An improved counting method for motile protozoan cultures

A simple counting method is described for routine cultures of highly motile organisms. It is based on dyeing the organisms with a suitable stain, filtering them through a Millipore filter and counting single organisms directly under a microscope after rendering the filter transparent.     

Photographic defocusing: a means to render recognizable line traced models of negatively stained biological specimens

A simple method of obtaining analogue images from traced models of biological specimens is presented. It consists of the photographic defocusing of traced models and it is illustrated with negatively stained cylindrical forms of the ASFV; the black lines of the trace in the model correspond to the negative stain surrounding the viral morphological subunits as seen in the electron micrograph. The photographic defocusing is the means by which the traced model is filtered and is used to introduce grey levels on an otherwise black and white image. The right amount of defocusing is attained when the width of the trace of the model equals the width of the rim of the negative stain appearing between the morphological subunits in the electron micrograph.     

Stevenel's Blue, an excellent stain for optical microscopical study of plastic embedded tissues

Stevenel's Blue is a reliable, rapid, and clean, one-step polychromatic stain for 1 micron thick epoxy sections. The staining solution, originally used by L. Stevenel (1918) to stain human parasites, is made by adding diluted potassium permanganate (2%) to an aqueous solution of the methylene blue (1.3%) and redissolving the precipitate thoroughly, by boiling in water bath and filtering. Staining is carried out in a Coplin jar at 60 degrees C for approximately 10 minutes for tissues embedded in Epon 812 or Poly 812, or 20 minutes for tissues embedded in Spurr's medium. The sections are rinsed, air dried, and mouted in Permount. The staining solution is very stable, and does not tend to form precipitates on the tissue. The stain brings excellent histological differentiation to nuclear, cytoplasmic, and extracellular components. Incorporation of the stain by elements within each tissue varies from intense to light with a subtle gradation of intermediate shades of purple and blue tones. For most cell structures the density of the stain parallels the electron density of that structure as seen under the electron microscope. For example, nucleoli and heterochromatin stain in dark purple while euterochromatin appear in a light blue shade. In all cases, the embedding media remains unstained. The bond between Stevenel's Blue and the tissues is stable, remaining unaltered by the mounting medium. It is also resistant to time-fading.     

在用润滑油斑点扩散性定量测试方法研究

本文基于图象检测原理，提出了一种新的在用润滑油斑点扩散性定量测试方法。在分析油扩散斑点图象特征的基础上，依据图象灰度直方图分析定义了平均污染水平、污染严重度、扩散环水平、污染指数、相对污染度和污染率等一组定量测试参数，用于评价在用润滑油的污染变质情况。车用发动机的台架试验和天然气发电机组的实际监测试验表明，这六种参数对于反映在用润滑油的污染变质状况变化较为敏感。本文所提出定量测试方法简单易行，在工业现场具有广阔的应用前景。     

Enhanced effects of nonisotopic hafnium chloride in methanol as a substitute for uranyl acetate in TEM contrast of ultrastructure of fungal and plant cells

This ultrastructural study showed that nonisotopic methanolic hafnium chloride and aqueous lead solution was an excellent new electron stain for enhancing TEM contrasts of fungal and plant cell structures. The ultrastructural definition provided by the new stain was often superior to that provided by conventional staining with uranyl acetate and lead. Definition of fine ultrastructure was also supported by quantitative data on TEM contrast ratios of organelles and components in fungal and plant cells. In particular, polysaccharides, which were localized in cell walls, glycogen particles, starch grains, and plant Golgi vesicle components, were much more reactive to the new stain than to the conventional one. The new nonisotopic stain is useful for enhancing the contrast of ultrastructure in biological tissues and is a safer alternative to uranyl acetate. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

MRT letter: a fast and easy method for general fluorescent staining of cultured cells on transparent or opaque supports

Development of cell-based therapy entails the use of different types of materials as support for cultured cells. Some of these materials are opaque. For a general microscope study of cell cultures prepared on transparent supports, Giemsa stain with bright field microscopy is useful. With opaque supports or scaffolds, epifluorescence microscopy is necessary. The method the authors describe uses eosin Y to stain cytoplasm and DAPI to stain nuclei under fluorescence microscopy. This method provides easy and fast fluorescent staining for a general morphological study of cultured cells on transparent or opaque supports.     

