bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructural description of glutamate‐, aspartate‐, taurine‐, and glycine‐like immunoreactive terminals from five rat brain regions
Authors: Clements J. R. , Magnusson K. R. , Beitz A. J.
Published: February 2005

The ultrastructural localization of putative excitatory (glutamate, aspartate) and inhibitory (taurine, glycine) amino acid neurotransmitters is described in several selected rat brain regions. In general, axon terminal profiles immunoreactive for excitatory amino acids formed asymmetric synapses with non‐immunoreactive small diameter dendritic profiles or dendritic spines. In the cerebellum, both mossy fiber terminals and parallel fiber terminals were immunoreactive for glutamate and aspartate. In the hippocampus, mossy fiber terminals within the stratum lucidum of the CA3 region were immunoreactive for glutamate. Localization of glutamate and aspartate to cerebellar parallel and mossy fibers, as well as the identification of glutamate in hippocampal mossy fibers, is consistent with the excitatory nature of these fibers as described in previous physiological studies. Glutamate‐like immunoreactive terminals were also identified in subnucleus caudalis of the spinal trigeminal nucleus and in the dorsal horn of the spinal cord.

Immunoreactive axon terminals for two putative inhibitory neurotransmitters, glycine and taurine, displayed a greater number of morphological variations in synaptic structure. In the cerebellum, taurine‐like immunoreactivity was present in both basket cell axon terminals which formed symmetric synapses with Purkinje cell neurons, and in a few mossy fiber terminals which formed asymmetric synapses with dendritic spines. In the area dentata of the hippocampus, taurine‐like immunoreactive profiles formed asymmetric synapses with dendritic elements. Glycine‐like immunoreactive terminals formed symmetric synapses with cell perikarya in both the ventral horn of the spinal cord and in the cochlear nuclei, and on axon terminals in the spinal trigeminal and cochlear nuclei. In contrast, some glycine‐like immunoreactive terminals formed asymmetric synapses with distal dendritic profiles in the spinal cord and spinal trigeminal nucleus. The localization of taurine to cerebellar basket cell axons and glycine to axon terminals that synapse on ventral horn motor neuron perikarya is consistent with the hypothesis that these amino acids are functioning as inhibitory neurotransmitters at these synapses. Taurine localization to cerebellar mossy fibers and to fibers in the molecular layer of the dentate gyrus may be more consistent with a proposed neuromodulator role of taurine.
Scanning electron microscopy of nerve fibers in human fetal cochlea
Authors: Hoshino Tomoyuki
Published: February 2005

The cochleas of four human fetuses ranging 22–25 weeks gestation were studied by scanning electron microscopy (SEM) for the purpose of obtaining a better understanding of the nerve fiber arrangement in the human ear. After critical point drying, the specimens were dissected and the floor of the tunnel of Corti and the outer wall of Nuel's space were exposed for observation. Upper cochlear turns, especially the apical turn, seemed to be still immature.

Observed nerve fibers were classified into three types:

Spiral fibers: Fibers traveling basalward and following the shape of the cochlea were found in both the tunnel of Corti and Nuel's space and believed to be the afferent nerves responsible for innervating the outer hair cells

Radial fibers: radiating outward from the osseous spiral lamina—one such radial fiber transversing high in the tunnel space (supposedly the efferent nerve servicing the outer hair cells), and another sort of radial fiber (found crossing the tunnel floor), the nature of which was uncertain.

Irregular fibers: Consisting of thin, randomly running fibers within the cochlea. The destination of these fibers was not determined, but possibly they represent transitory nerve branchings of afferent or more probably efferent nerves, which would later regress during maturation.
Auditory hair cells in human fetuses: Synaptogenesis and ciliogenesis
Authors: Lavigne‐Rebillard Mireille , Pujol Rémy
Published: February 2005

This paper brings together the most recent findings concerning the development of human fetus cochlear hair cells, as observed using transmission and scanning electron microscopy (TEM and SEM). Specific attention is drawn to the formation of synapses and the growth of stereocilia. In both types of hair cells, synaptogenesis begins before ciliogenesis (week 10 of gestation versus week 12). In the inner hair cell (IHC), an adultlike stage is seen around week 15 for synapses, but not before week 22 for stereocilia. In the outer hair cell (OHC), both synapses and stereocilia are not yet fully mature at week 22. Classic gradients of maturation are found: a base‐to‐apex gradient, and an IHC‐to‐OHC gradient. By comparing these results with the anatomical and functional data on cochlear development in laboratory mammals, the onset of cochlear function in the human fetus can be estimated to occur around week 18. The completion of cochlear maturation based upon the same anatomical criteria should occur about 10 weeks later.
Ultrastructure of the intercalated body, a novel organelle associated with fluid forming cells in the organ of Corti
Authors: Sobkowicz Hanna M. , Holy Jon , Scott Grayson L.
Published: February 2005

