bluuebunny/crossref_metadata_2025 huggingface dataset:
A temperature‐jump device for time‐resolved cryo‐transmission electron microscopy
Authors: Chestnut M. H. , Siegel D. P. , Burns J. L. , Talmon Y.
Published: February 2005

We describe a temperature‐jump device that permits time‐resolved studies of thin cryo‐transmission electron microscopy specimens. The specimen is rapidly heated to induce a change in microstructure just prior to cryo‐fixation. The apparatus consists of a xenon arc lamp equipped with a shutter controlled by timing circuitry, used in conjunction with an environmental specimen preparation chamber. The specimen is heated by exposure to focused light from the lamp, and then plunged into cryogen. Using a thermocouple constructed from an electron microscope grid, we show that temperature jumps of 30–60 K are achieved with exposure times of 150–450 milliseconds. Micrographs of dimyristoyl phosphatidylcholine (DMPC) vesicles and n‐docosane films, subjected to these exposures, shew that the specimens are still at least 20–30 K above their initial temperature when they contact the cryogen. This method could be applied to a variety of biological and chemical systems which undergo structural changes activated by a rise in temperature.
Utilization of electron microscopic techniques in the in vitro study of adenohypophysial function and regulation
Authors: Asa Sylvia L. , Kovacs Kalman
Published: February 2005

Morphologic studies of human adenohypophysial cells using immunocytochemistry and electron microscopy have characterized the hormone‐producing cell types of the normal gland and pituitary adenomas. The classifications which have emerged allow more accurate clinicopathologic correlations than ever before, but have also raised new questions concerning cytogenesis, pathogenesis, and structure‐function correlations. We report the results of studies which marry the conventional morphologic techniques of light microscopy, immunohistochemistry, electron microscopy, and ultrastructural immunocytology with functional analyses using tissue culture and radioimmunoassay of hormones released into culture media. The hormone secretory activity of nontumorous and adenomatous pituitary cells is correlated with their structural features; their secretory responses to several adenohypophysiotropic factors are compared with morphologic alterations which are characterized at the light and electron microscopic levels by morphometric analysis. These studies have shown that hypothalamic stimulating hormones increase hormone release by their target cells and alter the ultrastructural appearance of the affected cells by increasing organelles involved in hormone synthesis. Inhibitory drugs and adrenal and gonadal steroids are capable of suppressing hormone release by some tumors and also give rise to morphologic changes which correlate with the functional inhibition. Hormone release by clinically nonfunctioning adenomas has been characterized and the behavior of these tumor cells in vitro sheds some light on the reasons for lack of clinical symptomatology. The plurihormonal nature of several nontumorous and adenomatous pituitary cell types has been characterized in vitro. The results of these studies provide the basis for more accurate structure‐function correlations which can be used to study the hormonal milieu in vivo, to predict the role of pathogenetic factors in pituitary tumorigenesis, and to assess the therapeutic value of stimulating or inhibiting hormones and drugs.
Cytoskeletal elements in mammalian spermiogenesis and spermatozoa
Authors: Camatini Marina , Colombo Anita , Bonfanti Patrizia
Published: February 2005

Identification of the cytoskeletal elements and their role in the formation as well as the maintenance of head membrane compartmentalization is a much debated issue in mammalian spermatozoa. Data which have emerged during the last ten years are summarized. Those which have converged in a common opinion, such as the distribution of actin in mammalian spermiogenesis, are distinguished from those which have to be confirmed, such as the role of actin related proteins and actin in mature spermatozoa.
Recent studies of surface dynamic processes by reflection electron microscopy
Authors: Yagi Katsumichi , Yamanaka Akira , Homma Itsuro
Published: February 2005

Reflection electron microscope studies of surface dynamic processes are reviewed and illustrated with recent new observations. They include: surface electromigration and current dependent structures of Si surfaces; surface etching by oxidation of Si surface; and growth of two dimensional alloyed adsorbate by co‐deposition of metals on Si surface. The observations revealed details of the surface dynamic processes, which are difficult to obtain with other surface analysis techniques.
Reflection electron energy‐loss spectroscopy and imaging for surface studies in transmission electron microscopes
Authors: Wang Z. L. , Bentley J.
Published: February 2005

A review is given on the techniques and applications of high‐energy reflection electron energy‐loss spectroscopy (REELS) and reflection electron microscopy (REM) for surface studies in scanning transmission electron microscopes (STEM) and conventional transmission electron microscopes (TEM). A diffraction method is introduced to identify a surface orientation in the geometry of REM. The surface dielectric response theory is presented and applied for studying α‐alumina surfaces. Domains of the α‐alumina (012) surface initially terminated with oxygen can be reduced by an intense electron beam to produce Al metal; the resistance to beam damage of surface domains initially terminated with Al+3 ions is attributed to the screening effect of adsorbed oxygen. Surface energy‐loss near‐edge structure (ELNES), extended energy‐loss fine structure (EXELFS), and microanalysis using REELS are illustrated based on the studies of TiO2 and MgO. Effects of surface resonances (or channeling) on the REELS signal‐to‐background ratio are described. The REELS detection of a monolayer of oxygen adsorption on diamond (111) surfaces is reported.

