Combined quick freezing,freeze-drying,and embedding tissue at low temperature and in low viscosity resins

The techniques of quick freezing and freeze-drying provide an alternative to the more classical methodologies of chemical fixation and dehydration with organic solvents. It is possible to embed freeze-dried tissue in low viscosity resins, either at room temperature or at subzero temperatures in Spurr's resin or Lowicryl K4M, respectively. The choice of embedding medium affords additional flexibility in postdrying and embedding conditions, since Spurr's resin allows vapor fixation with osmium tetroxide and thermal polymerization. Osmium tetroxide is not recommended for Lowicryl resins, but these media permit polymerization at subzero temperatures with ultraviolet light. Both resins have unique advantages that may be utilized, depending upon the purpose of the embedding. In this paper, we discuss the details of preparing smooth muscle, from rabbit renal artery, by quick freezing and freeze-drying, as well as methods for the embedding of the freeze-dried tissue in both Spurr's resin and Lowicryl K4M. Although we have previously reported the ultrastructure of smooth muscle embedded in Spurr's low viscosity resin, the combination of freeze-drying and infiltration in Lowicryl K4M represents a new approach that allows the elimination of chemical fixation, dehydration with organic solvents, and heat polymerization of the embedding medium.     

Electron-microscope preparation techniques applied to the light microscopy of the cambium and its derivatives in Pinus radiataD. Don.

Epon 812 was used successfully as an embedding medium for the preparation of the secondary meristem and its derivatives in Pinus radiata for light microscopy. Specimens were fixed in glutaraldehyde followed by osmium tetroxide solution, using a schedule developed for the electron microscopy of these tissues. Sections from 2 to 5 μm thick were cut using an LKB Ultratome III and glass knives. Sections were evaluated without prior removal of the embedding medium using bright-field, phase-contrast and interference-contrast microscopy. Results for cambium were generally superior to those obtained by using the conventional fixatives for light microscopy and paraffin embedding. The advantages and disadvantages of this technique are discussed and it is concluded that the use of Epon for embedding this type of material is easier and gives better results than older established methods. It is suggested that methods of electron microscopy be considered when the preparation of a material for light microscopy has proved difficult or impossible by other means, or when particularly thin sections are required.     

Oleic acid‐added embedding medium for histological analysis of hard tissue

For the histological analysis of hard tissue such as bone, various acrylate‐based materials have been used as an embedding medium. However, commercial embedding media are expensive, and cutting the embedded block takes a long time. In this study, mixtures of methyl methacrylate (MMA), di‐butyl‐phthalate (DBP), and oleic acid (OA) were tested for possible application as an embedding medium for large and small undecalcified bone specimens. Mechanical properties were tested in a compressive mode. We investigated the change of hydrophilicity in the sectioned surface by measuring the contact angle depending on the OA. Crystallinity was analyzed using a X‐ray diffractometer (XRD). Surface analysis was performed using a confocal laser scanning microscope. To determine the staining efficiency of staining dyes, hamatoxylin‐eosin (H&E) and Masson's trichrome (MT) staining methods were performed for the histological analysis of bone‐implant complex. We confirmed that the investigated embedding media showed good properties such as optimal mechanical strength appropriate for cutting the embedded block and proper staining efficiency for histological analysis. Therefore, the MMA/DBP/OA mixtures can be used as an embedding media appropriate for various hard tissues and bone‐implant complex. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.     

Comparison of polyethylene glycol and diethylene glycol distearate embedding methods for the preservation of fungal cytoskeletons

Hyphae of the fungus Basidiobolus magnus were embedded in extractable polyethylene glycol (PEG) or diethylene glycol distearate (DGD) to prepare embedment-free sections in order to seek components of the cytoskeleton that may be obscured in epoxy-embedded sections. All methods showed that hyphae possess an intricate filamentous matrix, which is apparently unoriented. However, the appearance of the cytoskeleton depended on the embedding medium, the solvent used during embedding, and whether cells were fixed by conventional fixation or freeze-substitution. PEG proved to be the best embedding medium for fungal cells because DGD caused cell shrinkage and produced a more lamellar than filamentous matrix. Also, the cytoskeletal filaments were clearer in freeze-substituted cells than in conventionally-fixed cells. Since we failed to find microtubules in the embedment-free sections, we re-embedded cells in Epon to discern whether microtubules or other cytoplasmic components had changed. Neither PEG nor DGD adequately preserved microtubules as compared to regular Epon-embedded sections, and other cellular structures were significantly altered. Therefore, alternative methods need to be employed to further characterize fungal cytoskeletons.     