The intercalated body is a newly discovered organelle in the inner and outer spiral sulcus cells of the mouse organ of Corti. The organelle was found in the cochleas of 14‐day and older intact mice and in organs in culture of corresponding ages. The organelle consists of a stack of interconnected cisternae of endoplasmic reticulum and of membrane bound rodlets that are intercalated between, and run parallel to, the cisternae. The cisternal membranes are predominantly smooth, but some may display ribosomes. Most rodlets are from 1 to 2 μm long, about 0.1 μm wide, and contain electron dense material. Mitochondria are commonly associated with or incorporated into the organelle. Some electron micrographs suggest that the rodlets may originate from modified mitochondria. It is our impression that the formation of the organelle begins with the apposition of cisternae and mitochondria. Cisternal‐associated mitochondria appear to constrict, elongate, and lose their inner membranes. In both the intact animal and in culture, the cells of the inner and outer spiral sulci display microvilli, apical junctional complexes, lateral intercellular spaces containing interdigitating cell processes, and appear to be involved in fluid formation. Moreover, in culture, the cells of inner and outer spiral sulci as well as some cells proliferating in the outgrowth zone participate in fluid formation, producing large fluid pockets. All these cells commonly contain intercalated bodies. It is possible that in the intact animal, as in culture, intercalated bodies may play a role in fluid regulation in the immediate vicinity of the hair cells.
Amorphisation of MgO single crystal specimens prepared by ion milling for transmission electron microscopy studies
Authors: Khan M. Younis , Brown L. M. , Chaudhri M. M.
Published: February 2005

Single crystal MgO specimens having low load Vickers indentations were thinned in an ion milling machine employing a single ion gun, and their characteristics were investigated with optical microscopy and high voltage electron microscopy (HVEM). It was found that the state of cleanliness of the specimen chamber of the ion milling machine had a very marked influence on the quality of the thinned specimens. If the specimen chamber was not well cleaned before ion milling a fresh specimen, the latter tended to show amorphisation due to the deposition on the specimen of the debris left in the chamber from the previously ion‐milled specimens. Such observations were made from MgO specimens ion milled in several different types of commercial ion milling machine employing a single gun. It is proposed that to obtain good‐quality ion milled TEM specimens, it is important to clean the specimen chamber thoroughly prior to milling.
Renewal of the intestinal epithelium: New aspects as indicated by recent ultrastructural observations
Authors: Altmann G. G.
Published: February 2005

This article is a summary of our work of several years on the renewal of the intestinal epithelium. A combination of ultrastructural, radioautographic, and light microscopic analyses was carried out using normal tissue and tissue affected by inhibitors of RNA and protein synthesis. Measuring protein synthesis by 3H‐leucine radioautography showed that the life span of the columnar (absorptive) cells in the rat small intestine was divisible into two main phases: differentiation (from stem to functional cell) and maturation (from functional to extruding cell), each phase and its subdivisions being well defined morphologically. Differentiation involved a linear rise in the rate of protein synthesis per cell and showed at the same time heterochromatinization and silencing of RNA transcription. Data from various experiments indicated that the cells functioned from stored information (RNA), part of which came from the nucleolus, which underwent marked and characteristic ultrastructural changes. Although transcription from rDNA ceased, the nucleolus released its ribosomal material, which added to the existing protein synthesis, presumably by recruiting excess stored mRNAs. Maturation involved a nearly linear decrease of the rate of protein synthesis per cell to a characteristic low value at which extrusion took place. A gradual exhaustion of the stored RNA was indicated to be the key factor in this decrease. Ultrastructurally, maturation was associated with a gradually increasing vesiculation of rER and Golgi. The results thus imply a regulatory role of cellular protein synthesis level in renewal. This would be an epigenetic response after the genes are silenced. The nucleolus seems to play a central role in this process, and this in turn is reflected in its characteristic ultrastructural changes. The work also included new observations on the epithelium of the rat ascending colon describing a hitherto unrecognized deep crypt mucus‐secretory (“DCS”) cell which is a nongoblet mature cell type apparently arising from midcrypt mitoses. In between the DCS cells, occasional slender columnar cells were seen which displayed the ultrastructural features of stem cells. These were probably reserve stem cells. We also observed nongoblet deep crypt mucous cells in the human right colon although fewer in number than in the rat. Nucleolar regulation and the presence of reserve stem cells represent new dimensions in our understanding of renewal. Electron microscopy is an essential tool in this investigation.
Extrusion of colonic epithelial cells in vitro
Authors: Baron David A. , Miller Donald H.
Published: February 2005

Rat colonic mucosae fixed in situ in Ussing chambers provided a model of the extrusion of absorptive enterocytes and less commonly of goblet and enteroendocrine cells. The cells were lost at extrusion zones midway between crypt mouths. Even in mucosae in which the number of extruding cells was large, epithelial continuity was maintained as evaluated morphologically and electrophysiologically. Beneath points of remaining contact between desquamating cells and the epithelial sheet, microfilaments of the terminal web formed band‐like structures linking adjacent junctional complexes. Freeze‐fracture replicas disclosed extensive macular regions of tight junction strands in the plasma membranes of desquamating cells. Tight junctions between newly neighboring cells were often irregular and often occurred beneath the terminal web region. Dithio‐threitol enhanced cell loss and increased basal epithelial conductance, but histological continuity was maintained and the mucosae continued to respond typically to bradykinin. These observations suggest that during the loss of senescent enterocytes, tight junctions are maintained; old junctional elements are lost, and tight junctions are formed between remaining adjacent cells. This model offers a means to study the synthesis and turnover of tight junctions and the maintenance of the colonic epithelial barrier.
Striated brush border of intestinal absorptive epithelial cells: Stereological studies on microvillous morphology in different adaptive states
Authors: Mayhew Terry M.
Published: February 2005

Different regions of small bowel were examined in five groups of rats in three separate experiments. The effects on mucosal morphology of position along the bowel, induced hypoproliferation (due to fasting), and induced hyperproliferation (due to streptozotocin diabetes) were investigated.