It is shown that phase contrast REM image content can be significantly increased with the use of a field emission gun (FEG). Phase contrast effects close to the core of a screw dislocation are discussed and the associated Fresnel fringes around a surface step are observed. Finally, an in situ REM experiment is described for studying atomic desorption and diffusion processes on α‐alumina surfaces at temperatures of 1,300—1,400°C.
Observation of double line contrast in surface imaging
Authors: Yao Nan , Cowley John M.
Published: February 2005

The double line contrast of a single‐atom height step observed in surface imaging for a single crystal in reflection electron microscopy is studied under a variety of experimental conditions. It is suggested that this abnormal contrast is directly associated with the dynamical electron diffraction process. The behavior of the double line contrast is closely related to the order of the Bragg reflected beam, and can be observed mostly under one of the two commonly cited resonance conditions. This phenomenon clearly reveals the differences in the surface imaging for various resonance conditions.
Preparation and characterization of MgO surfaces by reflection electron microscopy
Authors: Crozier P. A. , Gajdardziska‐Josifovska M. , Cowley J. M.
Published: February 2005

We have employed several different methods to prepare (100) and (111) surfaces of MgO crystals. (100) surfaces prepared by simple cleaving give good reflection high energy electron diffraction (RHEED) patterns and surfaces with a high density of coarse steps. Chemical polishing of this surface results in a roughening of the topography whilst annealing in oxygen considerably smoothens the surfaces although they appear to be contaminated. Under certain conditions we find that the MgO crystals will cleave along the (111) plane. Both cleaved and mechanically polished (111) surfaces are atomically flat and reconstructed after oxygen annealing.
Scanning electron microscopy of negatively stained catalase on a silicon wafer
Authors: Furuno Taiji , Ulmer Kevin M. , Sasabe Hiroyuki
Published: February 2005

A high‐resolution scanning electron microscope capable of 7 Å spatial resolution at 30‐kV accelerating voltage was used to observe negatively stained protein molecules. Thin platelet crystals, densely packed monolayers, and low‐density deposits of beef liver catalase were prepared on the surface of silicon wafers and negatively stained with phosphotungstic acid. The tetrameric structure of the catalase molecule was observed for the first time by scanning electron microscopy on the surface of the smooth silicon wafer.
A combined pseudoreplica‐immunochemical technique for research and diagnostic Virology
Authors: Fritz Blayne , Moore Kenneth , Naides Stanley J.
Published: February 2005

Pseudoreplica and immunochemical techniques were combined in a single protocol for identification of virus in research and clinical specimens. Stock preparations of vesicular stomatitis virus (VSV), cytomegalovirus (CMV), and herpes simplex virus (HSV) were used to develop the technique. Traditional pseudoreplicas of viral stock solutions were prepared but not negatively stained. The virus was then immunolabeled in two stages. Virus‐specific polyclonal antisera were used in the first stage; colloidal gold congugated antibodies were used as indicator antibody in the second stage. After immunolabeling, the grids were negatigvely stained. As a demonstration of the clinical usefulness of this approach, it was employed to antenatally identify human parvovirus B19 particles in ascites from a 22 week gestational fetus with nonimmune hydrops fetalis. The combined pseudoreplica‐immunochemical approach offers several advantages over both the pseudoreplica and immunochemical methods when used in isolation. Advantages include relative purification of samples, concentration of virus, morphological preservation, and enhanced diagnostic specificity.
Ultrastructure of the mammalian pinealocyte under natural and experimental conditions: Quantitative aspects
Authors: Karasek Michal
Published: February 2005

This review briefly summarizes data accumulated on the quantitative aspects of the ultrastructure of the mammalian pinealocyte. Quantitative changes have been demonstrated under natural and experimental conditions in pinealocyte cell organelles in various species. Special attention is paid to two cytoplasmic components most frequently studied by means of quantitative electron microscopy, namely, dense‐core vesicles and “synaptic” ribbons.
The thickness determination of organic crystals under low dose conditions using electron energy loss spectroscopy
Authors: Rez Peter , Chiu Wah , Weiss J. K. , Brink J.
Published: February 2005

In the 3‐dimensional (3‐D) reconstruction of protein crystals with variable thicknesses the electron images and diffraction patterns can only be merged if the crystal thickness is known. Measurement of the thickness using the ratio of the number of inelastically scattered electrons to the number of electrons in the zero loss peak can be accomplished with parallel electron energy loss spectrometry (PEELS). A theoretical analysis of the accuracy of the technique on paraffin crystals of different thicknesses is presented. Our experimental studies with paraffin crystals show the feasibility of measuring a single layer of 47 Å with good accuracy under low dose and low temperature conditions. A simple experimental apparatus is proposed to obtain thicknesses from small regions of unstained protein crystals prior to collecting the 3‐D data sets from the unexposed area of the same crystal.
Computer‐assisted morphometry: Point, intersection, and profile counting and three‐dimensional reconstruction
Authors: Hyde Dallas M. , Magliano David J. , Reus Edward , Tyler Nancy K. , Nichols Steve , Tyler Walter S.
Published: February 2005

