bluuebunny/crossref_metadata_2025 huggingface dataset:
A review of recent observations concerning the synaptic organization of the basilar pontine nuclei
Authors: Mihailoff G. A. , Kosinski R. J. , Border B. G. , Lee H. S.
Published: February 2005

Ultrastructural studies are described that have identified in the basilar pontine nuclei (BPN), the synaptic boutons formed by the corticopontine, cerebellopontine, tectopontine, and dorsal column nucleipontine afferent projection systems. In addition, immunocytochemical studies visualized neuronal somata, dendrites, and synaptic boutons that contain immunoreactivity for GABA or the synthesizing enzyme glutamic acid decarboxylase (GAD).

Based upon differences in the mode of degeneration and postsynaptic locus of degenerative synaptic boutons in the BPN, it is suggested that two types of cortical neurons and three classes of deep cerebellar nuclear cells project to the BPN. For similar reasons, it appears that two types of neurons in the dorsal column nuclei project to the BPN while only one type of afferent synaptic bouton takes origin from the superior colliculus. Furthermore it appears that the population of BPN neurons projecting to the paramedian lobule receives convergent inputs from the cutaneous periphery and the corresponding region of sensorimotor cortex. Studies employing GAD immunohistochemistry indicate that GABA‐ergic neurons and axon terminals are present in the BPN and thus support the suggestion that a local inhibitory interneuron is present within the BPN. Taken together these observations suggest that basilar pontine neurons might play a more active role in the integration of various types of information destined for the cerebellar cortex than has previously been recognized.
Synaptic organization of the thalamic reticular nucleus
Authors: Ohara P. T.
Published: February 2005

This study describes the synaptic organization of the thalamic reticular nucleus (TRN) in the rat, cat, and monkey using electron microscopy combined with immunocytochemistry, degeneration, or horseradish peroxidase (HRP) tracing methods. Three morphological types of terminals are described in the TRN of the rat: a small terminal with densely packed spherical vesicles (D‐terminal), which originates from the cortex; a large terminal with loosely packed spherical vesicles (L‐terminal), which originates in the dorsal thalamus; and a terminal containing flattened synaptic vesicles (F‐terminal) that is probably a TRN recurrent collateral. The cortical input to the TRN has been shown by double‐labeling studies to terminate directly upon TRN projection neurons. Similar classes of terminals are found in the TRN of cat and monkey, but there is in addition a large terminal with spherical synaptic vesicles that is invaginated by dendritic spines. Also present in the cat and monkey, but not in the rat, are vesicle‐containing dendrites and dendritic appendages.

In the rat, degeneration experiments indicate that the terminals of TRN projection neurons in the dorsal thalamus are F‐terminals. These terminals contain flattened synaptic vesicles and exhibit GABA immunoreactivity.
High‐resolution electron microscopy image simulation on a Cray 1S/2300 computer
Authors: Spycher R. , Stadelmann P. , Buffat P. , Flueli M.
Published: February 2005

High‐resolution electron microscopy (HREM) image simulation by the multislice method has been implemented on a Cray 1S/2300 computer, allowing accurate full nonlinear image intensity calculations with possible sampling up to 2n × 2m samples of the transmission function (n + m = 20). As examples of applications, images have been obtained from an interface in PbTiO3 and of a small gold aggregate. HREM image simulation of perfect or faulted crystals can now be applied with reasonable computing times to problems involving a large number of atoms and thick or large supercells.
Image analysis of electron micrographs relating to mineralization in calcifying cartilage: Theoretical considerations
Authors: Hunziker E. B. , Herrmann W. , Cruz‐Orive L. M. , Arsenault A. L.
Published: February 2005

Biological mineralization is a cell‐mediated process which is believed to be triggered by a “nucleating agent.” Various matrix structures, such as matrix vesicles, collagen fibrils and macromolecules, have been claimed to be the source of this substance, since these components have been found by transmission electron microscopy (TEM) of thin sections to be associated with early mineral crystals. Systematic image analysis of the relationships revealed in electron micrographs between specific matrix components and early mineral deposits has shown that unequivocal image interpretation is not possible. This is due principally to the problems posed by overprojection and truncation phenomena, since the structures being analyzed lie within the same dimensional range as thin section thickness. Various examples are illustrated and discussed. The site at which mineral crystals are initially laid down thus cannot be identified with any matrix structure using thin section TEM. Possible technical approaches to resolve this problem of image analysis are discussed.
Optimum specimen positioning in the electron microscope using a double‐tilt stage
Authors: Turner J. N. , Rieder C. L. , Collins D. N. , Chang B. B.
Published: February 2005

Optimal imaging of complex structures requires proper alignment relative to the optic axis of the electron microscope. This is especially important for high‐voltage and intermediatevoltage microscopes, which form an in‐focus image throughout the entire thickness of the object. As a result, structures at different specimen heights form overlapping and confused images that severely curtail the usefulness of these instruments.

The work described here provides a generalized, flexible method for optimizing specimen orientation and eliminating or limiting image overlap by means of a commonly used double‐tilt stage. Analysis of the motion about the two axes provides accurate tilting for any azimuthal direction whether or not it corresponds to a mechanical axis of the stage. An object can be positioned to minimize image overlap, to record stereopairs for any parallax axis, and to record three‐dimensional data sets by the conical collection geometry.