Intestines fixed by in situ perfusion with buffered glutaraldehyde were sampled by strictly randomised procedures. Pieces of tissue from segments of roughly equal length were processed for electron microscopy and embedded in resin. Complete transverse sections were cut for light microscopy and estimates of villous surface areas were obtained by stereological methods devised for the purpose. Ultrathin sections from random sectors of the same tissue blocks were sampled systematically to obtain micrographs of the villous surface. These were analysed for quantitative information about microvilli (length, diameter, surface area, and number). Structural quantities from individual segments were pooled to provide values for the entire small bowel.

Significant regional differences in villous and microvillous dimensions were found in all groups. The numbers of microvilli per bowel were remarkably constant in all control groups. Other variables were estimated reproducibly in rats of the same sex, strain, and average body weight.

Effective absorptive surfaces did not show a linear gradient but tended to peak in middle segments. Neither fasting nor induced diabetes altered the mean length, diameter, or packing density of microvilli. However, surfaces due to villi and microvilli altered commensurately during fasting and induced diabetes. Therefore cell number seems to be the key quantity for determining villous and microvillous surface areas.

The findings are discussed in the context of kinetic, biochemical, and physiological changes found in different adaptive states.
Fast processing of black and white films used in the EM lab
Authors: Fermin Cesar D. , Diaz Ellis , Maldonado Aura , Martin Dale , Hruska Zuzana
Published: February 2005

Kodak black and while film emulsions 4489, 4162, 2415, TMAX‐100, TMAX‐400, MPD‐4 and RPC‐651 can be developed in less than 10 minutes on a film processor, quickly printed on resin coated paper and dried with auto‐processor and dryer. Adjustments to the ASA (ISO) to compensate for increased contrast with auto‐processing are described.
A review of echinoderm oogenesis
Authors: Smiley Scott
Published: February 2005

This review of the anatomical, histological, biochemical, and molecular biological literature on echinoderm oogenesis includes the entire developmental history of oocytes; from their inception to the time they become ova. This is done from a comparative perspective, with reference to members of the five extant echinoderm classes; crinoids, holothurians, asteroids, ophiuroids, and echinoids. I describe the anatomy and fine structure of the echinoderm ovary, with emphasis on both the cellular relationships of the germ line cells to the somatic cells of the inner epithelium, and on the neuromuscular systems. I review the literature on the growth of oogonia into fully formed oocytes, including the process of vitellogenesis, presenting an ultrastructural analysis of the organelles and extracellular structures found in fully formed echinoderm oocytes. Echinoderm oocyte maturation is reviewed and a description of the ultrastructural, biochemical and molecular biological changes thought to occur during this process is presented. Finally, I discuss oocyte ovulation, the severing of cellular connections between the oocyte and its surrounding somatic epithelial cells.
Ultrastructure of sperm, spermiogenesis, and sperm‐egg interactions in selected invertebrates and lower vertebrates which use external fertilization
Authors: Koch Robert A. , Lambert Charles C.
Published: February 2005

This review discusses the ultrastructure of sperm with reference to their development, the surface morphology of the egg, and the processes of sperm binding and penetration during fertilization. These topics are treated for selected invertebrates and lower vertebrates which live in aquatic environments and fertilize their eggs externally. Specifically, sperm and eggs from cnidarians, echinoderms, decapod crustaceans, ascidians, lampreys, bony fishes, and amphibians are discussed. Sperm from the majority of these groups exhibit the classical head‐midregion‐tail configuration characteristic of primitive sperm. Specific variations within this general morphology have been described. The notable exceptions to the primitive‐sperm paradigm are the sperm of decapod crustaceans and amphibians. Eggs from all of the animals considered are covered by complex vitelline envelopes except those of cnidarians. In general, the ultrastructural analysis of these egg envelopes shows that they are composed of fibrous subunits. Sperm bind to the vitelline envelope and then penetrate through it to fertilize the egg in all groups reviewed except fishes. In fishes, the sperm reaches the egg surface via micropyles. Finally, we discuss the specific changes in sperm ultrastructure which occur during penetration of the egg envelopes in both flagellated and non‐flagellated sperm. These changes, which involve membrane fusion and reorganization as well as movement of membranous organelles, aid the sperm in reaching the actual site of gamete fusion.
The preparation of cross‐sectional transmission electron microscopy specimens of Nb/Al multilayer thin films on sapphire substrates
Authors: Barmak Katayun , Rudman David A. , Foner Simon
Published: February 2005

