bluuebunny/crossref_metadata_2025_split huggingface dataset:
Application of an ultrahigh‐resolution scanning electron microscope (UHS‐T1) to biological specimens
Authors: Tanaka Keiichi , Mitsushima Akira , Kashima Yuzuru , Nakadera Takashi , Osatake Hitoshi 
Published: February 2005

AbstractIn 1985 we developed an ultrahigh‐resolution scanning electron microscope with a resolution of 0.5 nm. It is equipped with a field emission gun and an objective lens with a very short focal length. In this study we report a survey of some different preparation techniques and biological specimens using the new scanning electron microscope.Intracellular structures such as cell organelles were observed surprisingly sharper than those observed by ordinary scanning electron microscopes. However, at magnifications over 250,000 X, platinum particles could be discerned as scattered pebbles on the surface of all structures in coated materials. Using an uncoated but conductively stained specimen, we successfully observed ribosomes on a rough endoplasmic reticulum at a direct magnification of 1 million. In these images some protrusions were recognized on the ribosomes.Ferritin and immunoglobulin G were used as samples of biological macromolecules. These samples were observed without metal coating and conductive staining. The ferritin particles appeared as rounded bodies without any substructure on the surface and immunoglobulin G as complexes of three‐unit bodies. In the latter the central body might correspond to the Fc fragment and two side ones to Fab fragments.We assume that ultrahigh‐resolution scanning electron microscopy is an effective means for observation of the cell fine structures and biological macromolecules. It will open a new research field in biomedicine.
Cytoskeletal architecture of neuromuscular junction: Localization of vinculin
Authors: Yorifuji Hiroshi , Hirokawa Nobutaka 
Published: February 2005

AbstractCytoskeletons underneath the postsynaptic membrane of neuromuscular junctions were studied by using a quick‐freeze deep‐etched method and immunoelectron microscopy of ultrathin frozen sections. In a quick‐freeze deep‐etched replica of fresh, unfixed muscles, 8.9 ± 1.5‐nm particles were present on the true postsynaptic membrane surface. Underneath this receptor‐rich postsynaptic membrane, networks of fine filaments were observed. These cytoskeletal networks were more clearly observed in extracted samples. In these samples, diameters of the filaments which formed networks were measured. In the platinum replica, three kinds of filament were recognized—12 nm, 9 nm, and 7 nm in diameter. The 12‐nm filament seemed to correspond to the intermediate filament. The other two filaments formed meshworks between intermediate filaments and plasma membrane. In ultrathin frozen sections vinculin label was localized just beneath the plasma membrane. Thirty‐six percent of the label was within 18 nm from the cytoplasmic side of the plasma membrane and 50% was within 30 nm. Taking the size of the vinculin molecule into account, it was concluded that vinculin is localized just beneath the plasma membrane and might play some role in anchoring filaments which formed meshworks underneath the plasma membrane.
Structural study of nanometer‐sized iron crystallites in single crystalline iron–mgo composite films
Authors: Tanaka Nobuo , Nagao Masaru , Yoshizaki Fukio , Mihama Kazuhiro 
Published: February 2005

AbstractSingle crystalline composite films of iron and MgO are prepared by a simultaneous vacuum deposition technique. The structures of the composite films, especially of the iron crystallites embedded, are studied by high‐resolution electron microscopy and nanometer‐area electron diffraction. The α‐iron (b.c.c.) crystallites of 1 nm in size are epitaxially embedded in single crystalline MgO films, the orientation being (011)[100]Fe/(001)[100]Mgo and (001)[110]Fe//(001)[100]Mgo. A heat treatment of the as‐grown films at 500–1,000°C brings about a phase transformation of the crystallites from α‐iron to γ‐iron (f.c.c.), followed by a grain growth of α‐iron and finally the growth of the spinel, MgFe2O4. The γ‐iron crystallites transformed are circular plates and have strains at the periphery to accommodate the surrounding MgO‐matrix. The magnetic property of the composite films is also reported.
A high‐voltage scanning transmission electron microscope at Nagoya University
Authors: Hibino Michio , Shimoyama H. , Maruse S. 
Published: February 2005