Images of muscle paracrystals are shown after tilting about an axis perpendicular to a symmetry direction. The tilted image displays higher‐order symmetry, which is altered by changes of one degree. Precision double‐tilting for optimizing stereopairs is shown for a desmosome recorded using different parallax axes and pretilts. A tomographic conical data‐collection scheme is demonstrated by imaging a microtubule axoneme for a specific cone half‐angle and arbitrary azimuthal angles.
Preparation of cross‐sectional transmission electron microscopy samples by electron beam lithography and reactive ion etching
Authors: Wetzel J. T.
Published: February 2005

A cross‐sectional sample preparation technique is described that relies on lithographic and dry‐etching processing, thus avoiding metallographic polishing and ion milling. The method is capable of producing cross‐sectional transmission electron microscopy samples with a large amount of transparent area (1 μm × 2.5 mm) which allows the examination of many patterned test sites on the same sample from the same chip of a silicon wafer. An example of the application of the technique is given for localized oxidation through a mask.
Experimental conditions for surface microanalysis with reflection electron energy‐loss spectroscopy
Authors: Wang Z. L.
Published: February 2005

Experimental conditions for obtaining the optimum signal‐to‐background (S/B) ratio in reflection electron energy‐loss spectroscopy (REELS) are investigated. It is shown that the S/ B ratio can be improved by lowering the incident energy of the electrons. The spectra taken from the GaAs (660) specular reflection spot under the surface resonance condition is demonstrated to have the best S/B ratio and lowest surface plasma excitation, which is capable of providing structural information on the top few atomic layers.
Removal of background label in immunocytochemistry with the apolar lowicryls by using washed protein A–gold‐precoupled antibodies in a one‐step procedure
Authors: Dürrenberger Markus B.
Published: February 2005

Antiserum mixed with protein A–gold produces aggregates that can be washed by centrifugation and redissociated by ultrasonication. These precoupled antibodies are used for immunocytochemical labeling in a one‐step procedure and reveal a very low background on sections of material embedded in polar and apolar Lowicryls. Controls have shown that the background label stems from serum components with only one protein A binding site and is discarded during the wash steps of the precoupling procedure.
Quantitative analytical electron microscopy of multiphase alloys
Authors: Prybylowski J. , Ballinger R. , Elliott C.
Published: February 2005

In this paper, we present a technique for analysis of composition gradients, using an analytical electron microscope, within the primary phase of a two‐phase alloy for the case where the second‐phase particle size is similar to the size of the irradiated volume. If the composition difference between the two phases is large, the detected compositional fluctuations associated with varying phase fractions may mask any underlying composition gradient of the primary phase. The analysis technique was used to determine grain boundary chromium concentration gradients in a nickel‐base superalloy, alloy X‐750. The technique may also be of use in other alloy systems.
Study of airway epithelial permeability with dextran
Authors: Hulbert W. C. , Forster B. B. , Mehta J. G. , Man S. F. P. , Molday R. S. , Walker B. A. , Walker D. C. , Hogg J. C.
Published: February 2005

We studied the penetration of fluorescein isothiocyanate (FITC) T‐40 dextran into the paracellular spaces in the tracheal mucosa and its appearance in the blood plasma of guinea pigs exposed to cigarette smoke or (controls) breathing room air. Under general anesthesia, the dextran solution was instilled onto the tracheal surface via a tracheotomy tube, arterial blood was sampled serially for 40 min, and then the trachea was fixed by perfusion or immersion. Examination of the tracheal mucosa with light microscopy, with and without epifluorescence, and transmission electron microscopy, revealed dextran in the paracellular spaces in mucosa from experimental animals but not controls. In all control animals, the levels of the dextran in plasma was below the sensitivity of the assay. By contrast, dextran levels in the plasma from the experimental animals fell within the sensitivity range of the assay and increased in blood at a rate of 0.00125 ± 0.00023 (SE) expressed as a percentage of the instilled dose/min. We conclude that FITC T‐40 dextran provides a reliable, fairly simple, fast method for assessing major, but not subtle, changes in paracellular permeability of the tracheal mucosa.
Observation of microdiffraction patterns with a dedicated STEM instrument
Authors: Cowley J. M. , Ou H.‐J.
Published: February 2005

A two‐dimensional detector system, designed for the observation and recording of microdiffraction patterns formed in an HB 5 scanning transmission electron microscope (STEM) is described and discussed. Possibilities are described and demonstrated for the simultaneous or successive recording of microdiffraction patterns from regions of diameter 3 å or more, bright‐ or dark‐field STEM images, EELS spectra, secondary electron images, and in‐line holograms. Applications of the system have been made to studies of catalyst particles, reflection‐mode imaging of bulk surfaces, and image reconstruction from microdiffraction patterns obtained from each point of a STEM image.
Extraction of carbon 14‐labeled compounds from plant tissue during processing for electron microscopy
Authors: Coetzee J. , van der Merwe C. F.
Published: February 2005