We have developed a technique for preparation of cross‐sectional transmission electron microscopy samples of reacted and unreacted Nb/al multilayer thin films on sapphire substrates. The choice of substrate was found to be extremely important. Sapphire sputters more slowly than Nb and Nb‐compounds and therefore makes it possible to obtain the electron transparent regions in the thin films rather than in the substrate. However, the brittle nature of the sapphire restricts the types of thinning techniques that can be used, requiring extensive ion thinning as a final stage.
Occurrence and developmental consequences of aberrant cellular organization in meiotically mature human oocytes after exogenous ovarian hyperstimulation
Authors: Van Blerkom Jonathan
Published: February 2005

The extent to which fertilization failure in in vitro inseminated human oocytes obtained from hyperstimulated ovaries was associated with alterations in cellular structure and cytoplasmic organization was examined by a combination of DIC, fluorescence, time‐lapse video, and transmission electron microscopy. Detailed analysis of meiotically mature preovulatory (uninseminated) oocytes, and of oocytes that failed to fertilize in vitro, indicated that between 10% and 15% of grossly normal‐appearing MII‐stage oocytes displayed one of the following cellular perturbations: (1) a subtle change in organelle distribution, (2) a small region(s) of intracellular cytolysis, (3) a massive aggregation of SER tubules, (4) an accumulation of vesicles presumed to be of SER origin, (5) a change in the structural organization of the cortical cytoplasm and overlying plasma membrane, (6) a sudden and rapid internalization of perivitelline fluid by means of an apparently aberrant process of endocytosis, and (7) a premature and partial exocytosis of cortical granules. The description of these disorders is discussed with respect to the developmental consequences for the oocyte.
Use of microwave fixation in the preparation of cell cultures for observation with the scanning electron microscope
Authors: Argall Kristin , Armati Patricia
Published: February 2005

This paper describes a method for primary fixation of cultured cells for scanning (SEM) and transmission (TEM) electron microscopy using microwaves alone. This method of fixation takes 8 seconds and is therefore quicker and less expensive than conventional fixation techniques. The preservation of cell morphology is excellent and cultures of mammalian immune system cells and peripheral nervous tissue have been examined using this fixation.
Evaporation during preparation of unsupported thin vitrified aqueous layers for cryo‐electron microscopy
Authors: Cyrklaff Marek , Adrian Marc , Dubochet Jacques
Published: February 2005

Evaporation of water cannot be fully avoided when an unsupported thin vitrified film of an aqueous suspension is prepared for cryo‐electron microscopy. This results in increasing concentration of solute which could affect the observed material. We have quantitatively studied this effect by measuring the contrast of polystyrene spheres in a metrizamide solution. The drying effect is generally negligible when specimens are prepared on a hydrophilic perforated support but it is frequently important when hydrophobic films are used instead. A flow of humid air, double blotting with minimal exposure of the thin liquid film to the atmosphere, or an automatic plunger optimizing the blotting conditions are simple methods for reducing drying effects. With this third device acting on a hydrophilic supporting film, the increase of solute concentration is limited to less than 20%.
Comparisons of golgi structure and dynamics in plant and animal cells
Authors: Griffing Lawrence R.
Published: February 2005

The Golgi apparatus of both higher plant and animal cells sorts and packages macromolecules which are in transit to and from the cell surface and to the lysosome (vacuole). It is also the site of oligosaccharide and polysaccharide synthesis and modification. The underlying similarity of function of plant and animal Golgi is reflected in similar morphological features, such as cisternal stacking. There are, however, several fundamental differences between the Golgi of plant and animal cells, reflecting, in large part, the fact that the extracellular matrices and lysosomal systems differ between these kingdoms. These include (1) the form and replication of the Golgi during cell division; (2) the disposition of the Golgi in the interphase cell; (3) the nature of “anchoring” the Golgi in the cytoplasm; (4) the genesis, extent, and nature of membranes at the trans side of the stack; (5) targeting signals to the lysosome (vacuole); and (6) physiological regulation of secretion events (constitutive vs. regulated secretion). The degree of participation of the Golgi in endocytosis and membrane recycling is becoming clear for animal cells, but has yet to be explored in detail for plant cells.
Structure and function of the extracellular matrix of anuran eggs
Authors: Hedrick Jerry L. , Nishihara Tatsuro
Published: February 2005