The use of computers in morphometry can involve (1) automated image analysis, semiautomated image analysis and point, intersection, intercept and profile counts of two‐dimensional images on tissue sections with mathematical extrapolation to the third dimension, (2) direct measurement of volumes, surfaces, lengths, and curvature using x,y,z coordinates of serial sectioned images, or (3) stereologic techniques and serial sections which is a combination of 1 and 2 above. Automated and semiautomated image analysis are generally restricted to specimens that are characterized by differential contrast such as interalveolar septa in the lung or histochemically stained mucous granules in pulmonary epithelium. Point, intersection, and profile counts using hand‐held, notebook PCs, portable PCs, or standard PCs and MS‐DOS—based application programs are extremely efficient, precise, affordable, and convenient methods of quantitating average values of a population. When morphometric measurements of individual structures are required, computer‐assisted three‐dimensional reconstruction using x,y,z coordinates of the surface outline from serial sections is a tedious yet precise method. We describe a computer program that efficiently estimates mean caliper diameter, volume, and surface area with less than five percent error with five sections per structure. We also describe a program that does digital image subtraction on serial sections, superimposes digitally generated test systems on biological images, and accumulates point, intersection, and profile counts using a Macintosh II series computer. © 1992 Wiley‐Liss, Inc.
Application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver in conjunction with cytochemical staining with 3‐3′‐diaminobenzidine and immunocytochemistry
Authors: Beier Konstantin , Fahimi H. Dariush
Published: February 2005

We describe the application of automatic image analysis for quantitative morphological studies of peroxisomes in rat liver. For automatic detection by light and electron microscopy peroxisomes must be stained with the alkaline DAB procedure for catalase. There is a good agreement between the results obtained by conventional morphometric techniques and by automatic image analysis of DAB‐stained electron microscopic preparations. Moreover, the image analyzer may be used in conjunction with a light microscope for evaluation of semithin sections (1–0.25 μm), provided the section thickness factor is taken into consideration. This latter approach has proven highly efficient in estimation of peroxisome proliferation. The limitations of this method and the relevance of volume density as a reliable morphometric parameter for evaluation of peroxisome proliferation are discussed. In the second part of this study we present the application of image analysis for quantitation of alterations of individual peroxisomal enzyme proteins after treatment with bezafibrate in immunogold stained ultrathin sections. There is good agreement between the results of quantitative immunocytochemistry and Western (immuno) blot analysis of highly purified peroxisomal fractions. In our experience quantitative immunoelectron microscopy provides a versatile, highly sensitive, and efficient method for detection of modulations of various proteins in peroxisomes. Finally the limitations and prospects of quantitative immunocytochemistry for investigation of peroxisomal proteins are discussed. © 1992 Wiley‐Liss, Inc.
Computer assisted data collection for stereology: Rationale and description of point counting stereology (PCS) software
Authors: Pentcheff N. Dean , Bolender Robert P.
Published: February 2005

The paper describes microcomputer software for point counting stereology. Stereology includes a collection of statistical methods that quantify the images of light and transmission electron microscopy. The methods use test grids placed over images to collect raw data, which includes counts of points, intersections, transections, and profiles. In turn, the counts are included in stereological equations that give estimates of compartmental volumes, surfaces, lengths, or numbers. These parameters describe the composition of a structure in three‐dimensional space. The PCS (point counting stereology) System Software III serves as a data collection, storage, and management tool. Users set up point counting protocols without programming, enter data by pressing predefined function (MS‐DOS) or alphabetic keys (UNIX), store data in files, select files for analysis, and calculate results as stereological densities. The latest version of the PCS software includes a new user interface and is designed as a research “front end” that can feed data either into the calculation tools of a stereology tutorial (Bolender, 1992, this issue) or into the analysis routines of quantitative morphology databases (Bolender and Bluhm, 1992). © 1992 Wiley‐Liss, Inc.
From egg to pole cells: Ultrastructural aspects of early cleavage and germ cell determination in insects
Authors: Zissler Dieter
Published: February 2005

Insect eggs are giant and very complex cells covered by an extremely resistant shell. Both the egg cell and surrounding eggshell express anteroposterior and ventrodorsal polarity. The molecular and cytoplasmic organization of both axes originates during oogenesis and leads to the production of an ooplasmic system which consists of euplasm and deutoplasm (yolk) and contains a nucleus as well as extranuclear determinants of maternal origin. Both are part of the store of information for early embryogenesis. In addition, the deutoplasm serves as raw material and early nutrient supply for building the embryo.
The insect egg cell, which is arrested in the first maturation division when released from the ovary during oviposition, will be activated by different stimuli among different species to complete meiosis and start embryogenesis. The zygote nucleus undergoes a number of synchronous mitotic divisions leading to cleavage energids which initially form a syncytial blastoderm and subsequently the cellular blastoderm. In many insects, prior to blastoderm formation, polar granules (or oosome material) are incorporated in a single cell or a small number of cells which bud off at the posterior pole. These so called pole cells give rise to the primordial germ cells. Therefore, polar granules or the oosome material mark the germ line, and while structural counterparts of determinants of body pattern formation have so far not been found, the polar granules or oosome serve as an autonomous ooplasmic determinant for the pole or germ cells. Anteroposterior body polarity can arise independent of the germ plasm. © 1992 Wiley‐Liss, Inc.
Preservation and visualization of molecular structure in detergent‐extracted whole mounts of cultured cells
Authors: Lindroth Margaretha , Bell Paul B. , Fredriksson Bengt‐Arne , Liu Xiao‐Dong
Published: February 2005

