bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytochemical applications of X‐ray microanalysis
Authors: Sumner A. T.
Published: February 2005

X‐ray microanalysis (XRMA) has been applied to a wide variety of cytochemical problems, but the most valuable applications have been to the validation of cytochemical methods (by the qualitative or quantitative analysis of reaction products), and to the simultaneous localization of more than one substance, which cannot easily be achieved by using alternative methods. The latter applications involve stoichiometric studies (the quantitative relationships between reaction products and substrates), and distribution studies. Ultrastructural cytochemistry with XRMA is limited by the need to use high‐brightness electron sources. Apart from the limited availability of such sources, they may cause unacceptable damage to the specimen.

Preparation methods for cytochemistry using XRMA are reviewed; in principle these do not differ from those used for other cytochemical applications, but it is important not to introduce extraneous elements (from fixative, buffer, or embedding medium) into the specimen, where the additional X‐ray peaks may interfere with the analysis.

Quantification in XRMA of cytochemical preparations poses special problems, because the addition of the reaction product to the specimen alters the yield of continuum X rays, used for assessing the mass of the specimen, and also dilutes endogenous elements. However, measurement of ratios between characteristic elemental peaks is a useful method in X‐ray microanalytical cytochemistry, and it is concluded that one of the most important attributes of XRMA for cytochemical purposes is the ease with which the substances of interest can be measured.
Renal cell culture
Authors: Kreisberg Jeffrey I. , Wilson Patricia D.
Published: February 2005

Methods for the establishment and growth of renal cell types in culture are reviewed, with emphasis on current trends. General techniques available for the isolation and culture of glomerular cells have progressed from explant to enzyme dissociation and cloning techniques. The growth characteristics and properties of cultured glomerular endothelial, epithelial, mesangial, and bone‐marrow‐derived cells are discussed. Studies are described in which cultures of contractile mesangial cells have led to an elucidation of their role both in normally functioning glomeruli and in disease states. Renal tubule culture techniques also have progressed from mixed tissue explants and cell isolates to fractionation of enriched tubule populations and growth of specific, individually microdissected proximal convoluted, proximal straight, thick ascending limb of Henle's loop, and collecting tubules. The differentiated tubule epithelial‐specific properties of such primary cultures are discussed in relation to those of permanently growing cell lines such as MDCK and LLC‐PK1. Renal tubule cultures will be invaluable for the study of the role of hormones and extracellular matrix in epithelial growth and polarity of normal structure and function. In addition, in vitro models of cultured renal tubules have been established to study the effects of age, nephrotoxins, and anoxic injury.
Introduction to the computer image processing of electron micrographs of two‐dimensionally ordered biological structures
Authors: Stewart Murray
Published: February 2005

Methods are described for the analysis of electron micrographs of regular biological objects. Fourier‐based processing of one‐dimensionally ordered arrays is described by way of introduction, before analysing two‐dimensional crystals in projection with the aim of enhancing signal:noise ratio and thus of feint features that were initially obscured. This form of analysis is then extended to decomposing the moiré patterns formed when sheets overlap, thereby enabling the separation of interfering image patterns. Analogous forms of an analysis can also be applied to objects with rotational symmetry. Methods for treating the effect of the microscope imaging system and compensating for lattice disorder in crystalline specimens are also discussed.
Computer image processing of electron micrographs of biological structures with helical symmetry
Authors: Stewart Murray
Published: February 2005