AbstractA high‐voltage scanning transmission electron microscope (STEM) H‐1250ST of the maximum accelerating voltage of 1.25 MV was constructed at Nagoya University in 1983. The microscope, equipped with a field‐emission gun, is designed with high‐level STEM performance as well as conventional transmission microscopy mode operation. The aim of developing the microscope, basic design schemes, principal instrumentation, and techniques developed are described.
Measurement of lattice parameter and strain using convergent beam electron diffraction
Authors: Randle Valerie , Barker Ian , Ralph Brian 
Published: February 2005

AbstractA brief review is presented of the methods of measuring lattice parameters and strain using diffraction techniques. The presence of strain leads to broadening of diffraction maxima, which is normally separable from any broadening caused by size. The special advantages of the convergent beam electron diffraction (CBED) techniques in measuring lattice parameters and strain are given from studies of precipitation (including misfit measurements) and from investigations of partially recrystallised microstructures. These examples are used to illustrate the advantages and limitations of the CBED technique.
High‐pressure freezing for the preservation of biological structure: Theory and practice
Authors: Dahl Rolf , Staehelin L. Andrew 
Published: February 2005

AbstractThe two main advantages of cryofixation over chemical fixation methods are the simultaneous stabilization of all cellular components and the much faster rate of fixation. The main drawback pertains to the limited depth (<20 μm surface layer) to which samples can be well frozen when freezing is carried out under atmospheric conditions. High‐pressure freezing increases the depth close to 0.6 mm to which samples can be frozen without the formation of structurally distorting ice crystals. This review discusses the theory of high‐pressure freezing, the design of the first commercial high‐pressure freezing apparatus (the Balzers HPM 010), the operation of this instrument, the quality of freezing, and novel structural observations made on high‐pressure‐frozen cells and tissues.
Counting and measuring IMPs and pits: Why accurate counts are exceedingly rare
Authors: Rash John E. , Giddings F. Dennis 
Published: February 2005

AbstractParticle counting and measuring techniques are now widely used to characterize normal membranes and to identify molecular changes occurring during development, maturation, and aging during progression of disease and following pharmacological manipulation. However, the use of particle counting and measuring for the identification of molecular changes in membranes has been premature. We show that current procedures rarely yield replicas that are free of cryogenic or mechanical prefractures, and as a result, the “complementarity” of membrane faces is severely compromised. However, with simple alterations of procedure, combined with the resolve to recognize and discard images of pre‐fractured membrane faces, a high degree of “complementarity” may be obtained. Criteria for recognizing the occurrence and relative frequency of noncomplementarity are presented and a cleaving method for avoiding a primary source of water vapor contamination is described. In such replicas, membrane pits are found in equivalent numbers and near‐identical diameters as the intramembrane particles (IMPs) in the complementary‐type membrane faces. When conditions of “cold fracture” and immediate replication are demonstrated, fracture faces are minimally contaminated by frozen water vapor, yielding images where (1) diameters of IMPs vs. pits are very nearly identical, (2) large diameter IMPs are very rare, and (3) the numbers of IMPs and pits are increased substantially over the numbers currently reported. Thus, we reiterate previous proposals that complementarity of membrane faces is the single most important criterion that must be met before accepting the validity of IMP counts or for attributing perceived changes in IMP density or size to conditions of experimental manipulation, to normal developmental processes, or to disease etiology.
Protocol for 3‐D visualization of molecules on mica via the quick‐freeze, deep‐etch technique
Authors: Heuser John 
Published: February 2005

AbstractMolecular imaging by freeze‐drying of molecules adsorbed to a mica substrate often provides better images of molecules than those attainable with other methods; the images are easier to interpret than those obtained with frozen thin film or negative staining, and the 3‐dimensional information content is greater. The complete procedure for the production and examination of platinum‐carbon replicas of molecules is described. Topics include production of a mica flake suspension, chemical pretreatment of the flakes to enhance adsorption, quick‐freezing of the samples on mica, optimal operation of the freeze‐fracture equipment, and orientation of replica topography. The production of stereo micrographs is analyzed in detail, with emphasis on the photographic procedures necessary for interpretation and on the identification of correct micrograph orientation. Guidelines are provided for the extrapolation from observed molecular size in platinum replicas to expected molecular weight.
Applications of freeze‐fracture replication to problems in materials and colloid science
Authors: Zasadzinski Joseph A. N. , Bailey Stuart M. 
Published: February 2005