Today's electron microscopes have a resolution sufficient to resolve supramolecular structures. However, the methods used to prepare biological samples for electron microscopy often limit our ability to achieve the resolution that is theoretically possible. We use whole mounts of detergent‐extracted cells grown on Formvar‐coated gold grids as a model system to evaluate various steps in the preparation of biological samples for high resolution scanning electron microscopy (SEM)

Factors that are important in determining the structure and composition of detergent‐extracted cells include the nature of the detergent and the composition of the extraction vehicle. Chelation of calcium is extremely important to stabilize and preserve the cytoskeletal filaments. We have also demonstrated both morphologically and by gel electrophoresis that treatment of cells with bifunctional protein crosslinkers before or during extraction with detergent can significantly enhance the preservation of both proteins and supramolecular structures.

The methods used to dry samples are a major determinant of the quality of structural preservation. For cytoskeletons freeze‐drying (FD) is superior to critical point‐drying (CPD), one reason being that CPD samples have to be dehydrated, thereby causing more shrinkage as compared to FD samples. The high pressures to which samples are exposed during CPD may also cause increased shrinkage, and water contamination during CPD causes severe structural damage. We have obtained the best structural preservation of detergent‐extracted and fixed cells by manually plunging them into liquid propane and drying over night in a freeze‐drayer.

The factor that most limits achievement of high resolution in SEM is the metal coat, which has to be very thin, uniform, and free of grain in order not to hide structures or to create artifactual ones. We have found that sputter‐coating with 1–3 nm of tungsten (W) or niobium )Nb( gives extremely fine‐grained films as well as satisfactory emission of secondary electrons. These samples can also be examined at high resolution by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). The best preservation and visualization of supramolecular structures have been obtained using cryosputtering, in which the samples are freeze‐dried and then sputter‐coated within the freeze‐dryer while still frozen. © 1992 Wiley‐Liss, Inc.
Sample preparation for electron microscopy of internal cell structure
Authors: Haggis Geoffrey H.
Published: February 2005

Methods are reviewed for examination of internal cell structure by high‐resolution scanning electron microscopy and compared with the rapid‐freeze deep‐etch replica technique used in transmission electron microscopy. Rapid freezing of fresh material, followed by freezefracture, provides a theoretically attractive approach in ultrastructure studies, but the high protein and solute content of most cells prevents a deep three‐dimensional view for material frozen without some form of extraction. After discussion of other methods it is concluded that the most useful general approach, at least for cultured cells, is to first permeabilize or break open the cells in a medium which preserves the structure under study in a functional state as, for example, the movement of chromosomes along the division spindle, or transport of proteins within the Golgi region. After permeabilization, with attendant partial extraction, the preparation can be fixed, then viewed by either deep‐etch replication, or by high‐resolution scanning electron microscopy, with structure of interest revealed in deep view. © 1992 Wiley‐Liss, Inc.
Cortical ultrastructure and chemoreception in ciliated protists (ciliophora)
Authors: Hufnagel Linda A.
Published: February 2005

The ciliated protists (ciliates) offer a unique opportunity to explore the relationship between chemoreception and cell structure. Ciliates resemble chemosensory neurons in their responses to stimuli and presence of cilia. Ciliates have highly patterned surfaces that should permit precise localization of chemoreceptors in relation to effector organelles. Furthermore, ciliates are easy to grow and to manipulate genetically; they can also be readily studied biochemically and by electrophysiological techniques. This review contains a comparative description of the ultrastructural features of the ciliate cell surface relevant to chemoreception, examines the structural features of putative chemoreceptive cilia, and provides a summary of the electron microscopic information available so far bearing on chemoreceptive aspects of swimming, feeding, excretion, endocytosis, and sexual responses of ciliates. The electron microscopic identification and localization of specific chemoreceptive macromolecules and organelles at the molecular level have not yet been achieved in ciliates. These await the development of specific probes for chemoreceptor and transduction macromolecules. Nevertheless, the electron microscope has provided a wealth of information about the surface features of clliates where chemoreception is believed to take place. Such morphological information will prove essential to a complete understanding of reception and transduction at the molecular level. In the ciliates, major questions to be answered relate to the apportionment of chemoreceptive functions between the cilia and cell soma, the global distribution of receptors in relation to the anterior‐posterior, dorsal‐ventral, and left‐right axes of the cell, and the relationship of receptors to ultrastructural components of the cell coat, cell membrane, and cytoskeleton. © 1992 Wiley‐Liss, Inc.
Ultrastructural neurobiology of the olfactory mucosa of the brown trout, Salmo trutta
Authors: Moran David Taylor , Rowley J. Carter , Aiken George R. , Jafek Bruce W.
Published: February 2005