Loss of 14C‐labeled compounds from bean leaf tissue was monitored during all the stages of routine specimen preparation. No significant differences in extraction were associated with the use of acetone, ethanol, or dioxane as dehydration fluids. Fixation at low temperature increased the loss of label. Prolonged fixation in glutaraldehyde increased the loss, but fixation in osmium solutions for periods as long as 4 hr had no influence on extraction. Buffer rinses and dehydration fluids caused appreciable amounts of label to be extracted. The use of propylene oxide as transition fluid resulted in low extraction. Some embedding media caused the loss of small amounts of labeled compounds, but one of the media tested (LR‐white) extracted significant amounts of label.
A variation of transmission electron microscope sample preparation for VLSI analysis
Authors: Madden Michael C. , Crafard Penny
Published: February 2005

Sample preparation for VLSI analysis is often slow due to long ion milling time and because the location of the thin area of the sample is difficult to control. By modifying the standard techniques used with a VCR Group (and perhaps other) mechanical dimpler, the ion milling time can be reduced to less than 30 min. and the location on the thinned area reasonably controlled. These modifications involve the use of a radiused edge on the dimpling tool, a rubber O‐ring on the polishing tool, and not rotating the sample platen during polishing. The modifications to the dimpling and polishing tools allow more control of the geometry of the dimple, while not rotating the sample platen allows a thinner sample to be produced and permits the use of the sample translation micrometers to shift the location of the thinned area during polishing. The quality of samples produced using this modified procedure is equivalent to that obtained with the more standard methods.
An ultrathin sectioning alignment tool with application to cell monolayers
Authors: Cramer Clay T.
Published: February 2005

Details are given concerning the construction and use of a simple tool to help align a specimen block face in the vertical axis for subsequent ultrathin sectioning. Further instructions are given for the preparation of cell monolayers for ultrathin sectioning. The advantage is simple, repetitive and quick alignment of the specimen block face for ultrathin sectioning.
Study of local atomic order in amorphous materials in a computerized transmission electron microscope
Authors: Balossier G. , Garg R. K. , Bonhomme P. , Thomas X.
Published: February 2005

Experimental results obtained by electron diffraction (ED) and extended electron energy loss fine structure (EXELFS) techniques to study the local atomic order in amorphous materials such as carbon, silicon, and its oxides are described. Potential applications of ED and EXELFS techniques and their limitations are also discussed.
First stages of crystalzation in a silicate glass: Study by high‐resolution electron microscopy
Authors: Ramos Aline , Gandais Madeleine
Published: February 2005

The first stages of crystallization have been investigated for a glass in the system SiO2‐Al2O3‐Li2O with small amounts of titanium and zirconium as nucleating elements. The studies were performed by electron diffraction and electron microscopy in high‐resolution mode. It has been found that the two phases forming the final glass ceramic crystallize successively. The first one gives small crystallites (<5 nm) randomly dispersed. The lattice plane images in several orientations are in good agreement with TiZrO4 crystal lattice. At 780°C the average crystal size increases with increasing time of the heat treatments from 2.5 nm at 0.5 h to 4.5 nm at 2 h and then remains nearly constant. The crystallization of the second phase, a silicate solid solution with β‐quartz structure, occurs after complete crystallization of TiZrO4.
Direct observation of immunoreactive sites and antibody molecules by ultrahigh‐resolution scanning electron microscope
Authors: Yasuda Kenjiro , Aiso Sadakazu , Nagatani Takashi , Yamada Mitsuhiko
Published: February 2005

Using an ultrahigh‐resolution scanning electron microscope (SEM), an attempt was made to directly visualize antibody molecules at antigenic sites in rat pancreas or on silicone plates. Although individual antibody molecules could not be discerned, a fluffy meshwork, probably indicating several molecules, was seen. With further improvements in specimen preparation, the high‐resolution SEM promises to be an important tool in examining individual antibody‐antigen sites without the need of an electron‐dense label such as colloidal gold.
The research center for ultra‐high voltage electron microscopy at Osaka University
Authors: Fujita Hiroshi
Published: February 2005

High‐voltage electron microscopy has shown itself advantageous for the study of natural science, including biology, but especially for materials science. The most important advantage for materials science is for in situ experiments about the detailed processes of the phenomena that occur in bulk materials. The present paper is mainly concerned with several types of in situ experiments that have been carried out in the Research Center for Ultra‐High Voltage Electron Microscopy, Osaka University. The following subjects have been studied: (a) fundamental problems, such as the conditions necessary for in situ experiments, functional features of specimen treatment devices, and the effects of electron irradiation; (b) the dislocation behavior of crystals under various conditions; (c) high‐temperature behavior of refractory materials, mainly ceramic composites; (d) new applications of electron irradiation effects, such as amorphization of crystalline materials and electron‐irradiation‐induced foreign‐atom implantation; (e) environment‐matter interaction, mainly chemical amorphization of alloys; and (f) future trends of the in situ experiment, such as combinations with Auger valency electron spectroscopy and high‐resolution electron microscopy.
Studies of refractory carbides, nitrides, and borides as the thermionic emitters for electron microscopy
Authors: Yada Keiji , Masaoka Hideki , Shoji Yutaka , Tanji Takayoshi
Published: February 2005

