bluuebunny/crossref_metadata_2025 huggingface dataset:
Current basis for the ultrastructural clinical diagnosis of tumors: A review
Authors: Russo José , Tait Larry , Russo Irma H.
Published: February 2005

The ultrastructural diagnosis of tumors requires a careful analysis that should be done in an orderly fashion. It requires precise planning from the time of specimen collection to the selection of the area to be examined. Pictures must be taken systematically and every micrograph should allow to answer whether the number of cells photographed is adequate; whether mitoses are present, what is the pattern of the tumor; what is the appearance of the cell membrane; whether the cells are joined by junctional complexes; whether free surfaces possess microvilli or cilia; what organelles are present and how they are distributed; whether there are secretory granules, melanosomes, or other cytoplasmic elements. Nuclear and nucleolar size and shape have to be taken into consideration. The composition of the interstitial extracellular matrix is important in certain types of tumors. Although these questions are not the only ones to be addressed, their use in a logical fashion is helpful when it concerns the ultrastructural diagnosis.
Metal evaporation shadowing: A computer simulation
Authors: Colquhoun William , Sokol Roger , Davison Edwin , Cassimeris Lynne
Published: February 2005

This paper addresses the question of what is the very best deposition that can be achieved by metal evaporation shadowing. It deals, therefore, with the resolution attainable in metal shadowed specimens. The analysis disregards interatomic attractions (nucleation, crystallite formation, decoration) and generates depositions solely on the basis of probabilities of vacant, single, and multiple atom landings at different points on a substrate.

The treatment starts by showing that random number generation can be used to simulate reasonably the evaporation of metal atoms from a shadowing source and their deposit on a substrate. A Fortran program tabulates and displays these simulated atom landings. The analysis shows that one can never expect to obtain a perfectly uniform deposit; some areas will always be heavily filled while others will be lightly filled or vacant. Resolution is thereby limited. The analysis shows how a deposit becomes more uniform as the number of atoms deposited increases. It predicts optimal amounts of deposition for various shadowing angles. The analysis also provides a rationale for the way in which deposition grain coarsens and resolution worsens as the angle of shadow is made more steep.

Computer‐generated “ideal” depositions are compared with real shadow grains achieved under equivalent experimental conditions. The simulations are also compared with deposits achieved by novel shadowing methods. Ways to improved metal evaporation technique are suggested by the analysis and these are discussed.
Measurement of electron probe beam diameter by digital image processing
Authors: Oho Eisaku , Sasaki Toshihide , Adachi Koichi , Kanaya Koichi
Published: February 2005

The present report illustrates a computerized method for precise measurement of the diameter of an electron beam. The value of this measurement extends beyond simply providing an accurate estimate of resolution. Other salient areas which will benefit include quantitative X‐ray microanalysis, energy loss spectroscopy, diffraction studies, and electron beam lithography. The biological sciences as well as the material sciences will gain enormously from improved accuracy in measurement (control) of beam diameter. It is anticipated that most or all of the mathematical manipulations outlined in this paper will be incorporated into digital electronic packages which will perform the functions automatically for setting the electron beam diameter to the scientist's choice. The purpose of the present report is to indicate some of the principles involved so that as electron microscopy becomes more computerized and automated, the user will have some understanding of what the electronics are doing rather than simply depressing a button or two and ignoring the power of what resides within the walls of the instrument.

The performance of a scanning electron microscope (SEM) and a scanning transmission electron microscope (STEM) is roughly determined by the incident electron probe beam size (diameter) involving a sufficient electron current. In the present paper, the diameter of an ultrafine electron beam is measured indirectly from the information given by the blurring of an edge in a STEM or a SEM image of a crystalline specimen with fine, sharp edges. The obtained data were processed by digital image processing methods which give an accurate value of the beam diameter. For confirming the validity of this method, a suitable simulation based on the convolution theorem was performed. By using this measurement, we could measure the diameter of an ultrafine electron beam down to 2 nm, which could not be measured easily by previous techniques.
Transmission electron microscopic analysis of kidney glomeruli obtained by fine‐needle aspiration
Authors: Rowden Geoffrey , Dean Shirley , Campbell James , Belitsky Philip
Published: February 2005

Aspiration biopsy of tumors and transplanted organs is a popular atraumatic method for diagnosis and also for the management of patients. Apart from the cytological analyses, it is clear that large aggregates of cells are obtained and these may be examined by electron microscopy. A study was carried out with dogs to assess the yield of intact glomeruli and tubules, resulting from a standard fine‐needle aspiration procedure. Employing a 20‐gauge needle, yields of approximately ten intact glomeruli were obtained with a tissue processing protocol that utilized filtration through a nylon mesh and backwashing, to resuspend the trapped aggregates. Processing for electron microscopy was carried out in microcentrifuge tubes. The ultrastructural preservation was excellent and the extension of these studies to investigations of not only renal transplantation, but also for diagnosis in human renal diseases of a broad spectrum, is suggested.
CTEM versus STEM versus HVEM: Evaluation of the three‐dimensional morphology of arsenic inclusions
Authors: Ramirez‐Mitchell R. , Sorensen E. M. B.
Published: February 2005