This paper describes four investigations of the olfactory mucosa of the brown trout: 1) the ultrastructure of the olfactory mucosa as revealed by scanning (SEM), conventional transmission (TEM), and high voltage (HVEM) electron microscopy; 2) light and electron‐microscopic investigations of retrograde transport of the tracer macromolecule horseradish peroxidase (HRP) when applied to the cut olfactory nerve; 3) SEM and TEM investigations of the effects of olfactory nerve transection on cell populations within the olfactory epithelium; and 4) ultrastructural investigations of reversible degeneration of olfactery receptors caused by elevated copper concentrations. The trout lofactory epithelium contains five cell types: ciliated epithelial cells, ciliated olfactory receptor cells, microvillar olfactory receptor cells, supporting cells, and basal cells. The ciliated and microvillar olfactory receptor cells and a small number of basal cells are backfilled by HRP when the tracer is applied to the cut olfactory nerve. When the olfactory nerve is cut, both ciliated and microvillar olfactory receptor cells degenerate within 2 days and are morphologically intact again within 8 days. When wild trout are taken from their native stream and placed in tanks with elevated copper concentrations, ciliated and microvillar cells degenerate. Replacement of these trout into their stream of origin is followed by morphologic restoration of both types of olfactory receptor cells. Ciliated and microvillar receptor cells are primary sensory bipolar neurons whose dendrites make contact with the environment; their axons travel directly to the brain. Consequently, substances can be transported directly from the environment into the brain via these “naked neurons.” Since fish cannot escape from the water in which they swim, and since that water may occasionally contain brain‐toxic substances, the ability to close off—and later reopen—this anatomic gateway to the brain would confer a tremendous selective advantage upon animals that evolved the “brain‐sparing” capacity to do so. Consequently, the unique regenerative powers of vertebrate olfactory receptor neurons may have their evolutionary origin in fishes. © 1992 Wiley‐Liss, Inc.
Olfactory epithelium in young adult and aging rats as seen with high‐resolution scanning electron microscopy
Authors: Nagurok Tomonori , Iwashita Kazuto
Published: February 2005

The present study uses mainly scanning electron microscopy to demonstrate the three‐dimensional internal cell structures of rat olfactory epithelial cells. The aldehyde‐prefixed osmium‐DMSO‐osmium (AODO) method devised by Tanaka and Mitsushima (1984) was applied to the present study to disclose intracellular structures such as endoplasmic reticulum, mitochondria, Golgi apparatus, and lysosomes. The spatial distribution pattern of these structures in olfactory and supporting cells is discussed, paying special attention to the formation of lipofuscin‐like granules present in aged rats. © 1992 Wiley‐Liss, Inc.
The human primary olfactory pathway: Fine structural and cytochemical aspects during development and in adults
Authors: Chuah Meng Inn , Zheng Da Ren
Published: February 2005

Despite increasing knowledge about the biophysiology of the human olfactory system, understanding of the development of this pathway in humans lags considerably behind that of other vertebrates. Development studies have largely concentradted on the generation of cell types in the olfactory epithelium during the first trimester, while detailed ultrastructural observations usually describe the adult morphologh. In this review, we have shown that contrary to what has been generally assumed, the surface of the human olfactory epithelium is heterogeneous and that its olfactory nerves differ ultrastructurally from those of other vertebrates studied. The development of the human primary olfactory pathwayis discussed in terms of the appearance of olfactory bulb laminae, synaptogenesis and the expression of specific cell markers, such as the S‐100 protein and olfactory marker protein (OMP). Positive immunohistochemical staining for N‐cadherin in human fetuses suggests that growth ofolfactory axons to their target may be mediated by cell adhesion molecules. The overall data presented here indicate that this pathway develops more precociously in humans than in rodents. Whether this translates also to earlier functional maturity remains to be elecidated. © 1992 Wiley‐Liss, Inc.
Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse: Ultrastructure and uptake of tracers
Authors: Bannister Lawrence H. , Dodson Hilary C.
Published: February 2005

Mammalian olfactory neurons possess a well‐developed system of endocytic vesicles, endosomes, and lysosomes in their dendrites and perikarya. Vomeronasal neurons are similar and also contain much perikaryal agranular endoplasmic reticulum (AER). Olfactory supporting cells contain endocytic vesicles and endosomes associated closely with abundant fenestrated AER, and vesicles and numerous large dense vacuoles are present basally. Vomeronasal supporting cells have little AER, and few dense vacuoles occur in their bases. In olfactory neurons, ultrastructural tracers (0.08% horseradish peroxidase, thorium dioxide, ferritin) are endocytosed by olfactory receptor endings and transported to the cell body, where their movement is halted in lysosomes. Higher concentrations (1%) of horseradish peroxidase penetrate olfactory receptor plasma membranes and intercellular junctions. In olfactory supporting cells, endocytosed tracers pass through endosomes to accumulate in dense basal vacuoles. These observations indicate that olfactory sensory membranes are rapidly cycled and that endocytosed materials are trapped within the epithelium. It is proposed that in the olfactory epithelium, endocytosis presents redundant odorants to the enzymes of the supporting cell AER to prevent their accumulation, whereas in the vomeronasal epithelium the receptor cells carry out this activity. © 1992 Wiley‐Liss, Inc.
Structures of grain boundaries in long‐chain te crystals observed by high‐resolution transmission electron microscopy
Authors: Isshiki Toshiyuki , Hirota Yoshihiro , Saijo Hiroshi , Shiojiri Makoto
Published: February 2005