Thermionic emission properties of several kinds of refractory carbides, nitrides, and borides of the transition metals in the form of powder were investigated with a newly developed measuring device and evaluated by the figure of merit defined as the ratio of the effective work function to the working temperature at which the vapor pressure becomes 1 × 10−5 Torr. There are several materials whose thermionic emission properties are better than those of tungsten or compatible to those of tungsten among the carbides and borides, such as TaC, HfC, ZrC, LaB6, and CeB6, as judged by the figure of merit. New preparation methods for carburization, nitriding, and boriding of the wires of matrix metals and alloys were successfully developed for using these materials as the cathode of the electron microscope. Other necessary techniques such as spot welding and electrolytic etching were also developed. From the brightness characteristics, it was found that some of carbides, carbide solid solutions, and borides such as HfC, ZrC (Ta0.8–0.7Hf0.2–0.3)C, TaB2, and HfB2 are very good emitters comparable to LaB6. It is emphasized that the work functions of the carbide‐solid solutions (Ta0.8Hf0.2)C and (Ta0.7Hf0.3)C, which have low rates of evaporation at high temperature, show no remarkable rise as compared with that of HfC, so that their figures of merit are better than that of HfC. Feasibility of providing good cathodes with HfC and (Ta0.8Hf0.2)C tips was demonstrated by taking high‐resolution electron micrographs.
Study of temperature factors in cubic crystals by high‐voltage electron diffraction
Authors: Matsumura Syo , Tomokiyo Yoshitsugu , Oki Kensuke
Published: February 2005

The critical voltages for systematic reflections and splits of Kikuchi lines were measured using a high‐voltage electron microscope to investigate the atomic temperature factors in cubic crystals. The split of the Kikuchi line at the intersection with the forbidden 222 Kikuchi line as well as the critical voltage of the 333 reflection for Si and Ge decreased steeply with temperature. The temperature dependence showed that the anharmonic contribution to the atomic‐temperature factor for Si and Ge is extremely weak in the temperature range 300 ∼ 1078 K. On the contrary, the B factors obtained from the measured critical voltages for Al, Cu, and Fe varied nonlinearly with temperature, suggesting the importance of the anharmonic effect in the vibration of atoms. The observed temperature dependence of the critical voltages for the metals were compared with calculations based on harmonic, quasi‐harmonic, and anharmonic approximations. The quasi‐harmonic approximation that takes into account the thermal expansion modification reproduces well the observed values for Fe but not those for Al and Cu. The effect of intrinsic anharmonic vibration should be considered for reproducing the results for Al and Cu. Fitting the measured critical voltages with the calculated ones, we estimated the values for coefficients of the isolated atom potentials. The results are in good agreement with those obtained by neutron and X‐ray diffraction.
High‐resolution transmission electron microscopy of inorganic materials in cellular and topological random systems
Authors: Shiojiri Makoto , Hirota Y. , Isshikim T. , Okashita K. , Sekimoto S.
Published: February 2005

The structure of amorphous Se films, in the topological random system, has been studied by the computer‐simulation, electron diffraction (ED), and high‐resolution transmission electron microscopy (HRTEM). As an example of HRTEM of the cellular random system, our recent investigation on Ba‐ferrites is reviewed beforehand. In HRTEM images of spin‐glass BaTi2–xSnxFe4O11 (x < 0.6), magnetic clusters or clusters of FeO6‐octahedra surrounded by TiO6‐octahedra have been found. The structure of BaSn2Fe4O11 (x = 2) has been determined. The ordering of Sn (Ti) and Fe ions increases with increasing x, which interprets the change from the spin‐glass state to nonspin‐glass state at x = 0.6. It is shown that a cluster of polarized lattice ions is detectable in images of polar‐glass BaxK2–xFexTi6–xO13 (x ≥ 1.2). The structure models of amorphous Se films forming on a substrate have been constructed on a computer, and their radial distribution function (RDF) and HRTEM images have been calculated. Experimental RDF and HRTEM images have been obtained from vacuum‐deposited amorphous Se films and are compared with the calculated ones. It is concluded that in the as‐deposited films most of Se molecules may be composed of Se atoms as small as three and that by the electron‐beam irradiation the molecules link to form spiral chains in amorphous state and then arrange to have the hexagonal crystal structure.
Immunohistochemical distribution and colocalization of regulatory peptides in the carotid body
Authors: Heym Christine , Kummer Wolfgang
Published: February 2005