The extracellular matrix (ECM) surrounding the anuran egg is composed of jelly coat layers, an envelope, and the perivitelline space, which separates the envelope from the egg plasma membrane. Both the jelly coat layers and egg envelopes are required for fertilization in anurans. This paper reviews the current understanding of the structure‐function relations of the ECM, with emphasis on the egg envelope. The fibrous egg envelope exists in four related forms. The envelope forms differ in their ultrastructures, macromolecular compositions, and cellular functions. After the oocyte is released from the ovary, conversion of one envelope form to another is brought about by factors secreted by the oviduct prior to fertilization and by factors released from the egg in the sperm‐triggered cortical reaction. An additional extracellular matrix structure, located in the perivitelline space, has recently been identified in Xenopus laevis, as well as a previously undescribed reorganization of envelope fibers occurring at fertilization. The molecular changes in the ECM glycoproteins (limited proteolysis, lectin‐ligand binding, and conformational changes) and the oviductal and egg macromolecules responsible for the conversion of envelope forms are discussed. New experimental evidence that supports the lectin‐ligand hypothesis for the formation of the fertilization layer is presented. It is proposed that the molecular changes in the ECM are responsible for the ultrastructural alterations of the ECM and for modifications of the fertilization and developmental functions of the anuran egg ECM.
Discussions on the multiple‐input maximum a‐posteriori wave‐function restoration method in high‐resolution electron microscopy
Authors: Huang Xu , Ximen Jiye
Published: February 2005

The multiple‐input maximum a‐posteriori (MIMAP) wave‐function restoration method in high‐resolution electron microscopy proposed by Kirkland (1984) is further discussed. A modified MIMAP criterion that considers likely correlations between individual micrographs in a series of micrographs taken at different imaging conditions is presented. By performing wave‐function restoration for a multislice method‐simulated defocus series of MgO[110], the convergent properties of this modified MIMAP method are also described.
An easy method for orientated embedding tissue culture cell monolayers in LR white resin for postembedding immunocytochemistry
Authors: Hirst Elizabeth M. A.
Published: February 2005

Partial polymerization of LR White resin blocks is a frequently encountered problem if oxygen is present during the polymerization reaction. Instructions are given for a simple method to embed cell culture monolayer in LR White acrylic resin, which is suitable for immunocytochemistry. The advantage of this method is that it is easy and reliable. It also ensures accurate orientation of the cell monolayer in relation to the desired plane of sectioning.
Confocal fluorescence microscopy and three‐dimensional reconstruction
Authors: Wright S. J. , Schatten G.
Published: February 2005

Several recent technological advances have considerably improved the field of confocal fluorescence microscopy. Improvements in confocal microscope design, new fluorescent probes and indicators, more sensitive imaging devices, and computer advances which allow for data manipulation and storage provide a convenient method to acquire complex three‐dimensional (3‐D) architectural details which previously were difficult or impossible to obtain from biological specimens. Applications of the laser scanning and tandem scanning confocal microscopes offer the potential for gaining powerful insights into the complex relationship of cellular structure and function. Confocal microscopy generates optical sections free from out‐of‐focus blur. With the development of new visualization tools to render and display complex 3‐D data, a set of optical sections taken at different focal planes can be three‐dimensionally reconstructed to create an animated sequence which can reveal latent features of the specimen. The combination of confocal microscopy and 3‐D reconstruction provides a powerful new imaging tool to advance knowledge about structural and functional cellular properties as they occur dynamically in three dimensions.
Application of confocal scanning laser microscopy in experimental pathology
Authors: Smith Gary J. , Robert Bagnell C. , Bakewell William E. , Black Kurt A. , Bouldin Thomas W. , Earnhardt Todd S. , Hook Gary E. R. , Pryzwansky Katherine B.
Published: February 2005

Confocal scanning laser microscopy (CSLM) represents an exciting new tool for scientific disciplines which focus on mechanistic studies such as experimental pathology. Enhanced resolution in the specimen plane and rejection of out‐of‐focus fluorescence flare allow analysis of specific nucleic acid sequences, enzymes, structural macromolecules, and cellular homeostasis utilizing fluorescent probes. Four different experimental applications are discussed which utilize CSLM to evaluate pathological processes at the subcellular, cellular, and tissue levels. Programmed cell death, or apoptosis, is a natural process of significance both during development and as a response to toxic stimuli. CSLM‐imaging of nuclei of human B lymphoblastoid cells following exposure to a monofunctional alkylating agent suggests that the degradation of chromatin characteristic of apoptosis may occur in asymmetric patterns. Surfactant apoprotein‐A is the major non‐serum protein component of pulmonary surfactant and is essential for the extracellular function of surfactant. CSLM of alveolar type II cells suggests that apoprotein‐A is present in both the cytoplasm, predominantly in lamellar bodies, and in the nucleus. The tumor promoter, phorbol myristate acetate, rapidly stimulated the formation of vacuoles in human neutrophils. CSLM using Lucifer Yellow as a probe suggests that cylindrical vacuoles are formed by fluid‐phase pinocytosis. The blood‐nerve barrier (BNB) in peripheral nerves may be an important target during toxin‐induced neuropathies. Ricin‐induced permeability of the BNB in the rat was rapidly visualized by CSLM as leakage of fluorescein isothiocynate (FITC)‐dextran into the endoneurial compartment.
Large‐area plan‐view sample preparation for gaas‐based systems grown by molecular beam epitaxy
Authors: Howard David J. , Paine David C. , Sacks Robert N.
Published: February 2005