Electron‐dense arsenic inclusions appeared in the nucleus of parenchymal hepatocytes from fish exposed to arsenate, but were absent in fish exposed under identical conditions to solutions lacking arsenic. Images of these inclusions were compared using conventional transmission electron microscopy (CTEM), scanning transmission electron microscopy (STEM), and high‐voltage electron microscopy (HVEM). Stereo pairs from the same individual inclusions were examined using each of these methods to provide a more complete understanding of their three‐dimensional organization and to evaluate the relative merits of each technique in the study of similar electron‐dense structures.

Comparable results were obtained with the three types of instrumentation. Although HVEM is the technique of choice for the analysis of three‐dimensional images of such high electron‐dense structures, STEM proved to be a good alternate technique for the selection and general evaluation of samples in preparation for HVEM.
Ultrastructural morphometric image analysis applied to estimation of condensed chromatin in normal and neoplastic lymphocytes
Authors: Dardick Irving , Dardick Alexander M. , Jeans M. T. Diane , Rippstein Peter , Caldwell Douglas R.
Published: February 2005

Currently, quantitative studies of malignant lymphoma are being performed in an attempt to improve the classification of non‐Hodgkin's lymphoma (NHL) for diagnostic, prognostic, and therapeutic purposes. Morphometric image analysis is one method that can be employed in cases of NHL to obtain objective data of nuclear parameters; condensed chromatin being a compartment of the nucleus best measured at the ultrastructural level. This report assesses similarities or differences in the amount, distribution, and arrangement of condensed chromatin in nuclear profiles of normal and neoplastic lymphocytes in human surgical biopsy specimens. Morphometric data derived from electron micrographs of lymphocytes in germinal centers of lymph nodes with reactive hyperplasia (three cases) and small cell types of NHL two examples of malignant lymphoma, well differentiated lymphocytic type (ML, WDL) and three cases of malignant lymphoma, poorly differentiated lymphocytic type (ML, PDL) are compared. Results indicate that the distribution of condensed chromatin, i.e., the size of aggregates, and their spatial placement within the nucleus varies more than the amount (both mean area per profile or mean volume) of this nuclear parameter, and that this applies to normal as well as neoplastic lymphocytes. When a series of condensed chromatin parameters were statistically compared, no major differences could be detected between lymphocytes in normal tissues and those in ML, WDL and ML, PDL, but considerable differences were found in each of the nuclear morphotypes in the individual cases within the groups. This degree of variation in nuclear characteristics within normal tissues and the two lymphoma categories has not been previously recognized. Clearly, the technique of morphometric analysis, as applied to electron micrographs, can provide new and useful data that must be appreciated if classification schemes currently used in NHL are to improve and reflect biologic considerations.
A FORTRAN computer program for converting among atom, weight, and oxide percentages for use in analytical electron microscopy
Authors: Bigelow W. C.
Published: February 2005

A FORTRAN program is given which will (1) calculate atom and weight percentages from a given chemical formula: (2) calculate atom percentages from given weight percentages; (3) calculate oxide percentages from given weight percentages; and (4) calculate weight percentages from given oxide percentages. The program is interactive, easy to use, and very convenient for use in electron beam microanalytical work.
Electron diffraction phenomena observed with a high resolution STEM instrument
Authors: Cowley J. M.
Published: February 2005

Because of the high brightness of the cold field emission source used in a dedicated scanning transmission electron microscopy (STEM) instrument, it is possible to focus electrons to a cross‐over of width 3Å or less and, with a suitable detection system, to obtain diffraction patterns from specimen regions of this size or greater. Coherent interference effects are visible in shadow images (in‐line holograms) and in convergent beam diffraction patterns. Special techniques have been developed for gathering information from the diffraction patterns for application to the study of the structures of crystal defects, crystal surfaces and small particles. Possibilities have been explored for holographic reconstruction from shadow images.
Modern electron microscopy for materials characterization
Authors: Thomas Gareth
Published: February 2005

To be successful, today's electron microscopist must have modern, well‐maintained facilities and be part of a team of experts with specific skills for research. Furthermore, in an ideal situation specific instruments should be dedicated to specific applications (high resolution, analytical, conventional, etc.), since in general the efficiency of a microscope varies inversely as its operations (and operators?). This is partly a reason for establishing national or regional electron microscopy centers, so that expertise and expensive equipment may be made available in central facilities.