Current investigations on the immunohistochemical occurrence and co‐occurrence of biogenic polypeptides in the mammalian carotid body were reviewed and extended by our own recent findings. The family of chromogranins and related peptides in glomus cells appears to have a widespread interspecies distribution, whereas other peptides investigated occur in a species‐specific pattern. Immunoreactivity to antisera against opioids, which derive from the proenkephalin sequence, appears to be present in glomus cells of the rabbit, cat, dog, and a shrew. Conversely, glomus cells of pig and guinea pig predominantly are immunoreactive to cleavage products of prodynorphin, which co‐occur in some cells with substance P and met‐enkephalin‐arg‐phe, respectively. In the rat and Callithrix jacchus, opioid immunoreactivity is present in nerve fibres but not in glomus cells. Immunoreactivity to other peptides, such as neurotensin, cholecystokinin, neuropeptide Y, and galanin, is found only in one or two particular species. Neurotensin immunolabelling occurs in beagle dog glomus cells, which are known to lack substance P. Cholecystokinin immunoreactivity is present in glomus cells of dog and Callithrix, and co‐exists with chromogranin A, neuropeptide Y, and substance P. Substance P appears to exist in both carotid body glomus cells and nerve fibres. Substance P immunoreactivity is present in glomus cells of all species investigated, except dog. Coexistence of substance P and calcitonin gene‐related peptide (CGRP) is demonstrated in nerve fibres of the guinea pig carotid body, which originate in the petrosal and jugular ganglia. Other peptides visualized immunohistochemically in mammalian carotid body nerve fibres are vasoactive intestinal peptide and neuropeptide Y. The functional significance of the various peptides present in the carotid body is discussed.
Electron microscopic localization of enkephalin‐like immunoreactivity in the human adrenal medulla
Authors: Hervonen Antti , Linnoila Ilona , Vaalasti Annikki , Alho Hannu , Pelto‐Huikko Markku
Published: February 2005

Enkephalin‐like immunoreactivity in human adrenomedullary cells was studied at the light and electron microscopic levels. Enkephalin immunostaining was associated with chromaffin granules and, in a few cells, with the rough endoplasmic reticulum as well. The relative number of stained granules varied from cell to cell, and a correlation with a particular granular population was not noted. Both large and small granules were labelled. It is concluded that in the human the ability to store enkephalin immunoreactive peptides is a general property of chromaffin granules and, furthermore, is not correlated with specific granular subpopulations or the particular type of catecholamine stored within the cell.
Theory of zone axis electron diffraction
Authors: Bird D. M.
Published: February 2005

The theory of high‐energy electron diffraction is reviewed, with an emphasis on methods that help most in understanding the form of zone axis convergent‐beam patterns. A Bloch wave description is used throughout the paper, and the theory is formulated in real space, leading to general expressions for both zero‐layer and higher‐order Laue zone (HOLZ) diffraction amplitudes. HOLZ diffraction is discussed in detail, and it is shown that first‐order perturbation theory is usually applicable and provides a clear picture of the diffraction. The computation of diffracted intensities is discussed with reference to the many‐beam equations. Diffraction at the [0001] axis in 1T‐VSe2 is analysed both to illustrate the theory and to provide an example of the special effects that occur at well defined string zone axes.
Systematic procedure for indexing HOLZ Lines in convergent beam electron diffraction patterns of cubic crystal
Authors: Fournier Daniel , L'Esperance Gilles , Saint‐Jacques Robert G.
Published: February 2005

This article has been written for experimenters who are beginning to work with convergent beam electron diffraction patterns (CBED). The indexing of the higher‐order Laue zone (HOLZ) lines inside the central spot of a CBED pattern is the first step towards studying variations in lattice parameter and three‐dimensional (3D) symmetry. To index the HOLZ lines one must construct and index the HOLZ, find its interplanar spacing and the location of its projection on the zero order Laüe zone (ZOLZ) for a given zone axis, and finally find the radius of the HOLZ ring. A computer program has been developed to perform all these tasks for cubic crystals and is very useful, especially when one varies parameters such as operating voltage, zone axis, lattice parameter, and crystalline structure.
Composite replicas: Methodologies for direct evaluation of the relationship between intramembrane and extramembrane structures
Authors: Coleman Richard A. , Wade James B.
Published: February 2005

Electron microscopic studies of membrane structure have been facilitated by the recent development of the composite replica technique in which the membrane is freeze‐fractured, then inverted and the surface deep‐etched and replicated. Examination in stereo of this composite preparation of two replicas with interposed half‐membrane and associated surface elements reveals the physical relationship between structures on the surface and within the membrane. Composite replicas of the toad urinary bladder surface demonstrated connections of filamentous glycocalyx elements to intramembrane particles (IMPs). Using a bidirectional shadowing technique, many membrane surface particles also are shown to be associated with underlying IMPs, suggesting that these membrane surface particles are projections of the IMPs above the surface of the membrane. There is evidence that elements whose attachment sites relate to the half‐membrane fractured away can be displaced from the membrane surface and lost. Labelling studies using colloidal gold‐labelled antibodies were carried out to assess loss of surface mesh from fractured membrane. Gold distributions and amounts were similar in labelled surface replicas, label‐fracture specimens, and labelled composite replicas, yet the amount of mesh detected in the composite replicas was less than in the surface replicas. This suggests that while some unlabelled or lightly labelled surface elements can be lost from fractured membranes, ligands stabilize elements and reduce their loss apparently by cross‐linking them.
Monolayer freeze‐fracture and scanning tunneling microscopy
Authors: Fisher Knute A.
Published: February 2005