We describe a method for plan‐view transmission electron microscopy (TEM) sample preparation that takes advantage of extreme etch‐rate selectivity in GaAs and AlAs in HF/H2O solutions. GaAs/InxGa1‐xAs/GaAs strained‐layer films (x = 0.05, 0.10, 0.19, 0.22) were chemically lifted off using this technique and were mounted on Cu TEM grids such that TEM transparent areas of up to 1 × 2 mm of constant thickness (196.4 nm) could be viewed. This simple, large‐area plan‐view technique uses only chemical methods and significantly extends the usefulness of TEM for the evaluation of crystal quality in GaAs‐based epitaxial systems. The method requires the growth of a release layer of AlAs (10 nm thick) prior to the layered structure of interest.
Comparisons of the low‐resolution structures of ornithine decarboxylase by electron microscopy and X‐ray crystallography: The utility of methylamine tungstate stain and butvar support film in the study of macromolecules by transmission electron microscopy
Authors: Stoops James K. , Momany Cory , Ernst Stephen R. , Oliver Robert M. , Schroeter John P. , Bretaudiere Jean‐Pierre , Hackert Marvin L.
Published: February 2005

The structure of ornithine decarboxylase (Mr ≈︁ 1.04 × 106) from Lactobacillus 30a was investigated by electron microscopy and x‐ray crystallography. Electron micrographs showed the structure to be well preserved in methylamine tungstate stain. The molecules interacted little with the Butvar support film, yielding three unique projections: a hexagonal ring (front view) and two rod‐shaped projections (edge views). Stereo pairs revealed a novel feature of the Butvar film in that some molecules were suspended in the stain in random orientations. Consequently, the relatedness of the hexagonal ring and the rod‐shaped particles could be demonstrated since some particle shapes interconverted when the stage was tilted ± 45°. The two edge views were related by a 30° rotation about the sixfold axis. Image averaging of the three primary views suggested a dodecamer (point group symmetry 622) composed of two hexameric rings, apparently in an eclipsed configuration. To investigate the structural organization of the complex, the dissociation of the enzyme was studied by electron microscopy. The dissociation process involved the initial breakage of the ring followed by separation of dimers from the ring (one subunit from each of the two hexamers). Thus, the dodecamer forms as a hexamer of dimers rather than a dimer of hexamers. These structural studies were confirmed and extended by x‐ray crystallographic analysis. A 4.0‐Å resolution electron density map revealed two hexameric rings, consisting of six closely associated dimers, tilted approximately 10° with respect to the molecular twofold axis. Electron density projections of the three primary views of the molecule derived from the x‐ray data corresponded closely to those obtained from image averaging of the electron microscopy data, thereby establishing in a novel way the reliability of the electron microscopy studies. Methylamine tungstate stain and Butvar support film therefore offer unique advantages for investigating protein structures by electron microscopy.
A rapid method for the preparation of yeast for immunoelectron microscopy using lowicryl hm‐20
Authors: Anderson Wayne H. , Thompson Ed W. , Zwizinski Craig W.
Published: February 2005

We describe a fixation and embedding procedure for the yeast Saccharomyces cerevisiae using Lowicryl HM‐20 which is rapid, gives excellent fixation, and avoids the low temperature handling normally associated with embedding in this resin. This procedure yields superior structural preservation when compared to the commonly used rapid embedding procedure which employs Lowicryl K4M. We demonstrate that sections prepared using our rapid procedure are suitable for use in immunogold labelling experiments.
Preservation of EDTA‐expanded grid‐mounted chromosomes and nuclei for electron microscopy using a specially designed freeze‐dryer
Authors: Woods Philip S. , Ledbetter Myron C. , Tempel Neal
Published: February 2005

We describe methods for freezing and drying EDTA‐expanded, fixed metaphase chromosomes and nuclei, attached to grids as whole‐mounts, for transmission electron microscopy. These methods use a special apparatus that is simple to construct. While separate freezers and dryers are commercially available, one for freezing blocks of tissue by slamming them against a cold metal surface, and the other for vacuum drying the frozen tissue, our apparatus is designed for gentler, cryogenic liquid plunge freezing and drying, sequentially, in the same apparatus, thus avoiding any compression or damage to the sepcimen. Use of a cryoprotectant is not essential; however, good results are obtained more often when 20% ethanol is used. Freezing is accomplished by rapid propulsion of the grid, with specimens attached, into slushy N2 (‐210°C) within the drying chamber; drying is automatic, by either sublimation under vacuum or by solvent substitution using absolute ethanol followed by acetone, which, in turn, is removed with a critical‐point dryer. The apparatus offers a means of drying chromosomes and nuclei in an expanded state, and avoids the shrinkage of these structures that occurs during stepwise passage through increasing concentrations of ethanol or acetone.
The structure of the developing chick retinal pigment epithelium revealed by high resolution scanning electron microscopy
Authors: Weiser B. A. , Hollenberg M. J. , Mandelcorn M. , Temkin R. J. , Lea P. J.
Published: February 2005