Methods for electron microscopy of the lamellated osmiophilic bodies of the lung

A study has been made of methods of fixing, staining, and embedding for electron microscopy the lamellated osmiophilic bodies (LOPBs) of the type II cells of the lung, which are probably connected with the lung surfactant. The preferred method for fixing and staining uses successively 2% glutaraldehyde, 1% osmium tetroxide, and a mixture of 10 vol M/40 lead nitrate with 10.5 vol M/60 potassium ferricyanide: all reagents are in cacodylate buffer. Unless the ferri-cyanide is in excess, the sections show blotchy precipitation. The bodies are damaged by ethanol and 1:2 epoxypropane. Dioxan or (with a quick time schedule) acetone may be used as a dehydrant and thinner prior to Araldite embedding. Durcupan water-soluble resin may also be used for dehydration and embedding; the preferred embedding mixture is: 5 ml resin A, 11.7 ml hardener B, 1.5 ml hardener C, and 0.4 ml plasticizer D, cured for 24–48 h at 60°C. Examination of LOPBs on a goniometer stage shows that they are composed of layers spaced at 4 nm, each lamella containing several such layers. Further evidence is adduced linking the LOPBs with the surfactant, and the application of the new methods to various problems involving the LOPBs is discussed.     

Image tamper detection and recovery using adaptive embedding rules

Many image tamper detection methods implement authentication and recovery in units of blocks; however, these methods do not consider the characteristics of blocks to distinguish watermark embedding and detection modes, thus resulting in poor hiding effects. We suggest that embedding an excessive number of bits within a region with only a slight change in an image to record recovery information is unnecessary. In addition, more recovery information is required in a region with major changes to improve recovery quality. Therefore, this study used smoothness to distinguish the types of image blocks, and employ different watermark embedding, tamper detection, and recovery strategies for different block types to enhance hiding efficiency, authentication, and recovery effects. The experimental results regarding the authentication error rate and image quality showed that the proposed scheme has satisfactory performance.     

无线网络化微传感器及其嵌入式系统

首先对无线传感网络作了分类,描述了无线网络化微传感器的体系结构,提出了两种蓝牙无线网络化传感器的体系结构,其主要特征是蓝牙、RF-MEMS天线及嵌入式系统.然后,讨论了无线网络化微传感器的技术要求与关键技术,包括自适应跳频与自配置蓝牙组网等新思想;研究重点在于无线通信的稳定性与可靠性、无线组网方案及实时互联网连接.为实现环境监测并通过无线网络通信传递测量值,可设计无线网络化微传感器嵌入式系统;选用了适当型号的嵌入式处理器和uClinux操作系统,设计了系统软件、低层驱动程序、调度程序及蓝牙高层协议程序.最后,以无线远程图像监视系统作为实例加以说明.     

Preparation of hard particle powders for examination in the transmission electron microscope

A new technique for preparing electron-transparent specimens of hard particle powders is described. It consists of mechanically embedding the powder into a thin foil of soft metal followed by ion-beam etching. This has numerous advantages over other methods of examining powders, such as their direct examination when supported by a carbon film, or by using epoxy resin as an embedding medium. The metal foil can be chosen to minimize overlap of the spectrographic peaks of the metal and the hard particles.     

Freeze substitution followed by low melting point wax embedding preserves histomorphology and allows protein and mRNA localization techniques

Fixation and embedding are major steps in tissue preservation for histological analysis. However, conventional fixatives like aldehyde‐based solutions usually mask tissular epitopes preventing their immunolocalization. Alternative fixation methods used to avoid this drawback, such as cryopreservation, alcohol‐ or zinc salts‐based fixatives do not efficiently preserve tissue and cell morphology. Likewise, paraffin and resin embedding, commonly used for thin sectioning, frequently damage epitopes due to the clearing agents and high temperatures needed along the embedding procedure. Alternatives like cryosectioning avoid the embedding steps but yield sections of poorer quality and are not suitable for all kinds of samples. To overcome these handicaps, we have developed a method that preserves histoarchitecture as well as tissue antigenic properties. This method, which we have named CryoWax, involves freeze substitution of the samples in isopentane and methanol, followed by embedding in low melting point polyester wax. CryoWax has proven efficient in obtaining thin sections of embryos and adult tissues from different species, including amphioxus, zebrafish, and mouse. CryoWax sections displayed optimal preservation of tissue morphology and were successfully immunostained for fixation‐ and temperature‐sensitive antigens. Furthermore, CryoWax has been tested for in situ hybridization application, obtaining positive results. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Quantitative studies on the preservation of choline and ethanolamine phosphatides during tissue preparation for electron microscopy. II. Other preparative methods