AbstractUnderstanding the relationship between the molecular structure and the macroscopic properties of polymer solutions and gels, oil‐water‐surfactant emulsions, lyotropic and thermotropic liquid crystals, colloidal dispersions, detergents, and other such “microstructured fluids” is essential to the optimal use of these commercially important materials. Modern rapid‐freezing methods followed by freeze‐fracture replication techniques are ideally suited to allow the direct visualization of the three‐dimensional structure of the particles or units that make up the dispersion, while simultaneously revealing their orientation and distribution with molecular resolution. This paper reviews the necessary experimental conditions required to successfully exploit the freeze‐fracture technique as it applies to microstructured fluid systems. The benefits and limitations of structural studies by freeze‐fracture techniques as opposed to the more commonly used light, X‐ray, and neutron‐scattering methods are discussed. Freeze‐fracture replicas can also be imaged by scanning tunneling microscopy to reveal directly three‐dimensional fracture contours with improved resolution.
Fine structure and function of kupffer cells
Authors: McCuskey Robert S. , McCuskey Patricia A. 
Published: February 2005

AbstractKupffer cells are macrophages that are attached to the luminal surface or inserted in the endothelial lining of hepatic sinusoids. In this site, Kupffer cells play a key role in host defense by removing foreign, toxic and infective substances from the portal blood and by releasing beneficial mediators. Under some conditions, toxic and vasoactive substances also are released from Kupffer cells which are thought to play a role in a variety of liver diseases. Many of these activities may be modulated by the levels of gut derived endotoxin normally present in the portal blood.The ultrastructural aspects of Kupffer cell structure function in situ are best studied using perfused‐fixed livers. In fixed livers, transmission and scanning electron microscopy reveal Kupffer cells during health to be irregular in shape with their exposed surfaces presenting numerous microvilli, filopodia, and lamellopodia. Long filopodia penetrate endothelial fenestrae to secure Kupffer cells to the sinusoid lining. Specific membrane invaginations known as worm‐like bodies or vermiform processes are seen in the cytoplasm of Kupffer cells as are numerous endocytotic vesicles and lysosomes which vary in density, shape and size. Sometimes, annulate lamellae connected to the rough endoplasmic reticulum also are found. The principal endocytic mechanisms of Kupffer cells are phagocytosis of particulates and cells, and bristle‐coated micropinocytosis for fluid‐phase endocytosis of smaller substances. Many of these events are mediated by specific receptors. In some species, Kupffer cells can be distinguished from other sinusoidal lining cells and monocytes by specific cytoplasmic staining or monoclonal antibodies. Kupffer cells have been shown to be of monocytic origin as well as having the capacity for self‐replication.
Method for forming two‐dimensional paracrystals of biological filaments on lipid monolayers
Authors: Ward Richard John , Menetret Jean‐Francois , Pattus Franc , Leonard Kevin 
Published: February 2005

AbstractA method is described for electron microscopic observation of two‐dimensional paracrystals on unsupported lipid monolayers. The method uses a hydrophobic holey C‐coated grid placed on a monolayer made positively charged by the inclusion of stearylamine (SA) and has been used to align scallop thin filaments and reconstituted actin/tropomyosin filaments to form paracrystals. The use of unsupported monolayers allows the paracrystals to be viewed in either negative stain or with cryoelectron microscopy. Those paracrystals in frozen hydrated specimens have better order than those with negative stain.It was found that varying the lipid composition between the less fluid distearolyphosphotidylcholine/SA and the more fluid egg yolk phosphotidylcholine/SA alters the size and order of the paracrystals, the more fluid system having smaller, more ordered paracrystalline domains. The advantage of the technique for studying actin/thin filaments is the ability to form large two‐dimensional paracrystals under physiological conditions of [Mg2+] and pH.
Quick‐freeze, deep‐etch replication of cells in monolayers
Authors: Pumplin David W. , Luther Paul W. , Samuelsson Steven J. , Ursitti Jeanine A. , Strong John 
Published: February 2005