The retinal pigment epithelium (RPE) in the developing eye of chick embryos has been studied during the early stages of development by high resolution scanning electron microscopy (HRSEM). Specimen preparation techniques which involve removal of the cytoplasmic matrix permitted visualization of organelles and other subcellular structures within RPE cells in detail and in three dimensional (3‐D) stereo HRSEM. Using this technique, we were able to examine changes in melanosome structures during development and demonstrate that pigmentation in the RPE was present by day 4 of development. RPE plasma cell membranes showed extensive folding of the apical portion of the membrane closest to the developing neural retina by day 9. Examination of RPE photoreceptor junction revealed photoreceptor inner segments by day 6 and an outer segment by day 9. Mitochondria in the RPE were found to contain tubular cristae only. The ultrastructure in 3‐D of the Golgi apparatus, smooth and rough endoplasmic reticulum, lysosomes and nuclear chromatin of the RPE, and Bruch's layer was revealed by the HRSEM method.
A new computer‐assisted three‐dimensional reconstruction method provides accurate measurement of glomerular mesangial volume
Authors: Cho Carolyn R. , Winslow James , Whiteside Catharine , Lumsden Charles J.
Published: February 2005

Many glomerulopathies are characterized by progressive mesangial (interstitial) expansion which can be quantitated by morphometric analysis. The purpose of this study was to analyze mesangial and glomerular volumes using a new computer‐assisted reconstruction (CAR) method. CAR was compared to two standard planar methods, point‐counting and linear integration, for accuracy and time efficiency. In Phase I of the study, a computer‐based model of the mesangial space was created by placing spherical and ellipsoidal objects of known volume into an enclosing volume mimicking the glomerulus. The simulated mesangium occupied approximately 10 percent of the glomerular volume. The model glomerulus was sectioned serially into ten sections of equal thickness and the three morphometric methods applied to determine the mesangial/glomerular volume.

The complexity of the mesangial model was varied by increasing the number of mesangial regions from one to ten to 100. The CAR method estimated the model mesangial volume more accurately (1–9 percent error) through each level of complexity compared to point‐counting (3–17 percent error) and linear integration (3–18 percent error). The point‐counting method consistently overestimated (P < 0.05) the fractional mesangial volume for the ten‐ and 100‐region mesangium models.

In Phase II of the study, a normal rat glomerulus was sectioned serially (215 sections) and a transmission electron micrograph (TEM) of every fifth section (n = 43) was obtained. Each TEM image (2% of glomerular surface) was digitized for analysis by CAR. Point‐counting and linear integration were also performed on the whole glomerular TEMs (n = 10, randomly chosen). The estimated relative mesangial/glomerular volume was 6.6 ± 0.1 percent by CAR (x̄ ± SD), 9.7 ± 1.5 by linear integration, and 14.9 ± 3.4 by point‐counting. The point‐counting method was most efficient, requiring 40 ± 8 sec/section, followed by CAR at 85 ± 24 sec/section. Linear integration was least efficient (93 ± 23 sec/section).

We conclude that CAR is the most accurate morphometric method of the three compared for estimating mesangial and glomerular methods, although it is more time consuming than the point‐counting method and requires more complex instrumentation. CAR is the only method that will analyze the shape and three‐dimensional complexity of glomerular structures using TEMs.
The use of freeze‐substitution and lr gold in the study of rye grass (Lolium perenne) pollen
Authors: Martinez Liza B. , Wick Susan M.
Published: February 2005

Pollen grains of Lolium perenne (rye grass) were prepared for transmission electron microscopy by rapid freezing in liquid propane, substitution in acetone, methanol or diethyl ether, and embedment in the acrylic resin London Resin gold. These were compared to pollen chemically fixed (CF) in aldehyde/osmium tetroxide and embedded in the epoxy resin Quetol 651. Ultrastructural preservation was superior in freeze‐substituted (FS) pollen, particularly with the use of acetone or methanol. Optimally preserved FS pollen displayed a homogeneous aspect of the cytoplasm and nucleoplasm, and smooth, uninterrupted contour or organelles. A striking difference was also seen in the preservation of inclusions in the intine. Varied forms and sizes of intine inclusions were evident in FS pollen but these were not discernible in the CF image. The FS scheme studied here presents enormous potential for both ultrastructural and immunolabelling studies in rye grass pollen. Problems discussed include artifacts associated with each of the substitution solvents used, and a gradient of freezing damage observed within the pollen grain.
Primitive unit cell volumes obtained from unindexed convergent‐beam electron diffraction patterns
Authors: Le Page Y. , Downham David A.
Published: February 2005

Provided that multiple reflection is present, a common occurrence with electron diffraction on microcrystallites, a single unindexed convergent‐beam electron diffraction (CBED) pattern taken with the beam axis parallel to any direct lattice row allows the volume of the primitive cell to be calculated. The film measurements required are the diameters of the successive high‐order Laue zones (HOLZ), the lengths of any two coprime vectors in the zero‐order Laue zone (ZOLZ), and the angle between them. The primitive cell volume is an objective criterion allowing in a simplification in the identification of a phase under study by rapidly eliminating other possible phases. The computer program CELVOL for the calculation of the primitive cell volume from film measurements, or from literature cells, is available from the authors.
High resolution biological scanning electron microscopy: A comparative study of low temperature metal coating techniques
Authors: Hermann René , Müller Martin
Published: February 2005

Structural information on the surface of biological specimens can be resolved within molecular dimensions by “in‐lens” field emission scanning electron microscopes when cryo‐methods are used to adequately preserve the native state of the specimen. The visual definition of molecular surface structures depends largely on the metal coating. The thickness of the coating, as well as the temperature at which it is deposited, are among the most important parameters affecting visual definition. These were evaluated on T4 polyheads and T4D phages using chromium double‐axis rotary shadowing (DARS). Micrographs of optimally DARS coated T4 polyheads and T4D phages were compared with chromium planar‐magnetron sputtering (PMS) and unidirectional shadowing with platinum/carbon. Metal deposition was carried out at low temperatures during all three procedures.