Improving image quality by zero-loss energy filtering: quantitative assessment by means of image cross-correlation

Fourier ring correlation and root-mean-square contrast of pairs of images, taken under identical conditions, were used as criteria of image quality for comparing unfiltered with zero-loss energy-filtered imaging using a TEM equipped with a post-column energy filter. For three different specimens (amorphous carbon film, macromolecules in light negative stain, virus particles in deep negative stain) the dependence of these quantities on electron dose, specimen thickness and defocus was investigated. A model, based on simple assumptions, was used to describe quantitatively their dependence on electron dose and specimen thickness. It was found that energy filtering is most advantageous for low-dose imaging and small defocus values. The gain due to energy filtering strongly increases with specimen thickness, whereby the dependence is linear for light scattering elements. For thick specimens, the gain by energy filtering is more pronounced in the resolution range between 4 and 2 nm than for lower spatial frequencies.     

基于变形均匀性的锻造模具形状优化设计方法

以直接设计预成形模具形状为目标,提出并建立了一种控制变形均匀性的灵敏度分析理论和模具形状优化设计方法。以任意单元的等效应变与所有单元的平均等效应变的差值的平方和作为目标函数,Ｂ样条曲线表示模具形状,Ｂ样条曲线控制点坐标作为优化设计变量,优化设计的目标是通过设计预成形模具形状使目标函数最小,即使整个工件的变形尽可能均匀。给出了目标函数的表达形式,详细推导了目标函数、单元等效应变率和单元各应变率分量对优化设计变量的灵敏度,给出了优化设计的步骤。应用该方法对圆柱体平模镦粗工艺进行预成形优化设计,给出了相应的优化设计结果,并提出了一种检验优化结果的变形均匀因子,优化与未优化结果的比较表明优化取得了良好的效果。     

Method for forming two-dimensional paracrystals of biological filaments on lipid monolayers

A method is described for electron microscopic observation of two-dimensional paracrystals on unsupported lipid monolayers. The method uses a hydrophobic holey C-coated grid placed on a monolayer made positively charged by the inclusion of stearylamine (SA) and has been used to align scallop thin filaments and reconstituted actin/tropomyosin filaments to form paracrystals. The use of unsupported monolayers allows the paracrystals to be viewed in either negative stain or with cryoelectron microscopy. Those paracrystals in frozen hydrated specimens have better order than those with negative stain. It was found that varying the lipid composition between the less fluid distearolyphosphotidylcholine/SA and the more fluid egg yolk phosphotidylcholine/SA alters the size and order of the paracrystals, the more fluid system having smaller, more ordered paracrystalline domains. The advantage of the technique for studying actin/thin filaments is the ability to form large two-dimensional paracrystals under physiological conditions of [Mg²⁺] and pH.     

Hydrogen peroxide staining to visualize intracellular bacterial infections of seedling root cells

Visualization of bacteria in living plant cells and tissues is often problematic due to lack of stains that pass through living plant cell membranes and selectively stain bacterial cells. In this article, we report the use of 3,3′‐diaminobenzidine tetrachloride (DAB) to stain hydrogen peroxide associated with bacterial invasion of eukaryotic cells. Tissues were counterstained with aniline blue/lactophenol to stain protein in bacterial cells. Using this staining method to visualize intracellular bacterial (Burkholderia gladioli) colonization of seedling roots of switch grass (Panicum virgatum), we compared bacterial free seedling roots and those inoculated with the bacterium. To further assess application of the technique in multiple species of vascular plants, we examined vascular plants for seedling root colonization by naturally occurring seed‐transmitted bacteria. Colonization by bacteria was only observed to occur within epidermal (including root hairs) and cortical cells of root tissues, suggesting that bacteria may not be penetrating deeply into root tissues. DAB/peroxidase with counter stain aniline blue/lactophenol was effective in penetration of root cells to selectively stain bacteria. Furthermore, this stain combination permitted the visualization of the bacterial lysis process. Before any evidence of H₂O₂ staining, intracellular bacteria were seen to stain blue for protein content with aniline blue/lactophenol. After H₂O₂ staining became evident, bacteria were often swollen, without internal staining by aniline blue/lactophenol; this suggests loss of protein content. This staining method was effective for seedling root tissues; however, it was not effective at staining bacteria in shoot tissues due to poor penetration. Microsc. Res. Tech. 77:566–573, 2014. © 2014 Wiley Periodicals, Inc.     