AbstractWe have made several technical improvements for quick‐freeze, deep‐etch replication of monolayers of cells grown on, or attached to, glass coverslips. Cells studied include muscle cells of rat and Xenopus cultured on glass coverslips, and erythrocytes attached to coverslips coated with poly‐L‐lysine. We describe methods for identifying particular areas of cultures, e.g., clusters of acetylcholine receptors on muscle cells, by light microscopy and then relocating these areas after replication. For good preservation of structure by quick‐freezing, it is necessary to ensure that the surface to be frozen is covered by a minimum depth of water (< 10 μm). Insufficient or excess water left on the sample during freezing causes recognizable artifacts in its replica. We describe two ways to control the water table–one by improving visual control of water removal, the other by blowing excess water off the sample surface with a jet of nitrogen applied during its descent to the freezing block. Finally, we describe a new specimen holder that allows us to etch and replicate six samples at once with good thermal contact between the stage and samples.
Cholinergic synapses in the central nervous system: Studies of the immunocytochemical localization of choline acetyltransferase
Authors: Houser Carolyn R. 
Published: February 2005

AbstractCholinergic synapses can be identified in immunocytochemical preparations by the use of monoclonal antibodies and specific antisera to choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine (ACh) and a specific marker for cholinergic neurons. Electron microscopic studies demonstrate that the fibers and varicosities observed in light microscopic preparations of many brain regions are small‐diameter unmyelinated axons and vesicle‐containing boutons. The labeled boutons generally contain clear vesicles and one or more mitochondrial profiles. Many of these boutons form synaptic contacts, and the synapses are frequently of the symmetric type, displaying thin postsynaptic densities and relatively short contact zones. However, ChAT‐labeled synapses with asymmetric junctions are also observed, and their frequency varies among different brain regions. Unlabeled dendritic shafts are the most common postsynaptic elements in virtually all regions examined although other neuronal elements, including dendritic spines and neuronal somata, also receive some cholinergic innervation. ChAT‐labeled boutons form synaptic contacts with several different types of unlabeled neurons within the same brain region. Such findings are consistent with a generally diffuse pattern of cholinergic innervation in many parts of the central nervous system. Despite many similarities in the characteristics of ChAT‐labeled synapses, there appears to be some heterogeneity in the cholinergic innervation within as well as among brain regions. Differences are observed in the sizes of ChAT‐immunoreactive boutons, the types of synaptic contacts, and the predominant postsynaptic elements. Thus, the cholinergic system presents interesting challenges for future studies of the morphological organization and related function of cholinergic synapses.
GABAergic synapses in the brain identified with antisera to GABA and its synthesizing enzyme, glutamate decarboxylase
Authors: Ribak Charles E. , Roberts Rosalinda C. 
Published: February 2005

AbstractGABA is a known inhibitory neurotransmitter in the mammalian brain. The site of GABAergic synapses can be determined with immunocytochemical methods that localize either GABA or its synthesizing enzyme, glutamate decarboxylase (GAD). In general, GABAergic axon terminals contain pleomorphic synaptic vesicles and form symmetric synapses. However, a small number of GABAergic axon terminals in selected brain regions (spinal cord, cerebellum, superior colliculus, striatum, globus pallidus, inferior olive, and substantia nigra) form asymmetric synapses. GAD‐ and GABA‐immunoreactive processes that contain synaptic vesicles participate in every known morphological type of chemical synapse. These include axosomatic, axodendritic, axospinous, initial segment, axoaxonic, dendrodendritic, serial, reciprocal, and ribbon synapses. Although GABAergic synapses form a heterogeneous group, they most commonly form axosomatic, axodendritic, and initial segment synapses in the brain and spinal cord. These findings provide helpful guidelines for the identification of GABAergic synapses in future studies.
Ultrastructural identification of neuropeptides in the central nervous system
Authors: Maley Bruce E. 
Published: February 2005