Optimal visual definition of structural details on the surface of DARS coated T4 polyheads and T4D phages (capsomeres of T4 polyheads and their subunits with diameters of 8 and 3 nm; T4D phage tail fibres with a thickness of 3 nm) is achieved at a thickness of the chromium film greater than the minimum required for metal film coalescence. Chromium DARS coating at room temperature resulted in poor structural definition, whereas DARS at specimen temperatures of −85°C and −150°C, with the chromium thickness optimized for each temperature, yielded good visual detail of polyhead substructures. The visual definition was slightly reduced when DARS coating was carried out at a specimen temperature of −250°C. Adequate structural visibility of T4D phage and T4 polyhead surface structures was achieved with the three coating techniques tested. Smaller filamentous structures, however (e.g., phage tail fibres), were more clearly identified after chromium DARS coating or unidirectional platinum/carbon shadowing than after optimized PMS with chromium. Each method has its own merits.
Ultrastructure of the seminiferous epithelium and intertubular tissue of the human testis
Authors: Kerr J. B.
Published: February 2005

The ultrastructural features of the human testis are reviewed with emphasis upon the process of spermatogenesis and the cytology of the Leydig cells. The seminiferous epithelium is structurally partitioned by the Sertoli cells into basal and adluminal compartments via the specialized tight junctions between the Sertoli cells. Spermatogonia reside in the basal compartment, and, via a series of cell divisions, produce the primary spermatocytes, which at the commencement of their development move into the adluminal compartment, and thus the lengthy process of meiotic maturation is initiated. The fine structure of primary spermatocytes is described together with the complex transformation of the spermatids into spermatozoa during the process of spermiogenesis. Earlier studies of the organization of the human seminiferous epithelium showed that germ cells at different developmental stages formed identifiable collections termed cell associations or stages, but since several stages were seen in a single tubule cross‐section, this gave the impression of an extremely irregular pattern of spermatogenic development. When the topographic arrangement of germ cells was re‐examined with the aid of computer modelling, a highly ordered distribution was revealed, conforming to a helical pattern based on the geometry of spirals. Thus spermatogenesis in the human testis is subjected to a precise regulation in keeping with the ordered arrangement of the germ cells seen in other mammalian species. The intertubular tissue of the human testis is composed of loose connective tissue containing blood vessels, occasional lymph capillaries, macro‐phages, mast cells, and the Leydig cells which occur either as single cells or form small clusters. The Leydig cell cytoplasm contains an abundant supply of smooth endoplasmic reticulum and mitochondria with tubular cristae, both features being characteristic of steroidogenic cells. Human Leydig cells contain large Reinke crystalloids of variable size and number, but their function remains obscure. The frequent occurrence of paracrystalline inclusions within the cytoplasm of the human Leydig cell suggests that these elements are precursors of the Reinke crystalloids.
Preparation by ion milling and TEM investigation of embedded needle‐shaped crystals of H‐Nb2O5
Authors: Krumeich Frank , Mertin Wilhelm
Published: February 2005

A method for preparing needle‐shaped and platelike crystals for electron microscopical investigation was elaborated. Crystals of H‐Nb2O5 were embedded in a synthetic resin and disks were cut off perpendicular to the desired direction of observation. The thickness of the sample was reduced by planar grinding and then by using a dimple grinder and furthermore by ion milling with argon ions. With the precision ion milling system small crystal areas were selected and subsequently irradiated. The TEM investigations showed that the desired crystallographic orientation was reached and that the crystal structure has been preserved. The contrast of highly resolved images was reduced by an amorphous surface layer which was not removable.
Real time computer simulation of electron microscope images with tilted illumination: Grain boundary applications
Authors: Krakow W.
Published: February 2005

Computer programs have been developed to simulate electron microscope images from digitized graphically represented model structures. Via a television rate image processing system, these programs allow real time, interactive modification of the microscope objective lens parameters, incident beam inclination, and incident beam energy. In addition to explaining the computational methods, the need for using tilted beam illumination is explored to extend microscope resolution. For this study, the subject of grain boundary imaging is analyzed for a copper σ = 5,36.9°,(310) tilt boundary with a [001] common rotation axis. The Cu {200} lattice spacings of ∼1.8Å on both sides of the interface cannot be reliably resolved under axial illumination conditions in a 200 kV microscope. Therefore, either tilted beam modes or higher incident beam energies were explored and the types of image features correlated with atomic position data through the digital frame store system.