The loss of ¹⁴C-ethanolamine- and ³H-choline-labelled phospholipids from rat liver during preparation for electron microscopy by some less frequently used processing methods has been examined. Permanganate and formaldehyde-potassium dichromate fixation followed by Araldite embedding were investigated and five procedures involving embedding in water-miscible methacrylates (GMA). These procedures included a conventional method of dehydration and embedding in GMA, a low-temperature GMA embedding method, dehydration with ethylene glycol, freeze drying and freeze substitution. These results are compared with those obtained after conventional tissue preparation (presented previously, Cope & Williams, 1969). Formaldehyde-potassium dichromate compared favourably with the conventional procedures for the preservation of both phosphatides, especially phosphatidyl ethanolamine. Permanganate fixation was much less effective. Severe loss of both phosphatides occured after freeze drying and freeze substitution in glutaraldehyde-alcohol. GMA is shown to be a more potent phospholipid solvent than ethanol under the conditions employed. Low-temperature embedding reduced the loss of phosphatidyl choline during embedding. Results obtained by scintillation counting were confirmed by grain counts on thick-section autoradiographs. No direct relationship between extraction and the electron-microscopic appearance of membranes was discernible. It is believed that membrane prominence is largely dependent upon the electron density of the surrounding cytoplasm rather than on the degree of phospholipid extraction.     

Cryoultramicrotomy versus plastic embedding: comparative immunocytochemistry of rat anterior pituitary cells

The anterior pituitary of the rat is used as a model for the study of the effects of freezing or plastic embedding on the maintenance of antigenicity. Rat anterior pituitaries are fixed in 2.5% glutaraldehyde in 0.1 m phosphate buffer pH 7.4. Some of the blocks are post-fixed before being divided into two lots. One batch is frozen, while the other is dehydrated and embedded. The indirect antibody enzyme method is applied to ultrathin sections obtained by cryoultramicrotomy after freezing or by sectioning after embedding. All six pituitary hormones are detected by both methods. Comparison shows that the morphological characteristics are identical for both techniques, though ultrastructural preservation is better after embedding. Immunoreactivity is found in secretory granules and sometimes in the endoplasmic reticulum. Osmium postfixation may reduce or even abolish antigenicity in plastic-embedded tissue. After cryoultramicrotomy, however, even after osmium fixation, antibody may be used 1000 times more diluted than after plastic embedding. Embedding preserves ultrastructure and limited antigenicity while the use of cryoultramicrotomy is a far more sensitive technique.     

Heptane and isooctane as embedding fluids for high-pressure freezing of Petunia ovules followed by freeze-substitution

In comparison with other fixation methods, high-pressure freezing and freeze-substitution of Petunia ovules lead to improved ultrastructural preservation of all tissues. Crucial for adequate high-pressure freezing is the absence of air in the specimen sandwich; air has to be replaced by an embedding fluid. Frequently, 1-hexadecene is used for this purpose. Using 1-hexadecene as an embedding fluid resulted in only 5–10% of Petunia ovules being preserved without disturbance of the ultrastructure due to ice-crystal damage. Since 1-hexadecene is not soluble in acetone at − 90 °C, freeze-substitution is hindered when ovules remained completely surrounded by it; this results in recrystallization when the temperature is raised. We tested and compared the suitability of heptane and isooctane as embedding fluids for high-pressure freezing and freeze-substitution, reasoning that because of their low melting points and low relative densities, phase separation during freeze-substitution would result in complete exposure of the ovules to the substitution medium, leading to adequate freeze-substitution. Using either heptane or isooctane as an embedding fluid yielded up to 90% ice-crystal-free ovules. Both compounds, however, have some damaging effects on the outer one or two cell layers of the ovule, but not on the inner tissues.     

High pressure hermetic compression seals for embedding elastomeric membrane microvalves in polymer microfluidic devices

The application of elastomeric membrane microvalves with polymeric microfluidic devices is currently impeded by bonding methods which limit operating pressures. A novel compression sealing approach is presented for embedding elastomeric membranes between polymeric microchannel laminae through the use of sealing bosses. Sealing bosses are used effectively to concentrate clamping forces producing compression seals with higher operating pressures. The technique is capable of integrating elastomeric membrane microvalves within a wider variety of materials. Further, compression seals allow devices to be disassembled allowing for cleaning and analysis. Finite element methods are used to investigate the effects of sealing boss size and location on valve deformation as a function of clamping pressure. Experimental results are in good agreement with the model and show that the device can be configured to withstand operating pressure beyond 689 kPa.     

Low Temperature Biological Microscopy

We have tested a wide range of acrylate and methacrylate resin formulations and have concluded that embedding resins can be developed which are usable within a broad range of environmental conditions. To demonstrate this versatility, we have designed two highly-cross-linked resins, one polar and the other nonpolar, that are usable to temperatures from 243 to 223 K. Both of these resins formulations, which are now commercially available, show that systematic experiments can be easily done to study the effects of environmental parameters, such as water content, temperature, or resin polarity, on biological material during embedding. Using these resins and aldehyde-fixed protein crystals, it can be shown that low temperature minimizes the loss of molecular structure to an extent that is not often obtainable with conventional methods of dehydration and embedding. Embedded crystals of aspartate aminotransferase still retained molecular order to 0·6 nm. Embedded crystals of catalase show X-ray diffraction maxima out to 0·8 nm. When sectioned, catalase revealed stain-limited electron and optical diffraction patterns to 2·5 nm. Nevertheless, our work clearly demonstrates that low temperature embedding procedures are superior and that versatile, general purpose resins can be designed to take advantage of this fact.     