In this brief summary an attempt is made to illustrate how sophisticated methods of electron microscopy can be applied to practical problems by choosing representative examples from research at Berkeley using high resolution, high voltage, diffraction, and analytical techniques. More detailed examples have been given in some recent reviews (Thomas, 1984a, and Gronsky et al., 1985).
Reminiscences on the early observations of defects in crystals by electron microscopy
Authors: Whelan M. J.
Published: February 2005

A personal account is given of the development which led to the observation of dislocations in thin crystals by transmission electron microscopy in the mid‐1950s.
Three‐dimensional reconstruction of microtubule‐containing structures from electron micrographs
Authors: Murray John M.
Published: February 2005

Microtubules and associated structures are among the more difficult samples studied by 3D reconstruction techniques because of their size, complexity, and lability. Nevertheless, their importance in many cellular functions often justifies efforts to acquire 3D information up to the resolution limit of negatively stained specimens or beyond. A combined approach which utilizes methods appropriate to both helical symmetry and 2D crystal lattices seems to provide the surest route to 3D reconstruction. Images of unstained specimens obtained by newer techniques of microscopy present challenging problems in data analysis, but potentially offer higher‐resolution information.
A method for determining dislocation Burgers' vectors in ice
Authors: Baker I.
Published: February 2005

It is shown that the usual method of Burgers' vector analysis in the Transmission Electron Microscope (TEM) using invisibility criteria is unlikely to succeed for ice. A method is outlined using a novel application of computer‐simulated imaging which can be used to perform such an analysis.
Use of mixed glycosidase for the removal of mucus from the rat nasal epithelium in SEM studies
Authors: Monteiro‐Riviere Nancy A. , Jiang Xue‐Zhi
Published: February 2005

The surface layer of mucus, which obscures the epithelium from view, is a major obstacle when performing scanning electron microscopic studies of the nasal mucosa. Samples treated with a 1% mixed glycosidase solution for 1 or 2 minutes, followed by agitation, removed most of the mucus from the conchae without damaging the underlying epithelium. Removal of mucus allows the complete evaluation of the underlying epithelium in normal animals and the localization and characterization of lesions in animals exposed to nasal toxicants.
Monolayer freeze‐fracture—a modified procedure
Authors: Edwards Harold H. , Mueller Thomas J. , Morrison Martin
Published: February 2005

Monolayer freeze‐fracture of biological membranes is a valuable tool for integrating membrane morphology with biochemical analysis of membrane components. This correlation has been restricted by the purity of the biochemical sample. In this article, the method is reviewed, and an improved method is described. The essential modification was the use of a polysaccharide‐coated microscope slide, instead of a copper plate, to cover cells attached to a polylysine‐coated coverslip. It was found that proper freeze‐fracture will not occur unless there is a distinct temperature gradient, with its accompanying stresses, across the cell monolayer during the freezing process. This gradient is achieved by using glass slides of different thickness to cover each side of the monolayer. Comparison of the results with those obtained when using a copper‐glass system demonstrated a consistently purer sample for the glass‐glass system, with whole‐cell contamination of the external membrane leaflet being reduced to 0.4%. Problems associated with obtaining pure samples for biochemical analysis are discussed, and the results of freeze‐fracture with the glass‐glass and glass‐copper systems are compared. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of polypeptides associated with the separate halves of the erythrocyte membrane demonstrated that band 3, the anion transport protein, separates with the cytoplasmic face, whereas only sialoglycoproteins and their fragments are retained in the external face. This finding, obtained with the glass‐glass system, is consistent with results of our earlier freeze‐fracture study that used a copper‐glass system which showed that covalent bonds may be broken during this procedure.
Wheat germ agglutinin fracture‐label of lymphocyte plasma and intracellular membranes
Authors: Torrisi Maria Rosaria , Pavan Antonio , Lotti Lavinia Vittoria , Frati Luigi
Published: February 2005

Conventional lectin cytochemistry or immunocytochemistry can be associated with freeze‐fracture in fracture‐label techniques: The thin section fracture‐label and the critical point drying fracture‐label. In the first method, which is particularly useful for investigating intracellular membranes, cells or tissues are freeze‐fractured, thawed, labeled, and embedded in resin. Wheat germ agglutinin labeling on freeze‐fractured human lymphocytes confirms the existence of two compartments of intracellular membranes, based on the sialoglycocomponent content.
Digital image processing for nonperiodic objects and an on‐line image processing system for high resolution electron microscopy
Authors: Baba Norio , Oho Eisaku , Mukai Makio , Kanaya Koichi
Published: February 2005

A digital image processing method for noise removal and image enhancement in nonperiodic structural images is described. The method for noise removal uses a reversible transform between an image and image autocorrelation function. The Laplacian filter is then employed for image enhancement. Furthermore, an on‐line image processing system for highresolution TEM is presented.
Detection and identification of viruses by electron microscopy
Authors: Miller Sara E.
Published: February 2005