The use of silver nitrate as a stain for scanning electron microscopy of arterial intima and paraffin sections of kidney

The suitability of silver nitrate as a stain for scanning electron microscopy was investigated. Accordingly en face preparations of arterial intima were impregnated with silver nitrate, fixed with formalin, and coated with platinum-palladium alloy. In SEM images, the silver lines surrounding the endothelial cells, and the deposits on the intima appear as white lines or dots on a darker background. Similar results were noted for nitrocellulose-embedded endothelium. Paraffin sections of kidney treated with silver nitrate (von Kossa's stain) were also examined. Deposits of the calcium salts of phosphates and carbonates (stained with silver nitrate) were easily differentiated from other tissue components. Results were compared with those obtained with the light microscope. The scanning electron microscopic examination provided a finer definition of the silver granules relative to the surrounding architecture. The limitations and advantages of these techniques and possible further applications of silver nitrate as a stain for scanning electron microscopy are discussed. The use of silver nitrate as a stain for some SEM preparations is recommended.     

支气管镜检查在不典型肺结核诊断中的价值

目的探讨支气管镜检查对不典型肺结核的诊断,尤其是支气管镜下多项指标联合检测对不典型肺结核诊断的价值。方法对112例行常规痰涂片抗酸染色阴性但不能排外结核的患者,行电子支气管镜下肺泡灌洗液抗酸染色检查、支气管刷片抗酸染色检查、支气管镜下活检,了解不同检测方法的阳性检出率。结果 36例患者行活检16例病理确诊为肺结核,阳性率为44.4%,行肺泡灌洗液抗酸染色、支气管刷检抗酸染色检查阳性率分别为21.4%、25.0%,3种方法联合检测阳性率为44.6%。结论行支气管镜下灌洗、刷检、活检对不典型肺结核诊断具有一定价值,多项指标联合检测可提高肺结核的诊断率。     

Micro-structural tissue analysis for automatic histopathological image annotation

This article presents a new approach for extracting high level semantic concepts from digital histopathological images. This strategy provides not only annotation of several biological concepts, but also a coarse location of these concepts. The proposed approach is composed of five main steps: (1) a stain decomposition stage, which separates the contribution of hematoxylin and eosin dyes, (2) a color standardization that corrects color image differences, (3) a part-based representation, which describes the image in terms of the conditional probability of relevant local patches, selected by their stain contributions, (4) a discriminative classification model, which bridges out the found patterns and the biological concepts, (5) a block-based annotation strategy that identifies the multiple biological concepts within an image. A set of 655 skin images, containing 10 biological concepts of skin tissues were used for assessing the proposed approach, obtaining a sensitivity of 84% and a specificity of 67% when annotating images with multiple concepts.     

Quantitative method for estimating stain density in electron microscopy of conventionally prepared biological specimens

Stain density is an important parameter for optimising the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including serial block-face electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). Here, we show how some straightforward measurements in the TEM can be used to determine the stain density based on a simple expression that we derive. Numbers of stain atoms per unit volume are determined from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. The method is tested on specimens of embedded blood platelets, brain tissue and liver tissue.     

Microwave-accelerated cytochemical stains for the image analysis and the electron microscopic examination of light microscopy diagnostic slides