This article reviews research on planar monolayer methods, application of the methods to analyses of transmembrane signaling, and the combination of these methods with scanning tunneling microscopy (STM). Past research has involved the development of monolayer freeze‐fracture methods. These include monolayer freeze‐fracture autoradiography (MONOFARG), an electron microscopic cytochemical method to analyze in‐plane distributions of radioisotopes, and double‐labeled membrane splitting (DBLAMS) and single‐membrane monolayer splitting (SMMS), methods to analyze transmembrane distributions of native and radiolabeled proteins and lipids. Present research has focussed on using these methods to investigate mechanisms of transmembrane signaling mediated by protein kinase C (PKC), including the transbilayer distribution of the tumor promoter TPA, a lipophilic activator of PKC, and the transbilayer distribution of peripheral membrane proteins phosphorylated by PKC. Future work will involve the combination of planar sample preparation with STM. The principles and applications of biological STM are briefly reviewed and a low‐resolution STM image of a planar purple‐membrane monolayer is included. The combination of planar methods and STM can provide the chemical information lacking in STM images enabling microscopists to investigate biochemical phenomena at nanometer resolution.
Surface microanalysis by reflection electron energy‐loss spectroscopy
Authors: Wang Z. L.
Published: February 2005

Several basic physical concepts of applying eq. Ik = IσNxt to surface microanalysis by reflection electron energy‐loss spectroscopy (REELS) are clarified. Here Ik and I are the integrated intensities of the core ionization edge and the low loss part, σ is the scattering cross section of element x with atomic concentration Nx, and t is the specimen thickness. The reflected inelastic electrons are found to be distributed almost symmetrically around the Bragg sports and can be reasonably described by a Lorentzian function. EELS microanalysis can be performed by using the diffracted sports. The ω correction, arising from the angular contributions of the neighbouring spots into the spectrometer collecting aperture, is required to be considered.
Use of peldri II as a sublimation dehydrant in place of critical‐point drying in fracture‐label cytochemistry and in backscattered electron imaging fracture‐label
Authors: Kan Frederick W. K.
Published: February 2005

Peldri II, a fluorocarbon compound solid at room temperature, was used as a sublimation dehydrant in place of critical‐point drying (CPD) in freeze‐fractured cytochemistry. Its applicability to the fracture‐label replica method was demonstrated using freeze‐fractured rat pancreas labeled with Helix pomatia lectin‐gold complex and phospholipase A2‐gold complex for the detection of glycoconjugates and phospholipids, respectively. No difference in morphology and labeling pattern was detected in CPD‐treated and Peldri II‐treated tissue samples. The use of Peldri II was further extended to freeze‐fractured rat duodenum labeled with wheat germ agglutininovomucoid‐gold complex. Again comparable results were obtained when duodenal samples were prepared according either to the conventional CPD method or to the Peldri II sublimation method. Freeze‐fractured replicas of hamster ovaries labeled with Ricinus communis I‐gold complex revealed that Peldri II could also be used as a sublimation dehydrant for preparing tissue samples for examination in the scanning electron microscope by the backscattered electron imaging mode.
Counting cells with stereology: Random versus serial sectioning
Authors: Bertram John F. , Bolender Robert P.
Published: February 2005

Counts of cells and nuclei from sections provide information central to studying structural changes in cells, tissues, and organs. This study considers some of the practical problems associated with counting cells with the newer random and serial sectioning methods of stereology and tests the hypothesis that similar cell counts can be obtained with both random and serial sectioning methods.

Using irregularly shaped nuclei from alveolar cells of the goat lung, we compared cell counts derived from random (electron microscopic) and serial sectioning (light microscopic) methods. The results showed that both sectioning methods gave similar cell counts (107/cm3 of parenchyma) for type 1 epithelial cells (5.0 vs. 5.0; P=1.0), type 2 epithelial cells (8.6 vs. 9.8; P=0.42) and interstitial cells (34.6 vs. 33.4; P=0.64), provided that corrections were introduced for sectionrelated biases and that the nuclei of the random sectioning method were corrected for shape. We found counting biases of 5%–7% for nuclear shape and 16% for section compression. These observations support the hypothesis that similar cell counts can be obtained with random and serial sectioning, even when nuclei have irregular shapes.
High resolution SE‐I SEM study of enamel crystal morphology
Authors: Apkarian Robert P. , Gutekunst Marc D. , Joy David C.
Published: February 2005

Until recently high resolution TEM was the only imaging mode capable of probing the atomic lattice structure of crystals composing tooth enamel. Studies designed to determine the polyhedral shape of normal enamel crystals and initiation of carious lesions in enamel crystals were hampered and limited by interpretation of two‐dimensional TEM images from thin section and freeze fracture replica specimens lacking depth of field. The newly developed SE‐I signal mode for SEM (SE‐I/SE‐II ratio) can produce images of enamel crystals approaching beam diameter dimensions (0.7–2.0 nm), rivaling the resolution of the TEM technique and generating topographic contrasts for three dimensional imaging at very high magnification (≈︁1,000,000 X). Ultrathin chromium (Cr) films generate enriched high resolution SE‐I contrasts of enamel crystal surfaces and when imaged using an immersion lens field emission SEM operated at high voltage (20–30 KeV) produce unsurpassed topographic contrasts. Since the grain size of Cr is below the resolution of any SEM and is ultrathin (≈︁1 nm), then SE‐I images can provide a more accurate representation of enamel crystal structure than TEM methodologies.