Electron microscopy can aid in the rapid diagnosis of viral diseases, as it can be performed in a matter of hours, but on a routine basis it should be used in conjunction with other techniques. Initially, the specimen source and patient symptoms should be ascertained, as these will lend suggestions of possible agents while eliminating others; however, this information should not be allowed to prejudice observation in such a way as to cause oversight of an unlikely pathogen. Second, selection of the method of preparation should be based on sample consistency; extraction, debris clarification, concentration, tissue culture amplification, or embedment may be necessary. Finally, false‐positive results must be avoided by differentiating viruses from cell organelles or debris, mycoplasmal or bacterial contamination, and bacteriophages.
Freezing and drying of biological tissues with a toggle‐link helium freezer and an improved freeze‐drying apparatus: Application to neuropeptide immunocytochemistry
Authors: Coulter H. David
Published: February 2005

A freezing apparatus has been developed for bringing blocks of tissue into contact with a block of sapphire chilled to 17°K. A toggle linkage minimizes rebound by slowing the rate of approach of the tissue to the cold surface to a velocity of zero. A glove box limits condensation on the surface of the sapphire, and a miniature moist chamber protects the specimen from drying and premature freezing. About 50 blocks of tissue can be frozen in an hour and a half by using 5 liters of liquid helium. The tissue is then frozendried at controlled temperature, fixed with OsO4 vapor, and infiltrated with epoxy resin in a simple bench‐top freeze‐drier without breaking vacuum. About two‐thirds of the blocks are useful for electron microscopy. Brain tissue frozen and dried by using these methods retains enough immunoreactivity for enkephalin in plastic sections to permit its detection with immunohistochemistry by using both the light microscope (with immunofluorescence) and the electron microscope (with colloidal gold).
Preparation of cross‐sectional TEM samples with observable thin sections at desired regions of non‐uniform surfaced semiconducting devices
Authors: Rai A. K. , Rashid M. H. , Pronko P. P. , Ezis A. , Langer D. W.
Published: February 2005

Transmission electron microscopy of cross‐sectional samples offers an attractive means to study process evaluation and failure analysis of many semiconducting devices. In the present work, a technique to prepare cross‐sectional TEM samples, containing thin sections of specifically desired regions within nonuniform surfaced semiconducting devices, is described.
Biological variation in cryo‐microanalytical data from mouse small intestinal villi
Authors: Carr K. E. , Hayes T. L. , Watt A. , Klein S. , McKoon M. , Bastacky S. J. , Ellis S.
Published: February 2005

Microanalysis data obtained using low temperature scanning electron microscopy of frozen hydrated etched mouse duodenum gives information about peak/background ratios of various elements in different cellular compartments. The present paper describes ways in which the data can be analyzed to minimize artifactual variations and to make clear where there are genuine differences in peak/background ratios when readings from different animals are compared. Using these methods, it is shown that in some areas of villous epithelium there are measurable differences in the ratios of sodium, calcium, and sulphur.
Transmission and scanning electron microscopic examination of rat vaginal epithelium in the normal estrous cycle and in persistent estrous
Authors: Khosho Francis K. , Kaufmann Robert C. , Amankwah Kofi S.
Published: February 2005

The rat vaginal epithelium in both normal and persistent estrous (PE) cycles was examined by transmission and scanning electron microscopy utilizing cardiac perfusion and ruthenium red staining. It was demonstrated that surface vaginal epithelium in persistent estrous exhibited irregularly shaped cells. Borders were not well defined as is the case in normal estrous, and cells lacked the complex, interconnecting microridges and pits. Vaginal surfaces were completely smooth or covered with few microvilli. In the control specimens the fine structure en block stained with ruthenium red showed distinct cell membranes bordering the intercellular spaces between epithelial cells. With PE rats, this finding was absent, and there was instead a significant expansion of intercellular space with villiform processes of the cytoplasm projecting into the space. Utilization of the combination of techniques described provided the investigators with a better definition of vaginal tissue ultrastructure and revealed significant morphological differences between the normal estrous cycle and persistent estrous in vaginal specimens from rat models.
Preparation of cross sections of thin‐coated sheet steel for analytical electron microscopy observation
Authors: Selverian John H. , Marder Arnold R. , Notis Michael R.
Published: February 2005

A method of preparing thinned cross sections of coated sheet steel (Galvalume) for observation in an analytical electron microscope (AEM) was developed. Steel panels dipped for 4 sec in baths of 55% by weight (wt pct) Al‐1.7 wt pct Si‐balance Zn, and 55 wt pct Al‐6.88 wt pct Si‐balance Zn at 610°C were examined. The ratio of the intensity of the K and L x‐ray lines from the same element was used in determining the foil thickness. The AEM results compared favorably to results obtained using electron microprobe analysis (EMPA).
Transmission and scanning electron microscopic examination of intracellular organelles in freeze‐substituted Kloeckera and Saccharomyces cerevisiae Yeast Cells
Authors: Baba Misuzu , Osumi Masako
Published: February 2005