Vacuum‐deposited Te crystals, composed of endless chains of the right‐handed or left‐handed spiral, have been investigated by high‐resolution electron microscopy with the aid of image simulation. A (010) grain boundary, which accompanies edge dislocations having an extra layer of the width of one Te chain, has been observed. A through‐focal series of images reveal that it is not a reflectional nor a rotational twin boundary but a small angle grain boundary in a single crystal or an inversion twin. The lattice on one side of the boundary is shifted from that on the other side by [001]c/3 + [120]a/8, and inclined at 1.1° around the c‐axis of the other side. Also found between crystallites of [100] and [011] orientation is a grain boundary which is built with the (011) facets on one side of the boundary and the (211) or (0,1,10) facets on another side. © 1992 Wiley‐Liss, Inc.
Effect of crystal and beam tilt on simulated high‐resolution TEM images of interfaces
Authors: Howe J. M. , Rozeveld S. J.
Published: February 2005

The effects of crystal and beam tilt on high‐resolution transmission electron microscope (HRTEM) images of planar coherent interfaces were investigated by multislice image simulations. It was found that a beam tilt of 0.5 Bragg angle (θB) was sufficient to introduce detrimental artifacts into most images of interfaces in crystals only 1/8ξ000 thick, while crystal tilt had a much smaller effect even for crystals 1ξ000 thick. Effects produced in HRTEM images of interfaces by crystal and beam tilt included the introduction of additional periodicities and loss of compositional detail across a boundary, translation of a boundary from its actual position, and apparent mismatch of atomic planes across a perfectly coherent interface. These results indicate that alignment of the electron beam parallel to the optic axis is critical for reliable HRTEM imaging of interfaces in materials. Techniques for obtaining accurate alignment are also discussed. © 1992 Wiley‐Liss, Inc.
Improved method for the preparation of TiN‐coated WC‐Co‐based samples for cross‐sectional AEM investigation
Authors: Hefter J. , Ostreicher K. , Sung C.
Published: February 2005

An improved method for the preparation of cross‐sectional thin foils of coated WC‐Co samples for studies by analytical electron microscopy is described. A braze alloy is used to join the sections of the sample together and the resulting sample is stable during subsequent grinding, dimpling, and milling operations. Cross‐sectional micrographs provide examples of the efficacy of this method. No microstructural alteration associated with the brazing operation was observed. © 1992 Wiley‐Liss, Inc.
Intracellular injection in fixed slices in combination with neuroanatomical tracing techniques and electron microscopy to determine multisynaptic pathways in the brain
Authors: Buhl Eberhard H.
Published: February 2005

Intracellular Lucifer Yellow filling in fixed tissue has been recently introduced as a novel neuroanatomical approach to reveal the detailed morphology of individual neurons in isolated preparations of the central nervous system. Since dye injections are performed under visual control, the method is characterized by a high degree of inherent staining selectivity, thus circumventing the element of randomness often considered to be the crux of classical Golgi‐impregnation techniques. Moreover, the opportunity to optically monitor the injection procedure renders fixed slice preparations highly advantageous to be used in combination with retrograde fluorescent tracing. Subsequently, dye‐filled neurons may be subjected to a simple photoconversion procedure leading to the intracellular formation of a stable polymer thus obtaining permanent specimens for light microscopy purposes. Due to the osmiophilic nature of the precipitate the photoconverted material is equally suitable for correlated electron microscopy, thus enabling the analysis of neuronal microcircuitry. At the ultrastructural level, sources of afferent input to identified projection neurons may be revealed by lesion‐induced anterograde degeneration of synaptic terminals, therefore enabling the direct demonstration of multisynaptic links. Finally, morphologically identified neurons may be immunocytochemically characterized at the pre‐ and postembedding levels. It is therefore suggested that their methodological versatility and relative technical ease render intracellular fixed‐slice injections a promising complement to the catalogue of anatomical techniques. © 1993 Wiley‐Liss, Inc.
Electron microscopy of intracellularly labeled neurons in the hippocampal slice preparation
Authors: Kunkel Dennis D. , Scharfman Helen E. , Schmiege Donna L. , Schwartzkroin Philip A.
Published: February 2005