Our SE‐I SEM observations of normal human enamel crystals reveal fractured spicules which contain angled flat surfaces delineated by a prominent 2 nm wide SE‐I edge brightness contrast. Although microscopic observations often show crystals which are hexagonal in cross‐section, in both SEM and TEM many other growth habits, including rectangular or irregular crystals (30–40 nm in width) which contain “notches,” are also observed. More detailed morphological studies are therefore required to determine the most likely habit planes and their relevance to the function of the enamel crystals. The granular appearing fine structural contrast imposed onto <100> lattice planes of sectioned enamel in TEM micrographs is also resolved with topographic contrasts in SE‐I micrographs. These granules probably represent one or both of the enamel protein classes.
A specimen holder for low‐temperature scanning electron microscopy
Authors: Bastacky Jacob , Goodman Christian , Hayes Thomas L.
Published: February 2005

A miniature vise built into a copper stub is described that holds bulk, pre‐frozen, hydrated biological specimens during examination under the electron beam of the scanning electron microscope.
Letter to the Editor: Comment on “Stereo‐Imaging Made Easy”
Authors: Woodhouse J. B. , Eades J. A.
Published: February 2005

We believe that the paper in question (JEMT 11, 174, 1989) is presented in a way that is misleading.
Fine structure of hepatic sinusoids and sinusoidal cells in disease
Authors: Le Bail B. , Bioulac‐Sage P. , Senuita R. , Quinton A. , Saric J. , Balabaud C.
Published: February 2005

Liver sinusoids are special capillaries that are limited by fenestrated endothelial cells, without a genuine basement membrane, surrounded by perisinusoidal cells storing vitamin A, and harbouring Kupffer cells and pit cells, resident macrophages, and large granular lymphocytes, respectively. Each nonparenchymal cell and parenchymal cell of the liver interacts with all others and with the extracellular matrix. Therefore, the functional ability of each cell is constantly being modified by the metabolic activity of the others.

Human liver biopsies (132), needle or surgical, perfusion‐fixed with glutaraldehyde and processed for transmission electron microscopy (TEM), and occasionally for scanning electron microscopy (SEM), were examined. The study included liver diseases (such as alcoholic liver diseases, benign and malignant liver tumors, cholestasis of various origins, fulminant hepatitis, acute rejection after orthotopic liver transplantation, Budd‐Chiari syndrome), as well as general or extrahepatic diseases (such as diabetes, hemochromatosis, hypervitaminosis A, various hematological disorders), and normal controls.

Ultrastructural abnormalities are described and illustrated under two different headings: (1) elementary lesions of sinusoidal cells (endothelial, Kupffer, perisinusoidal and pit cells), nonsinusoidal cells (in the space of Disse and/or in the lumen), the extracellular matrix; and (2) the major pathological entities including perisinusoidal fibrosis, capillarization of sinusoids, sinusoidal dilatation, and peliosis. In the discussion, an overview of the major abnormalities reported in the literature is presented, and some specific questions regarding (1) perisinusoidal fibrosis in liver with normal histology, (2) the overload of perisinusoidal cells with lipids in non‐hypervitaminosis A intoxication and (3) the etiological relationship of sinusoidal dilatation, peliosis, perisinusoidal fibrosis, or sinusoidal tumors with drugs and toxic compounds are discussed. In the event that lesions are not specific to any diagnosis, the knowledge of the ultrastructure of sinusoids is extremely useful from the perspective of the liver as an ecosystem.
Lanthanum probing of cell membrane permeability in the rat heart: Pathological versus artefactual alterations
Authors: Harper I. S. , Williams K. , Lochner A.
Published: February 2005

The influence of fixation on membrane permeability has been examined in the isolated rat heart using lanthanum as a permeability probe. Normal and ischaemic hearts were probed at various stages during a conventional fixation programme with either ionic or colloidal lanthanum and compared with lanthanum saline administered prior to fixation. Fixation of the myocardium coincident with or followed by lanthanum probing resulted in an influx of the probe into most myocytes in normal tissue. Alterations in permeability after ischaemic episodes could not be distinguished from this artefact. However, lanthanum saline prior to fixation showed exclusion of the probe from normal tissue, while the increased permeability demonstrated after ischaemia was associated with declining myocardial performance during subsequent reperfusion. These results illustrate the need for caution in the application and evaluation of methods determining permeability in fixed tissue. Probes of differing size and charge permeated fixed tissue to varying degrees thereby implicating the formation of specific lesions during chemical fixation.
Comparison of osmium/sonication and edta/sonication microdissection techniques in exposing the adepithelial basal lamina surface of developing rat colon
Authors: McCarthy Kevin J. , Kaye Gordon I.
Published: February 2005

We have used two epithelial‐stripping techniques in our studies of the basal lamina in the developing rat colon. The first involves prolonged osmication followed by sonication; the second uses chelation of calcium by EDTA followed by sonication. Both techniques remove the epithelium from the basal lamina; however, the EDTA/sonication technique appears to produce a cleaner adepithelial surface of the basal lamina. In addition, the fine structure of the basal lamina appears to be better preserved in specimens prepared by the EDTA/sonication technique. In contrast, the basal lamina of specimens prepared by the osmium/sonication technique has a shattered appearance that we believe is due to an increase in the fragility of the delicate fetal basal lamina.
Monoamine synaptic structure and localization in the central nervous system
Authors: Maley Bruce E. , Engle Mary Gail , Humphreys Suzanne , Vascik Debra A. , Howes Kimberly A. , Newton Bruce W. , Elde Robert P.
Published: February 2005