The application of the conventional double‐fixation method (glutaraldehyde and osmium tetroxide) to whole yeast cells is difficult because the thick cell wall of the yeast prevents the penetration of osmium tetroxide. However, this problem was solved by using the freeze‐substitution fixation method. Therefore, it was possible to examine the intracellular structures of the yeast cells without digestion of the cell wall. In the present method, specimens for transmission electron microscopy and for scanning electron microscopy were prepared simultaneously. By scanning electron microscopic observation, three‐dimensional information about internal structures was obtained. In the cytological analysis of the yeasts, intracellular structures were well preserved by using the freeze‐substitution fixation method. On the outer leaflet of the nuclear envelope, many ribosomes were attached. The rough endoplasmic reticulum and Golgi apparatus were clearly seen in the yeast cytoplasm. The Golgi stack appeared to consist of smooth membranes, and small vesicles were present beside it. The details of other structures such as the nuclear division apparatus, actinlike filaments, and viruslike particles were also revealed. The present technique can be applied to most species of yeast cells. With this new information, the previous model of a yeast cell was modified.
Natural propane cryogen for frozen‐hydrated biological specimens
Authors: Ward Rob , Murray John M.
Published: February 2005

The properties of natural propane, mixed with 0–4% isopentane, as a cryogen suitable for rapid freezing of this layers of aqueous biological specimen suspensions are discussed. Although natural propane has rather variable properties, its freezing point can be depressed below the temperature of liquid nitrogen by adding a smaller amount of isopentane than is required for depressing the freezing point of pure propane.
Introduction of the protein G—gold complex for high‐resolution immunocytochemistry
Authors: Bendayan Moise
Published: February 2005

Protein G, an immunoglobulin G‐binding molecule, was tagged with colloidal gold particles and was applied in immunocytochemistry. The present study reports the conditions required for the preparation of the protein G–gold complex and for its application in electron microscopy. Once used in combination with specific primary antibodies, the protein G–gold complex was able to yield highly specific labelings over particular tissue compartments. Various control experiments confirmed the specificity of the labelings obtained. Since the IgG‐binding properties of protein G are superior to those displayed by protein A, the protein G–gold appears a far better tool for high‐resolution immunocytochemistry.
Construction of 35‐millimeter camera adaptors for scanning electron microscopes
Authors: Miller Marcia M. , Hardy John , Revel Jean Paul , Rusnak Miro
Published: February 2005

Described here is the construction of two 35‐mm camera adaptors for attachment to the recording monitors of scanning electron microscopes (SEM). The designs are simple and readily adaptable to almost any SEM. The choice of this camera format for recording SEM images is one of convenience as well as economy and does not sacrifice micrograph quality.
Correlation of some mechanical properties of embedding resins with their behaviour in microtomy
Authors: Acetarin Jean‐Dominique , Carlemalm Eric , Kellenberger Eduard , Villiger Werner
Published: February 2005

The technology of ultramicrotomy is now well established, but the properties of the resin that determine the different forces needed to generate a section have been neglected, although this process could introduce artefacts in the thin sections. We have investigated the principal resin dependent factors involved in the sectioning process and determined the related mechanical properties. Tensile experiments have given the best correlation with the sectioning quality of the resin: the elastic (Young's) modulus value (depending on polymer structure or hardening mode), the presence of a short plastic flow for a controlled fracture and enough flexibility to minimize shearing, and internal cracks, appear to be the main characteristic parameters. The ultrathin section seems to be generated by a process close to cleavage, favoured by the relative hardness of the embedding media, while machining and “true” sectioning requires softer resins.

Consequently, the rupture follows the path of least resistance in the specimen‐resin composite, providing sections with a surface relief. Embedded biological material copolymerizes with polycondensed matrix (epoxy resins), and, by reducing the heterogeneity, gives smoother sections. Embedments hardened by radical polymerization provide a rougher relief, since almost no copolymerization occurs, offering to the microtome a heterogeneous block with two constituents of very different mechanical properties. The surface relief seems to be an important factor in labelling, staining, and imaging, and more attention has to be paid for some improvements of the quality of the information provided by electron microscopy.
The study of 3‐dimensional structure at the node of ranvier and specimen contrast enhancement techniques
Authors: Ellisman Mark H. , Deerinck Thomas J. , Anderson Karen L.
Published: February 2005

In determining what to write about for this volume to honor Keith Porter, we thought about our projects and considered which ones Keith had either stimulated or otherwise influenced with his technological wizardry or intuition about cell structure and function. Perhaps, not surprisingly, we found something of Keith in everything we are working on. We therefore chose a topic that we presently are very interested in, and that we hope he will enjoy reading about and following in the years to come.
Dynamic aspects of the interaction between microtubules and actin filaments
Authors: Euteneuer Ursula , Cooper Mark C. , Pryzwansky Katherine B. , Schliwa Manfred
Published: February 2005

This paper summarizes observations on the motility and behavior of microtubule asters in polymorphonuclear leukocytes under a variety of experimental conditions. These observations suggest that the location and organization of microtubules is influenced, at least in part, by the activity of the cortical actin network. Aster motility may therefore serve as an indicator of the interaction between the centrosomal microtubules and the cell cortex, and it is speculated that this interaction is of importance for leukocyte translocation.
Three‐dimensional reconstructions from serial micrographs using the IBM PC
Authors: Young Stephen J. , Royer Suzanne M. , Groves Philip M. , Kinnamon John C.
Published: February 2005

We have prepared software for producing three‐dimensional reconstructions from serial micrographs using an IBM PC or compatible. The software can be configured for a variety of graphics board and digitizing tablet combinations.