Methods are described for the analysis of electron micrographs of biological objects with helical symmetry and for the production of three‐dimensional models of these structures using computer image reconstruction methods. Fourier‐based processing of one‐ and two‐dimensionally ordered planar arrays is described by way of introduction, before analysing the special properties of helices and their transforms. Conceiving helical objects as a sum of helical waves (analogous to the sum of planar waves used to describe a planar crystal) is shown to facilitate analysis and enable three‐dimensional models to be produced, often from a single view of the object. The corresponding Fourier transform of such a sum of helical waves consists of a sum of Bessel function terms along layer lines. Special problems deriving from the overlapping along layer lines of terms of different Bessel order are discussed, and methods to separate these terms, based on analysing a number of different azimuthal views of the object by least squares, are described. Corrections to alleviate many technical and specimen‐related problems are discussed in conjunction with a consideration of the computer methods used to actually process an image. A range of examples of helical objects, including viruses, microtubules, flagella, actin, and myosin filaments, are discussed to illustrate the range of problems that can be addressed by computer reconstruction methods.
Three‐Dimensional reconstruction of single particles from random and nonrandom tilt series
Authors: Radermacher Michael
Published: February 2005

To overcome the radiation damage‐induced limitations to the resolution of three‐dimensional reconstructions from electron microscopic tilt series, novel reconstruction schemes have been developed that require only a single exposure of the specimen. The tilt series collected with these methods have random projection directions. First, three‐dimensional reconstruction techniques are described that are applicable to data obtained from tilt series with regular tilt geometry, followed by the extensions of these techniques to permit analysis of projection series with randomly spaced tilts. The main emphasis is placed on the weighted back‐projection methods, which have recently been extended so as to be applicable to random tilt series. Besides a description of the algorithms, the complete procedure for a threedimensional reconstruction from a single‐exposure, random conical tilt series is explained, including the determination of the azimuthal angles, the alignment scheme for conical tilt series, the dependence of the achievable resolution on the number of projections for regular conical and single‐axis geometries, and the method to calculate the actual resolution of two‐dimensional image averages and of three‐dimensional reconstructions using the phase residual and Fourier ring correlation criteria. Examples are given of biological specimens to which these three‐dimensional reconstruction methods have been applied.
High‐resolution rlectron microscopic investigation of supported platinum particles reduced at high temperatures
Authors: Wang L. , Qiao G. W. , Ye H. Q. , Kuo K. H. , Chen Y. X.
Published: February 2005

High‐resolution electron microscopy has been used to characterize the platinum particles supported on TiO2 or ZnO. After reduction at elevated temperatures, the metallic particles display a regular, faceted shape, and several superstructures, Pt3 Ti(C), Pt3 Ti(H), PtTi, and PtZn, have been found. These results, which may involve strong metal‐support interaction, have been confirmed by optical diffraction and image simulation.
Preparation of cultured mammalian cells for transmission and scanning electron microscopy using aclar film
Authors: Kingsley R. E. , Cole N. L.
Published: February 2005

Common methods for the preparation of cultured cells for concurrent light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are not completely satisfactory. This article describes how we grow mammalian cells on plastic disks made from Aclar film. Aclar is a transparent fluorinated‐chlorinated thermoplastic that contains no volatile components and is, for all practical purposes, chemically inert. Cells adhere to it readily and remain attached after fixation, dehydration, and critical‐point drying or embedding. The film also accepts heavy metal coating by ionic bombardment and is extremely stable in the vacuum of the SEM. LM observations are unhindered by Aclar, since the film is as transparent as glass. Fluorescence microscopy is possible with this film, since it exhibits no detectable autofluorescence. During SEM observation, the film has great dimensional stability, and the cells and heavy metal coating remain attached to the Aclar even under high‐resolution operating conditions. TEM processing of specimens grown on Aclar is simplified by the fact that Aclar does not stick to the epoxy resins used in EM. Furthermore, Aclar is easily sectioned and does not damage knives used in ultramicrotomy. The use of Aclar film considerably simplifies the preparation of cultured cells for all types of microscopy. This method is particularly useful in correlating surface features between SEM and TEM observations.
Chemical synapses in the mammalian central nervous system: An introduction
Authors: King James S.
Published: February 2005

This paper provides a brief review of the historical development of our understanding of synaptic structure in the central nervous system. The basic structure of the synapse is reviewed as well as the development of ultrastructural techniques that have allowed the details of synaptic organization to be elucidated.
Synaptic organization of the globus pallidus
Authors: Difiglia Marian , Rafols Jose A.
Published: February 2005