We have assessed the properties of three intracellular markers, horseradish peroxidase, biocytin/Neurobiotin, and Lucifer Yellow, and have compared their usefulness as neuronal markers for light and electron microscopic visualization. Neurons in the acute slice preparation of rat hippocampus were filled with one of these markers, and the marker was converted to an optical and electron‐dense reaction product. Dimethylsulfoxide (DMSO) greatly facilitated penetration of recognition reagents while preserving membrane integrity. The markers were compared with respect to injection parameters, mobility and recognition, stability and visibility, and ultrastructural clarity. Horseradish peroxidase (HRP)‐labeled neurons, recognized histochemically with diaminobenzedine (DAB), were easily visualized by the density of the DAB reaction product; however, the electron density was often so great as to obscure ultrastructural details. Biocytin (BC)‐/Neurobiotin (NB)‐labeled neurons were recognized by avidin‐HRP, followed by histochemical localization of HRP with DAB. The optically dense reaction product gave complete visualization of the soma and processes at the light microscopic level. The electron density was homogeneously distributed throughout the cell, so that ultrastructural features were easily identified. Lucifer Yellow (LY), a fluorescent marker, was converted to an optical and electron‐dense reaction product via immunocytochemical staining with a rabbit anti‐LY antibody, followed by goat anti‐rabbit IgG‐HRP and DAB histochemical localization. Similar to BC/NB, the reaction product was evenly dispersed, providing good light microscopic and ultrastructural clarity. Under our experimental conditions, BC/NB and LY were superior markers that could be used routinely to label neurons, and give excellent visualization not only at the light but also at the electron microscopic level. © 1993 Wiley‐Liss, Inc.
Molecular and cellular properties of mammalian primary olfactory axons
Authors: Mori Kensaku
Published: February 2005

How are the axonal projections of olfactory and vomeronasal receptor neurons to the olfactory bulb formed during development? How are the primary olfactory axonal connections functionally organized? With progress in molecular biological techniques and histochemical methods, it became possible to study cellular strategies and molecular mechanisms which guide the primary olfactory axons of the main and accessory olfactory systems to the target glomeruli in the bulb. In addition, new methodologies have begun to elucidate various subsets of the primary olfactory axons with distinctive central connections. The aim of the present paper is to review (1) the characteristic organization of the projection of the primary olfactory axons, (2) projection patterns of histochemically defined subsets of primary olfactory axons, and (3) information on molecules expressed by the surface membrane of the primary olfactory axons. This knowledge gives insight into the functional organization of the primary olfactory axon projection, which is indispensable for understanding signal processing in the olfactory system. This knowledge also underscores the notion that the primary olfactory axon projection provides an excellent model system in which to study axonal guidance and the formation of specific synaptic connections. © 1993 Wiley‐Liss, Inc.
Characterization of the suprachiasmatic nucleus in organotypic slice explant cultures
Authors: Wray S. , Castel M. , Gainer H.
Published: February 2005

Suprachiasmatic nuclei (SCN) from hypothalami of postnatal rats were maintained for 18–39 days in vitro as organotypic slice explants. Neuronal subtypes containing vasopressin (VP), vasoactive intestinal polypeptide (VIP), gastrin releasing hormone (GRP), and GABA were immunocytochemically identifiable in these cultures. In situ hybridization histochemistry was compatible with these SCN slice explant cultures, and mRNA encoding for VP was detected bilaterally within these nuclei. After 18 days in vitro, both VP mRNA and VP immunoreactivity increased from levels present on postnatal days 4 (the earliest age from which the explanted tissue was derived) to levels typical of adult SCNs. In contrast, the GRP expression remained low, characteristic of early postnatal animals and far lower than adult levels. This suggests that the developmental cues or programs necessary for enhanced VP expression are maintained in these cultures, while those affecting GRP expression are absent or inhibited. VIP‐containing neurons were numerous in the cultures. Culture slices appeared healthy, and similar numbers and distributions of identifiable neurons within the SCN were observed, whether or not the slices were grown in the presence of serum. EM analysis revealed that the SCN in vitro is composed of tightly packed neurons, processes, and abundant synapses containing both clear and dense core vesicles, closely resembling the SCN in vivo. Vasopressinergic neuronal somata contained extensive Golgi systems and labeled secretory granules, the latter organelle being present also within processes and synaptic terminals. GABA‐immunopositive processes and synaptic profiles were abundant, with labeling occurring particularly over secretory vesicles and mitochondria. This slice culture system effectively maintained much of the intrinsic organization and cellular components of the SCN for long periods in vitro and should be an excellent model system for studying the intrinsic molecular mechanisms and extrinsic cues which regulate neuronal phenotype in this circadian pacemaker. Published 1993 Wiley‐Liss, Inc.
Granulated metrial gland cells: A natural killer cell subset of the pregnant murine uterus
Authors: Croy B. Anne , Kiso Yasuo
Published: February 2005

The metrial gland develops in the uterus of many rodent species during normal pregnancy. It is a maternally‐derived tissue that contains stromal and vascular elements plus a population of large cells, striking in their light microscopic appearance due to the presence of numerous cytoplasmic granules. These cells, which have become known in mice and rats as granulated metrial gland (GMG) cells, are derived from bone marrow precursors and recent work suggests they are a subset of lymphocytes belonging to the natural killer (NK) cell lineage. The functions of GMG cells during normal gestation have not been clearly defined. In vitro, GMG cells have been shown to produce cytokines and their cytokine profile is altered upon addition of medium containing the T cell growth factor interleukin‐2 (IL‐2). GMG cell granules contain the cytolytic protein perforin but GMG cells have a very limited capacity to kill in vitro unless they have been stimulated by IL‐2 or interferon‐gamma. Histological study of GMG cells has suggested they preferentially associate with fetal trophoblast. Since trophoblast appears resistant to immune lysis, except by IL‐2‐activated effector lymphocytes, and because resorbing murine embryos become infiltrated by lytic cells of the NK cell lineage, it is important to establish whether GMG cells are activated by pregnancy‐associated events to play a major lytic role in vivo. © 1993 Wiley‐Liss, Inc.
Controlled‐Depth and cross‐section preparation techniques for transmission electron microscopy subsurface studies in metals
Authors: Garcia‐Borquez A. , Kesternich W.
Published: February 2005