Data is entered into the program by digitizing contours directly from micrographs. The program can handle up to 2,000 contours per data file, of up to 255 object types. Morphometric information such as line length, perimeter, and area are generated for each contour. The reconstruction program aligns the plane information from each section and displays the final reconstruction on a high resolution (640 × 400 pixels) color monitor. Object types can be differentiated by line width, line color, and fill color. Hidden line processing and conditional fill routines make it possible to produce reconstructions with either a solid or semi‐transparent appearance. Reconstructions can be generated quite rapidly from any viewing angle in the X, Y, and Z axes. The program has proven valuable for the elucidation of the three‐dimensional nature of biological structures.
Immunolocalization of Alpha‐Actinin in Adult Chicken Skeletal Muscles
Authors: Zimmer Danna B. , Goldstein Margaret A.
Published: February 2005

Immunoelectron microscopy techniques were used to localize alpha‐actinin within the Z lattice of adult skeletal muscles. Analysis of electron micrographs by direct visualization demonstrated that anti‐alpha‐actinin Fab fragments bound throughout the Z lattice. A low‐resolution scanning densitometry technique was developed to quantitate the visual increase in the density of the Z lattice. These techniques did not allow determination of the particular component of the Z lattice, amorphous matrix, axial filaments, or cross‐connecting filaments with which the antibody was associated. Therefore, additional techniques, including direct measurement of filament diameters and optical diffraction, were utilized in determining which components of the Z lattice bound anti‐alpha‐actinin Fab fragments. These analyses suggest that the antibody binding is distributed evenly throughout the lattice, along the filaments, and between them and is confined to the region of double overlap of the ends of the thin filaments.
Preparation of neural tissues and sensory structures for electron microscopy
Authors: Song Jindan
Published: February 2005

This paper describes the preparation of specimens of neural tissues and sensory structures for electron microscopy. A solution of 2% paraformaldehyde and 2.5% glutaraldehyde was used as fixative. The fixative entered the spinal cord of the rhesus monkey through the aorta abdominalis and emptied into the internal ear of the guinea pig via the arteria vertebralis by the vascular perfusion method. After perfusion, the spinal cord and internal ear were removed from the columna vertebralis and temporal bone, respectively. The tissues were postfixed in 1% OsO4. If a desired direction of the section was required, directional embedding was applied. The application of vascular perfusion fixation, excising tissues on orientation, and embedding specimens in a certain direction makes it possible to obtain the desired tissue section and intact cellular ultrastucture.
Ultrastructural study of the morphogenesis of herpes simplex virus type 2 in organ cultured human uterine cervix and the interactions between virus and host cell
Authors: Lee Kun , Bao Jiaju , Wang Jiedong , Zhao Weiming , Liu Shide , Si Jingyi , Wang Yanyun , Zhang Wei , Jiang Jingwu
Published: February 2005

To determine the pathogenesis of herpes cervicitis and the relationship between herpes simplex virus type 2 (HSV‐2) and the carcinogenesis of human uterine cervix, the ultrastructural changes of 15 cases of long‐term organ cultures of human uterine cervix infected with HSV‐2 were studied. In order to demonstrate the morphogenesis of the virus and the interactions between virus and host cells, EM autoradiography was performed. After viral inoculation, the course of viral DNA replication, and the processes of maturation of virus were examined. Pathological alterations of infected nuclei‐including two kinds of granular matrix aggregations, proliferative changes in nuclei and nuclear envelope, multi‐lobular nucleated giant cell formation, intranuclear microtubules, and some evidence of replication of HSV‐2 in cytoplasm‐were observed. Because of the similarity of the pathologic alterations and the course and duration of infection in the clinic cases and our experimental subjects, this system appears to be a good model to investigate the pathogenesis of herpes cervicitis and the relationship between human cervical cancer and HSV‐2, other viruses, or other co‐carcinogens.
Effect of antibodies against gap junction protein on gap junction formation in early amphibian embryogenesis
Authors: Zeng (Mi‐Pai Tseng) Mibai
Published: February 2005