AbstractA number of different neuropeptides have been described within presynaptic terminals at the ultrastructural level in the central nervous system. The majority of these neuropeptides share a common morphology with one another. Terminals containing neuropeptides have a population of small, clear vesicles associated with the active zone of the synapse and a lesser number of large, granular vesicles that are located at a distance from the active site of the synapse. It is believed that the large, granular vesicles act as a mechanism for the transport/storage of the neuropeptides, while the small, clear vesicles are thought to be acting as structures responsible for the release of the neurotransmitter/neuropeptide into the synaptic cleft. The neuropeptide containing terminals most often have asymmetrical junctions associated with their presynaptic membranes, although symmetrical junctions have been described with peptide containing terminals in a number of areas in the central nervous system. Neuropeptide containing terminals contact every part of the neuronal membrane; however, the majority of synaptic contacts involve portions of the dendritic shafts. Evidence is beginning to accumulate to indicate that for certain neuropeptides there is a specific spatial arrangement to their termination along the neuronal membrane.
Biochemical and morphological differentiation of acetylcholinesterase‐positive efferent fibers in the mouse cochlea
Authors: Emmerling M. R. , Sobkowicz H. M. , Levenick C. V. , Scott G. L. , Slapnick S. M. , Rose J. E. 
Published: February 2005

AbstractWe have compared the biochemical expression of cholinergic enzymes with the morphological differentiation of efferent nerve fibers and endings in the cochlea of the postnatally developing mouse. Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are present in the newborn cochlea at specific activities 63% and 25%, respectively, of their mature levels. The relative increases in ChAT, in AChE, and in its molecular forms over the newborn values start about day 4 and reach maturity by about day 10. The biochemical results correlate well with the massive presence of nerve fibers stained immunocytochemically for ChAT and AChE or enzymatically for AChE in the inner and outer hair cell regions. Ultrastructural studies, however, indicate the presence of only few vesiculated fibers and endings in the inner and outer hair cell regions. The appearance of large, cytologically mature endings occurs only toward the end of the third postnatal week. The discrepancy may be resolved in the electron microscope using the enzymatic staining for AChE. Labeling is seen on many nonvesiculated fibers and endings in the hair cell regions, suggesting that the majority of the efferent fibers in the perinatal organ may be biochemically differentiated but morphologically immature. The results may imply that the efferents to inner and outer hair cells develop earlier than indicated by previous ultrastructural studies. Moreover, the pattern of development suggests that in the cochlea, as in other tissues, the biochemical differentiation of the efferent innervation may precede the morphological maturation.
Organization of microtubules in cochlear hair cells
Authors: Furness David N. , Hackney Carole M. , Steyger Peter S. 
Published: February 2005

AbstractThe organization of microtubules in hair cells of the guinea‐pig cochlea has been investigated using transmission electron microscopy and correlated with the location of tubulin‐associated immunofluorescence in surface preparations of the organ of Corti. Results from both techniques reveal consistent distributions of microtubules in inner and outer hair cells.In the inner hair cells, microtubules are most concentrated in the apex. Reconstruction from serial sections shows three main groups: firstly, in channels through the cuticular plate and in a discontinuous belt around its upper perimeter; secondly, forming a ring inside a rim extending down from the lower perimeter of the plate; and thirdly, in a meshwork underlying the main body of the plate. In the cell body, microtubules line the inner face of the subsurface cistern and extend longitudinally through a tubulo‐vesicular track between the apex and base.In outer hair cells, the pattern of microtubules associated with the cuticular plate is similar, although there are fewer present than in inner hair cells. In outer hair cells from the apex of the cochlea, microtubules occur around an infracuticular protrusion of cuticular plate material. In the cell body, many more microtubules occur in the region below the nucleus compared with inner hair cells.The possible functions of microtubules in hair cells are discussed by comparison with those found in other systems. These include morphogenesis and maintenance of cell shape; intracellular transport, e.g., of neurotransmitter vesicles; providing a possible substrate for motility; mechanical support of structures associated with sensory transduction.