Recent studies in our laboratories have shown how microwave (MW) irradiation can accelerate a number of tissue-processing techniques, especially staining, to aid in the preparation of single specimens on glass microscope slides or coverslips for examination by light microscopy (and electron microscopy, if required) for diagnostic purposes. Techniques have been developed, which give permanently stained preparations, that can be studied initially by light microscopy, their areas of interest mapped, and computer-automated image analysis performed to obtain quantitative information. This is readily performed after MW-accelerated staining with silver methenamine by the Giammara-Hanker PATS or PATS-TS reaction. This variation of the PAS reaction gives excellent markers for specific infectious agents such as lipopolysaccharides for gram-negative bacteria or mannans for fungi. It is also an excellent stain for glycogen and basement membranes and an excellent marker for type III collagen or reticulin in the endoneurium or perineurium of peripheral nerve or in the capillary walls. Our improved MW-accelerated Feulgen reaction with silver methenamine for nuclear DNA is useful to show the nuclei of bacteria and fungi as well as of cells they are infecting. Improved coating and penetration of tissue surfaces by thiocarbohydrazide bridging of ruthenium red, applied under MW-acceleration, render biologic specimens sufficiently conductive for SEM so that sputter coating with gold is unnecessary. The specimens treated with these highly visible electron-opaque stains can be screened with the light microscope after mounting in polyethylene glycol(PEG) and the structures or areas selected for EM study are mapped with a Micro-Locator? slide. After removal of the water soluble PEG the specimens are remounted in the usual EM media for scanning electron microscopy (SEM) or transmission electron microscopy (TEM) study of the mapped areas. By comparing duplicate smears from areas of infection, such as two coverslips of buffy coat smears of blood from a patient with septicemia, the microorganisms responsible can occasionally be classified for antimicrobial therapy long before culture results are available; gram-negative bacteria are positive with the Giammara-Hanker PATS-TS stain, and gram-positive bacteria are positive with the SIGMA HT40 Gram stain. The gram-positive as well as gram-negative bacteria are both initially stained by the crystal violet component of the Gram stain. The crystal violet stain is readily removed from the gram-negative (but not the gram-positive) bacteria when the specimens are rinsed with alcohol/acetone. If this rinse step is omitted, the crystal violet remains attached to both gram-negative and gram-positive bacteria. It can then be rendered insoluble, electron-opaque, and conductive by treatment with silver methenamine solution under MW-irradiation. This metallized crystal violet is a more effective silver stain than the PATS-TS stain for a number of gram-negative spirochetes such as Treponema pallidurn, the microbe that causes syphilis     

电测法在雷达天线有限元建模中的应用

为了正确地建立雷达天线有限元模型,得出雷达天线的最大应力和应变,防止材料失效,提出了雷达天线的建模原理.给出了雷达天线的测试理论基础,并分析了电测系统原理.介绍了直角应变花的测试过程和计算原理,得出雷达天线测试位置点的应力和应变.计算分析实例有效地将电测法应用在雷达天线有限元建模中.     

Polychromatic Giemsa staining for Epon semi-thin sections

A polychromatic staining procedure which uses the Giemsa stain and differentiates between several cell and tissue structures with distinct colours has been developed for semi-thin sections of glutaraldehyde-fixed, Epon-embedded animal and plant tissues. The method does not require removal of the plastic and can be applied in a simple and rapid way for routine staining of semi-thin sections with good results.     

基于DSP的光纤法珀应变仪数据采集技术研究

光纤法珀应变传感是20世纪80年代末发展起来的一种新型应变测量技术。相应的应变测量仪逐渐成为国内外研究的热点。针对现有的应变测量仪，提出了基于DSP的一体化光纤法珀应变仪的思路，对VC33与应变测量仪之间的数据采集进行了探讨，重点介绍了VC33与应变测量仪之间数据采集的硬件和软件设计，并做了采集验证实验。实验结果与理论上传感器的输出结果基本一致，验证了该采集技术的可行性，为后面的数据处理及实现一体化打下了良好的基础。     

Pollen viability of <i>Polygala paniculata</i> L. (Polygalaceae) using different staining methods

Polygala paniculata L. is a medicinal plant that grows in the Brazilian Atlantic coast, known as ‘barba-de-São-João’, ‘barba-de-bode’, ‘vassourinha branca’, and ‘mimosa’. In this study, pollen viability was estimated by three different staining methods: 2% acetic orcein, 2% acetic carmine, and Alexander’s stain. The young inflorescences of twenty accessions were collected and fixed in a solution of ethanol: acetic acid (3:1) for 24 hours, then stored in ethanol 70% under refrigeration. Six slides per plant, two for each stain, were prepared by squashing, and 300 pollen grains per slide were analyzed. Pollen viability was high (>70%) for most accessions of P. paniculata using the Alexander’s stain, which proved the most adequate method to estimate pollen viability.     

Three-dimensional molecular shape determination from a limited number of projections

A real space method allowing the reconstruction of negatively stained crystalline objects from a limited number of projections is presented. The method is based on the assumption that only two density levels are required to describe an ideally negatively stained object (that of the volume occupied by the object and that of the volume occupied by the stain). The method is illustrated by the reconstruction of the asymmetric unit of catalase microcrystals using only the three principal projections. It is shown that other projections can then be predicted to a good approximation.