By electron microscopic observations of freeze‐etching replicas, the gap junctions of ectoderm cells of early gastrulae of Xenopus leavis, injected with antibodies against gap‐junction protein at an earlier stage, were compared with those of normal gastrulae. Most of the gap junctions found in the antibody‐injected series were of very small size and consisted of fewer than 40 connexons, while those of the normal gastrulae usually have gap junctions with over 100 connexons. It was concluded that the normal assembly of intramembranous particles to form gap junctions was attenuated by microinjection of the antibodies.
Distribution of lead in renal proximal convoluted tubules of lead‐poisoned rats
Authors: Zhong Cisheng , Ling Yiping , Wu Zhengquan , Zhu Shiya , Hu Rongfang
Published: February 2005

Intranuclear inclusions have been found in epithelial cells of renal proximal convoluted tubules (RPCT) in lead poisoned animals. It has been suggested previously that the inclusions contained lead. The present report gives evidence to the contrary. Samples of renal cortex were taken from adult rats fed laboratory chow containing 2% lead acetate for 1 month. Under the electron microscope (EM), intranuclear inclusions were observed in epithelial cells of RPCT. They were round or ovoid, and not delineated by a membrane. They consisted of two parts: Peripheral and central. Lysosomes increased in number, and many myelin figures appeared in the cytoplasm. In the lumen of the tubules, high electron‐dense concretions were found. By x‐ray microranalysis, neither the central part nor the peripheral part of the intranuclear inclusions produced a lead peak in the spectrum. Lysosomes and the cytoplasmic matrix also showed no lead peak. However, when the electron beam was focused on the concretion in the lumen of the RPCT, the Lα peak of lead (10.551 keV) and Kα peak of calcium (3.691 keV) were evident. From these findings it is suggested that intranuclear inclusions in the cells of RPCT are not lead deposits. Rather, it is the concretions found in the lumen of tubule that contain lead.
Ultrastructural and cytochemical characteristics of megakaryoblastic leukemic cells
Authors: Tang Xueming , Chu Shaoyou , Zhang Huixin
Published: February 2005

Transmission electron microscopy, scanning electron microscopy, and ultrastructural cytochemistry were utilized to study megakaryoblastic cells from four patients suffering from megakaryoblastic leukemia. The results show that megakaryoblastic leukemic cells have a unique ultrastructural appearance, surface architecture, and cytochemical activity. The cells are positive for platelet peroxidase cytochemical reaction, which is localized in the perinuclear space and endoplastic reticulum, but not in the Golgi apparatus and cytoplasmic granules. They have a rather smooth surface and display blebs or tuberculi which are different from those in other types of leukemic cells as seen under the scanning electron microscope. The megakaryoblastic leukemic cells also show a special appearance under the transmission electron microscope, such as a cytoplasm which contains numerous small mitochondria, mostly concentrated in one pole of the cell. These ultrastructural and cytochemical characteristics of the megakaryoblastic leukemic cells revealed by the combined techniques are very useful in the diagnosis of megakaryoblastic leukemia.
Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high‐resolution scanning and scanning transmission electron microscopy
Authors: Bell Paul B. , Lindroth Margaretha , Fredriksson Bengt‐Arne
Published: February 2005

This paper describes the use of sputter coating to prepare detergent‐extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1–2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low‐angle rotary shadowing with platinum and carbon. However, the sputter‐coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter‐coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal‐coated cytoskeletal structures and electron‐dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.
Electron diffraction identification of structure types of martensite in Cu‐Zn‐Al alloys
Authors: Wang Renhui , Zhao Yuechao , Gui Jianian
Published: February 2005

Electron diffraction technique for identifying structure types of martensite in β‐Hume‐Rothery alloys is described. It includes determination of the stacking sequence of the martensite of the basal planes, differentiation of the long‐range ordering in martensite inherited from its parent phase (A2 type disordered; B2 type ordered; and DO3, or Heusler, type ordered), and distinguishing between normal‐type and modified‐type martensite. In addition to the 18R1‐type martensite, 12R, 6R, and 2H martensites were found in quenched Cu‐Zn‐Al alloys using this technique.
Comparison of TEM and APFIM in microstructural characterization and interpretation: An overview
Authors: Burke M. G. , Miller M. K.
Published: February 2005

A comparison of transmission electron microscopy (TEM) and atom probe field‐ion microscopy (APFIM) is presented with respect to the interpretation of complex microstructures, phase identification, determination of crystallographic order, and analysis of interfaces. The capabilities, spatial resolutions, and limitations of each technique are discussed with examples taken from combined analytical electron microscopy (AEM) and APFIM studies. Both techniques are extremely powerful for routine characterization of a wide range of materials, although care must be exercised in experimentation and interpretation. The combined use of TEM and APFIM is synergistic and extends their individual capabilities from the macro scale to the atomic level.
A study of section wrinkling on single‐hole coated grids using TEM and SEM
Authors: Abad Andre
Published: February 2005