Thin film specimen preparation from bulk material at a controlled depth below the surface and cross‐section thin film preparation for transmission electron microscope investigations of electrically conducting materials are described. Both techniques are illustrated by austenitic stainless steel, where they have been used complementary to each other for microstructural studies of subsurface ion irradiation damage. The advantages and limitations of both techniques are discussed. © 1993 Wiley‐Liss, Inc.
Subcellular calcium localization in Toxoplasma gondii by electron microscopy and by X‐ray and electron energy loss spectroscopies
Authors: Bonhomme A. , Pingret L. , Bonhomme P. , Michel J. , Balossier G. , Lhotel M. , Pluot M. , Pinon J. M.
Published: February 2005

The localization of calcium in Toxoplasma gondii tachyzoites was studied at the ultrastructural level, with a cytochemical pyroantimonate precipitation method (PA) and controlled by EGTA chelating and EDX and EELS microanalyses. Appropriate conditions for material preparation, fixation and embedding, were defined. The proportion of precipitates that were either free or inside vacuoles and their distribution inside Toxoplasma appeared to be PA dose‐dependent. Precipitation mainly occurred in the anterior pole of the Epon‐embedded tachyzoites. EDX and EELS analyses showed that out of 30 PA precipitates inside tachyzoites, 78% contained Ca. In Melamine sections, 96% of the tachyzoites had intracellular precipitates and the membrane complex was stained; 25% of the tachyzoites inside host cells contained PA‐Ca precipitates, but most of them were retained in the reticular network of the parasitophorous vacuole. Melamine embedding appeared to improve the preservation of calcium pyroantimonate precipitates. © 1993 Wiley‐Liss, Inc.
High‐resolution transmission electron microscopy of hexagonal and rhombohedral molybdenum disulfide crystals
Authors: Isshiki Toshiyuki , Nishio Koji , Saijo Hiroshi , Shiojiri Makoto , Yabuuchi Yasufumi , Takahashi Noboru
Published: February 2005

Natural (molybdenite) and synthesized molybdenum disulfide crystals have been studied by high‐resolution transmission electron microscopy. The image simulation demonstrates that the [0001] and [0110] HRTEM images of hexagonal and rhombohedral MoS2 crystals hardly disclose their stacking sequences, and that the [2110] images can distinguish the Mo and S columns along the incident electron beam and enable one to determine not only the crystal structure but also the fault structure. Observed [0001] images of cleaved molybdenite and synthesized MoS2 crystals, however, reveal the strain field around partial dislocations limiting an extended dislocation. A cross‐sectional image of a single molecular (S‐Mo‐S) layer cleaved from molybdenite has been observed. Synthesized MoS2 flakes which were prepared by grinding have been found to be rhombohedral crystals containing many stacking faults caused by glides between S/S layers. © 1993 Wiley‐Liss, Inc.
Effects of four different processing techniques on the microstructure of potatoes: Comparison with fresh samples in the ESEM
Authors: Uwins Philippa J. R. , Murray Margaret , Gould Robert J.
Published: February 2005

Four common scanning electron microscope (SEM) processing techniques involving freeze‐substitution and chemical fixation were compared with fresh unprocessed samples imaged in an environmental scanning electron microscope (ESEM) using small pieces of potato tubers as test specimens. Potato tubers were chosen for this investigation because of their high moisture content and, consequently, the common need for extensive processing for conventional, high vacuum SEM imaging. ESEM results showed that the fresh unprocessed specimens were essentially unaltered, showing clear potato cell structure, morphology, and cell content. However, processed samples showed strong differences to the fresh samples: freeze‐substituted specimens showed fine networks of material stretching across the surface of cells. These structures may represent fibrillar material or may be artifact caused during processing. Chemical fixation almost entirely destroyed the microstructure of these potato samples. © 1993 Wiley‐Liss, Inc.
Using ESEM and SEM to compare the performance of dentin conditioners
Authors: Gilbert Lynn C. , Doherty Robert E.
Published: February 2005

A comparison of four dentinal conditioners was performed utilizing a traditional scanning electron microscope (SEM) and a new technology, the ElectroScan™ environmental scanning electron microscope (ESEM). Both ESEM and SEM analysis verified current theorized mechanisms of adhesion to dentin surfaces. ESEM images appeared to more closely approximate what one would expect cut and treated dentin surfaces look like. Increases in information from the “surfaces” of uncoated specimens and the reduction in specimen preparation time were associated with ESEM analysis. © 1993 Wiley‐Liss, Inc.