Multiple manifolds analysis and its application to fault diagnosis

A novel approach to fault diagnosis is proposed using multiple manifolds analysis (MMA) to extract manifold information from the vibration signals collected from a mechanical system. The basic idea of MMA is to reconstruct a manifold by embedding time series into a high-dimensional phase space. The tangent direction of the neighborhood for each point is then used to approximate its local geometry. The variation of the multiple manifolds representing different states of the mechanical system can be revealed by performing multi-way principal component analysis. The vibration signals acquired from roller bearings are employed to validate the proposed algorithms. Test results show that the proposed MMA-based approach can interpret different machine conditions and is effective to the fault diagnosis, and the MMA-based fault clustering and trend analysis algorithms have outperformed the conventional fault diagnosis methods.     

Low temperature embedding with Lowicryl resins: two new formulations and some applications

Lowicryl K4M and HM20 are methacrylate/acrylate based low temperature embedding resins for biological material which can be used in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze-substitution. K4M and HM20 are applicable over a very extended temperature range, approximately 220 K to 340 K. With two new resins, K11M and HM23, one can reach even lower temperatures, c. 200 K. Freeze-substitution combined with low temperature embedding allows for very mild or no chemical fixation which seems to increase the sensitivity of immunocytochemical localization of antigens on sections.     

基于小波及非线性预测的轴承故障诊断方法

在非线性时间序列预测研究的基础上,提出非线性预测效果的特征提取方法.首先对采集到的足够长轴承数据采用小波变换进行消噪处理及边界延拓,使其满足预测需要的无限长、无噪声的条件,这样延迟时间取任意值均能重构原系统相空间;然后采用基于可预测性的选取嵌入维数的方法确定轴承各种状态信号的嵌入维数,进行相空间重构.应用实验结果表明:该方法提取的特征值能明显地区分轴承各种状态信号,且对数据分段长度的稳定性好,可以作为识别轴承故障的一种新途径.     

基于局部约束字典学习的数据降维和重构方法

针对目前已有的非线性降维算法存在计算复杂度高、难以处理大型数据集和增量化降维问题,本文提出了一种基于局部约束字典学习的非线性降维算法。该方法通过重构一些潜在标志点的局部内在流形,并在数据处理过程中将训练数据和未知数据一起嵌入到内在流形中,使得数据的内在几何结构特征得以保持。与已有非线性降维方法相比,该算法具有计算复杂度低、存储空间小和通用性强的特点,可以很好地解决增量化降维问题,易于处理大型数据集。另外,该算法也可以解决高维数据的重构问题,与已有重构方法相比具有计算简单、重构误差较低的特点。实验结果表明了算法的有效性。     

Low temperature techniques applied for CTEM and STEM analysis of cellular components at a molecular level

One of the most important problems in tissue preparation for electron microscopic analysis at a molecular level involves the preservation of the tissue without introducing extensive denaturation of the proteins. Low temperature is a most efficient condition for the inhibition of protein denaturation and freeze-drying offers favourable conditions for transferring proteins to a dry state with minimal denaturation of the proteins. However, the embedding of the dried tissue in a plastic leads to extensive denaturation of the proteins when performed in the conventional way. This eliminates very efficiently the advantages of the method. The situation becomes even worse when subjecting the tissue to freeze-substitution. To eliminate as far as possible the denaturing effect of plastic embedding, freeze-drying can be combined with low temperature embedding in a plastic. Freeze-fracturing allows a most efficient use of low temperature to reduce conformation changes in proteins. The value of the freeze-fracturing technique depends entirely on a precise knowledge of the location of the fracture planes. Since this location is not known, it must be determined on the basis of a deduction. If this deduction is wrong, the method becomes misleading. Two methods which allow a certain testing of the correctness of the deduced location of the fracture planes are mentioned.     

Preparation of tough,fibrous material for examination in the scanning electron microscope

A simple method has been devised which minimizes distortion of fibrous material during the cutting needed for examination of cross-sections in the SEM. Camphene, suggested for use in ‘freeze-drying’ at room temperature (Watters & Buck, 1971) is used as an embedding material with a surrounding layer of ice for reinforcement and sectioning can then be carried out. The technique has been successfully applied to hides and skins in several stages of processing to leather, i.e. in both wet and dry initial states.