The synaptic organization of the globus pallidus is reviewed with respect to present knowledge about neurons, fibers, axon terminals, and their intrinsic synaptic relationships. Information derived from studies employing Nissl stains, Golgi impregnations, lesion degeneration techniques, immunohistochemistry, and anterograde axonal labeling in various species are presented along with ultrastructural data. Studies indicate that the globus pallidus contains a principal efferent neuron with smooth or spiny dendrites and simple or complex terminal dendritic arborizations. This cell type receives convergent inputs from intrinsic and extrinsic sources and uses γ‐aminobutyric acid as a transmitter. A smaller and separate population of pallidal projection neurons contains acetylcholine. Two other less frequent neuronal types, of small and medium size, have also been recognized. Three to six types of axonal boutons forming synaptic contacts with pallidal neurons have been recognized in various studies. Among these, three types (types I, II, and III) are the most prevalent. Studies indicate that the most frequent category (type I) originates from neostriatal neurons via radial fiber projections and contains immunoreactive GABA and enkephalins. The synaptic architecture of the globus pallidus is dominated by a mosaic‐like arrangement of long dendrites that are ensheathed by longitudinally oriented axons making synapses en passant. Triadic synapses involving dendrites that are pre‐ and postsynaptic are encountered infrequently. Because both striatopallidal and pallidothalamic connections are inhibitory, pallidal target neurons in the thalamus may be “disinhibited” when the neostriatum is activated.
Synaptic organization of the striatum
Authors: Gerfen Charles R.
Published: February 2005

The striatum, the main component of the basal ganglia, is composed of mainly one type of neuron, the so‐called medium spiny neuron. This neuron cell type, which constitutes over 90% of striatal neurons, is the major output neuron of the striatum. Combined ultrastructural neuroanatomical methods have elucidated the organization of afferent connectivity to these neurons. The major physiologic function of striatal efferent activity appears to be inhibition of tonically active GABAergic neurons in the globus pallidus and substantia nigra pars reticulata. Thus, the excitatory input from the cerebral cortex, whose afferents make asymmetric synapses with the spines of medium spiny neurons, appears to drive the efferent activity of the striatum. Other extrinsic and intrinsic afferent synapses are situated in a position to regulate the effect of the corticostriatal excitatory input to the medium spiny neurons. For example, dopaminergic afferents from the midbrain make mainly symmetric synapses with the spine necks and dendritic shafts of the medium spiny neurons. Medium spiny neurons themselves have local axon collaterals, in addition to their efferent axon that exits the striatum, which serve to link together local clusters of medium spiny neurons. These local axon collaterals, which contain either GABA, substance P, or enkephalin, also make mainly symmetric synapses with the necks of spines or dendritic shafts of medium spiny neurons. Other afferents with similar synaptic connections to these neurons arise from cholinergic or somatostatinergic striatal intrisinic neurons. Additionally, the patterns of extrinsic and intrinsic afferents to medium spiny neurons and their extrinsic projections are related to the organization of medium spiny neurons into two mosaically organized macroscopic compartments, the striatal patches and matrix.
Determination of experimental and theoretical kASi factors for a 200‐kV analytical electron microscope
Authors: Sheridan Patrick J.
Published: February 2005

The relative sensitivity of an analytical electron microscope and energy‐dispersive x‐ray detector to x‐rays of various elements is investigated through an extensive kASi factor study. Elemental standards, primarily National Bureau of Standards multielement research glasses, were dry‐ground into submicrometer‐sized particles and analyzed at 200 kV accelerating potential. The effect of self‐absorption of x‐rays by the particle has been corrected for, allowing the experimental kASi factors from this study to approximate those that could be obtained from “infinitely thin” specimens.

