm tomographic images resolved sufficient structural detail such that the calculated anisotropy in the elastic modulus was within the uncertainties of the experimental measurements in all cases. The tissue modulus of each specimen was then estimated by comparing the calculated mean stiffness of the specimen, averaged over the three anatomical directions, with the experimental measurement. The absolute values of the experimental elastic constants could be fitted, again within the uncertainties of the experimental measurements, by a single tissue modulus of 6.6 GPa, which was the average tissue modulus of the five specimens. These observations suggest that a combination of mechanical testing, three-dimensional imaging, and finite-element modeling might enable the physiological variations in tissue moduli to be determined as a function of age and gender.     

Value of examining multiple sputum specimens in the diagnosis of pulmonary tuberculosis

To objectively assess the value of examining multiple sputum specimens in maximizing the sensitivity of detection of Mycobacterium tuberculosis, we retrospectively reviewed the acid-fast bacillus smear and culture results of patients diagnosed with culture-proven pulmonary tuberculosis (TB) at Hennepin County Medical Center between 1986 and 1996. Two hundred and forty six persons were diagnosed with pulmonary TB in the time period analyzed. In 93% of these cases (229 of 246) the laboratory diagnosis was made by detection of M. tuberculosis in sputum specimens; however, only 52% (120 of 229) of these patients had at least three sputum specimens submitted to the laboratory at the time of diagnosis. Of the patients from whom at least three specimens were collected, 47% (56 of 120) had at least one smear-positive specimen; the third or later specimen submitted was the first smear-positive specimen for 13% (7 of 56) of these persons but was the first culture-positive specimen for only 7% (4 of 56). Of the 64 patients with smear-negative specimens, for only 5% (3 of 64) was the third or subsequent specimen submitted the first from which M. tuberculosis was recovered. This data indicates that, in our institution, the overwhelming majority of culture-proven pulmonary TB cases are diagnosed from the first or second sputum specimen submitted to the laboratory and that only rarely is a third specimen of diagnostic value.     

Arrest of root surface caries in situ

This study tests the hypothesis that daily oral hygiene combined with topical fluoride arrests active root-surface caries lesions without changing the mineral content of the lesions. Therefore, changes in mineral content and distribution were studied in root surfaces during caries lesion development and subsequent arrest of lesion progression in situ. In 18 subjects, lesions were developed during 3 months in sound root-surface specimens inserted into lower partial dentures. After 3 months, ground sections were prepared from each lesion prior to re-insertion of the specimens into the dentures. In addition, one sound root specimen was added per subject. During the following 3 months, half of the subjects cleaned both sound and carious specimens once a day with an 1100-ppm fluoride toothpaste, and the specimens were treated twice with 2% NaF for 2 min in situ. The other half of the subjects continued the experiment without cleaning. During the initial three-month period, all specimens developed subsurface lesions extending 187 to 583 microm into the dentin. Lesion depth increased somewhat in both experimental groups during the following 3 months (P > or = 0.1). There was a non-significant increase in mineral loss in the plaque-covered specimens (P = 0.08). However, the total mineral content of specimens subjected to plaque removal and topical fluoride did not change. This treatment resulted in an increased mineral content in the surface layer (P < 0.01) and formation of a zone of higher mineral content within the body of the lesion. The sound root surfaces which had been cleaned for a three-month period showed mineral uptake in the surface layer, occasionally associated with subsurface demineralization extending 20 to 70 microm into the tissue. The mineral loss of these specimens was significantly smaller than that of plaque-covered surfaces (P < 0.001). It is concluded that daily plaque removal and topical fluoride use influence the distribution of mineral in sound and carious root surfaces and may arrest lesion progression without affecting the total mineral content.     

关于红外吸收法测定钢铁中硫的误差分析

从红外法定硫的原理、标定方法、空白的产生等方面入手分析了红外法定硫的误差。结果表明,采用纯物质标定仪器可能造成低含量硫的测定结果偏低。不同的试样由于产生不同的燃烧微区环境,产生的粉尘空白难以用空白试验来消除。     

Comparison of direct and standardized disk diffusion susceptibility testing of urine cultures

A comparison between direct and standardized disk diffusion tests was made on a total of 300 urine specimens containing >/=10(5) organisms/ml. Of these, 246 represented pure cultures and 54 represented mixed cultures. The number of major discrepancies per organism tested in pure culture was 18 (7.3%) and in mixed cultures it was 23 (42.6%). The percentage of major discrepancies per total number of antimicrobial drug comparisons made was 1.4%. Although this procedure may be of value in selected cases with pure cultures of organisms present in quantities >/=10(5)/ml, its use on a routine basis is not recommended.