The monoamines dopamine, noradrenaline, adrenaline, and serotonin as well as the diamine histamine have a widespread distribution in the central nervous system within synaptic terminals and nonsynaptic varicosities. In certain regions of the central nervous system the monoamines are contained in varicosities that have no synaptic specialization associated with them, suggesting a possible neuromodulatory role for some of the monoamines. The majority of monoamine labelled structures are synaptic terminals which are characterized by the presence of small, clear vesicles (40–60 nm) and large, granular vesicles (70–120 nm) within the terminal. A third population of vesicles—small, granular vesicles—which are visible only after histochemical staining, are probably the equivalent of the small, clear vesicles present after either autoradiographic or immunohistochemical labelling. Most monoamine containing terminals contact dendrites and dendritic spines and, less frequently, neuronal somata and other axons. Both asymmetrical and symmetrical membrane specializations are associated with monoaminergic terminals; however, asymmetrical contacts are the most frequent type found. These ultrastructural results indicate that monoamine containing terminals and varicosities in general share many common morphological features, but still have diverse functions.
Freeze‐fracture organization of hair cell synapses in the sensory epithelium of guinea pig organ of Corti
Authors: Saito Kogaku
Published: February 2005

The fine structure of both the afferent and efferent hair cell synapses in the sensory epithelium of guinea pig organ of Corti was examined by freeze‐fracture electron microscopy. In the afferent synapse, barlike aggregates of intramembrane particles (IMPs) of about 10 nm in diameter were seen on the P‐face of the afferent presynaptic membrane directly beneath the presynaptic dense projection which is located in the active zone of the presynaptic membrane. Small and large depressions have been seen on the presynaptic membrane. The former were observed in the proximity of the barlike aggregates, while the latter were observed some distance from the aggregate. In outer hair cells, IMPs of about 10 nm in diameter were seen on the P‐face of the afferent postsynaptic membrane at a density of 3,000/μm2.

In the efferent synapse, many aggregates composed of from several to tens of large IMPs of 13 nm in diameter were observed on the presynaptic membrane. These aggregates were localized to small membrane depressions, which tended to be deeper as particle number per aggregate increased.

Dense populations of IMPs of about 9 nm in diameter were observed on the P‐face of the efferent postsynaptic membrane at a density of 4,000/μm2. A fenestrated subsynaptic cistern completely covers the efferent postsynaptic membrane. Moreover, the subsynaptic cistern spans several efferent postsynaptic membranes when efferent synapses are gathered in a group.

In the afferent and efferent synapses of hair cells, specializations of the synaptic membranes were represented by marked aggregates characteristic of IMPs. In the efferent synapse, IMP movement inside the synaptic membrane was proposed in relationship to retrival of synaptic vesicle membrane. Structural relationship between the subsynaptic cistern and efferent postsynaptic membrane was revealed.
High resolution scanning electron microscopy of stereocilia in the cochlea of normal, postmortem, and drug‐treated guinea pigs
Authors: Osborne Michael P. , Comis Spiro D.
Published: February 2005

The morphology of hair bundles has been studied by high resolution scanning electron microscopy using a variety of fixatives, including glutaraldehyde, glutaraldehyde‐picrate, glutaraldehyde‐tannic acid, glutaraldehyde followed by post‐fixation in osmium tetroxide, and the osmium thiocarbohydrazide technique. Critical evaluation of several metal coatings, gold, gold‐palladium, and platinum has been carried out.

Both the surface texture of stereocilia and their cross‐links are sensitive to fixation and metal coating. We are of the opinion that glutaraldehyde gives the best general quality of fixation and preservation for all types of cross‐links. We have described three major sets of cross‐links: first, lateral links connecting stereocilia within the same row; second, lateral links connecting stereocilia of adjacent rows; and third, upward‐pointing links, one per stereocilium, connecting the tip of each shorter stereocilium to the lateral surface of the adjacent taller stereocilium. Current physiological and anatomical evidence suggests that the lateral links couple the individual stereocilia within the hair bundle so that they function as a single mechanical unit. The upward‐pointing tip links are ideally placed to respond to mechanical deformation of the hair bundle, being stretched when the stereocilia are deflected in the excitatory direction towards the tallest row and relaxed when deflected in the opposite, inhibitory direction.

Postmortem morphological changes are detected within 15 minutes of cardiac arrest and become progressively more pronounced in time. These results enabled us to distinguish specific druginduced changes which could not be attributed simply to cell death. Effects of cisplatin and kanamycin upon hair bundles are described. The work reported here is based on studies using the guinea pig cochlea. Some of the postmortem changes described have also been confirmed in human cochleas.

It is stressed that many of the postmortem and drug‐induced effects can only reliably be studied by high resolution scanning electron microscopy coupled with appropriate prearation procedures.