To prevent section wrinkles usually encountered with the use of coated single‐hole grids, a simple method was developed. Formvar film resting on a platform with holes (3.5 mm diameter) was heated with a slide warmer (60–65°C). The bottom of a glass petri dish was inverted over the platform to keep the ambient air at the desired temperature. Sections were picked up from the boat of the diamond knife with a single‐hole grid and deposited at the orifice of the platform and allowed to dry. The grids were then carefully pushed through the orifice of the platform with the blunt head of a nail (3 mm diameter).
Defect structure of superconducting YBa2Cu3O7−δ
Authors: Van Tendeloo G. , Amelinckx S.
Published: February 2005

The superconducting material Y1Ba2Cu3O7−δ has been investigated by electron microscopy. Special attention has been paid to the defects occurring in the material. Twinning on (110) or (110) planes is intrinsically related to the orthorhombicity, and when cooled slowly the twin bands are pseudoperiodic with an average width of ˜ 50 nm. The orthorhombic‐tetragonal transition is reversible and diffusion controlled. Under particular conditions and in slightly reduced material a 2a0 X b0 superstructure resulting from vacancy ordering is formed. When kept in air under powder form the material seems to be unstable. Planar defects along (001) accompany the degeneration of the material.
A procedure for making phosphor viewing screens for the transmission electron microscope
Authors: King Murray Vernon
Published: February 2005

A technique is described for preparing uniform, durable phosphor layers for viewing screens suitable for the transmission electron microscope; a settling procedure is used. The example described here is for a high‐voltage instrument, but with adjustment of the coating density, the technique should be equally suitable for screen preparation for transmission electron microscopes that operate at lower acceleration voltages.
Critical underfocus in low‐magnification electron microscopy: Rationale and practice
Authors: Fahrenbach Wolf H. , Luft John H.
Published: February 2005

Focus in transmission electron microscopy, especially at low‐to‐moderate powers, is usually achieved by an empirical, though largely haphazard, process of defocusing the objective below what is known to be perfect focus as defined by the absence of Fresnel diffraction fringes at the edges of holes. In this article we provide a precise, empirical method of focus, whose rationale resides in the less‐than‐perfect image transfer from the image space of the objective to the negative, i.e., the modulation transfer function. In practice, “perfect focus,” as defined above, is established with a beam deflector (“wobbler”), to which underfocus is then applied routinely by reference to a table. Such “critical underfocus” values have to be calibrated, as described here, for each microscope and depend on all factors that influence contrast, that is, accelerating voltage, condenser defocus and coherence of illumination, focal length of the objective, aperture sizes, combination of intermediate and projection lenses, and the properties of the photographic emulsion.
Progress in quantitative X‐ray microanalysis of frozen‐hydrated bulk biological samples
Authors: Marshall A. T.
Published: February 2005

The analysis of bulk frozen‐hydrated biological samples has developed now to a level where practical application of the technique is possible. Provided the sample is carefully coated with a conductive metal, the development of a space charge capable of causing a significant distortion of the electron diffusion volume does not seem to occur, and analytical resolution can be conveniently held to approximately 2 μm (both depth and lateral resolution). Two valid quantitative methods are available, and two methods of determining dry weight fractions are also available. An area where further research could lead to improvement in analysis of frozen‐hydrated bulk samples is in the investigation of fracturing methods. If fracture planes that were flat and reproducible could be easily obtained, some of the difficulties of analysing frozen‐hydrated bulk samples would be considerably reduced.
X‐ray microanalysis of freeze‐dried and frozen‐hydrated cryosections
Authors: Zierold Karl
Published: February 2005

The elemental composition and the ultrastructure of biological cells were studied by scanning transmission electron microscopy (STEM) combined with energy dispersive X‐ray microanalysis. The preparation technique involves cryofixation, cryoultramicrotomy, cryotransfer, and freeze‐drying of samples. Freeze‐dried cryosections 100‐nm thick appeared to be appropriate for measuring the distribution of diffusible elements and water in different compartments of the cells. The lateral analytical resolution was less than 50 nm, depending on ice crystal damage and section thickness. The detection limit was in the range of 10 mmol/kg dry weight for all elements with an atomic number higher than 12; for sodium and magnesium the detection limits were about 30 and 20 mmol/kg dry weight, respectively.

The darkfield intensity in STEM is linearly related to the mass thickness. Thus, it becomes possible to measure the water content in intracellular compartments by using the darkfield signal of the dry mass remaining after freeze‐drying. By combining the X‐ray microanalytical data expressed as dry weight concentrations with the measurements of the water content, physiologically more meaningful wet weight concentrations of elements were determined.

In comparison to freeze‐dried cryosections frozen‐hydrated sections showed poor contrast and were very sensitive against radiation damage, resulting in mass loss. The high electron exposure required for recording X‐ray spectra made reproducible microanalysis of ultrathin (about 100‐nm thick) frozen‐hydrated sections impossible. The mass loss could be reduced by carbon coating; however, the improvement achieved thus far is still insufficient for applications in X‐ray microanalysis. Therefore, at present only bulk specimens or at least 1‐μm thick sections can be used for X‐ray microanalysis of frozen‐hydrated biological samples.