Whenever possible, elemental k‐factors were determined by the analysis of many (up to a maximum of nine) different standard materials. Experimental kASi factors were calculated for a wide range of Kα, Lα, and Mα x‐ray lines. For comparison, theoretical kASi factors, employing a variety of ionization cross sections, were computed. Good agreement is obtained between several of the theoretical k‐factor models and the experimental results. Mass volatilization of Na and K from the small glass particles during analysis is discussed, as are observations that the grinding and/or dispersing of standard materials in a liquid (such as ethanol) may promote leaching of certain elements from the particle matrix.
Use of Peldri II (a fluorocarbon solid at room temperature) as an alternative to critical point drying for biological tissues
Authors: Kennedy John R. , Williams Richard W. , Gray John P.
Published: February 2005

A new chemical, Peldri II, is evaluated as a compound for drying soft biological tissues for scanning electron microscopy. Peldri II, a fluorocarbon, is a solid at room temperature and is a liquid above 25°C. Cells or tissues are embedded in Peldri II by immersing them in the liquid form and allowing it to solidify. Once solidified, Peldri II will sublime with or without vacuum to dry tissues, probably without introducing surface tension. Several types of cells and tissues have been examined to compare preservation with Peldri II and critical point drying techniques. No differences were detected between the two techniques when normal surface structures were examined. Peldri II appears to be a significant improvement over hexamethyldisilazane as a drying agent for scanning electron microscopy. It is also very convenient for drying large numbers of samples.
Improved preservation of bacterila capsule for electron microscopy
Authors: Jacques Mario , Graham Lori
Published: February 2005

Conventional fixation for thin‐section electron microscopy is insufficient to preserve bacterial capsular material, a highly hydrated structure which collapses during dehydration and embedding. Boyles et al. (1984, 1985) have recently introduced a modified aldehyde fixation process in which the addition of a primary amine was found to improve the preservation of actin filaments, the basement membrane material and cell surface glycocalyx of mouse tissues. To the best of our knowledge, the application of diamines to the analysis of bacterial ultrastructure has not been reported in the literature. In the present study, excellent preservation of bacterial capsular material was seen when a diamine (lysine) was used together with glutaraldehyde in conventional embedding procedures.
Stereo‐imaging made easy
Authors: Zurek Anna K.
Published: February 2005

A new stereo–imaging technique is described. This new technique uses both secondary electron and back scatter electron detectors simultaneously for imaging the fracture surfaces, pictures of which are used in stereo‒imaging.
Magnetic field emission gun with zirconiated emitter
Authors: Troyon M.
Published: February 2005

A magnetic‐field‐superimposed field emission gun with low aberrations and equipped with a zirconiated tungsten emitter has been developed for applications where very stable high probe currents are required. It has been tested on a conventional electron microscope at 10 kV and on an electron beam testing system at 1 kV. Probe current i = 250 nA in a probe size d = 0.4 μm is obtained at 10 kV; at 1 kV the resolution is 0.1 μm with i = 5 nA, and 0.4 μm with i = 30 nA. For these probe currents, the spatial broadening effect due to electron–electron interactions in the beam is the preponderant factor limiting the probe size.
Ultrastructural localization of DNA on ultrathin sections of resin‐embedded tissues by the lactoferrin‐gold complex
Authors: Benhamou Nicole
Published: February 2005

Lactoferrin, a DNA‐binding protein, was complexed to colloidal gold and applied on ultrathin sections of resin‐embedded plant tissues and bacterial cultures. Optimal results were obtained when lactoferrin was tagged to colloidal gold particles at pH 9.2. Postfixation with osmium tetroxide and embedding with Epon did not prevent the accessibility of the protein towards its corresponding binding sites. In plant nuclei, labeling was observed over the dense chromatin and to a lesser extent over the dispersed chromatin. Nucleolar labeling was preferentially located over the dense fibrillar component. Gold particles were also found to be associated with chloroplasts and mitochondria. In prokaryotic cells, a dispersed labeling was noted over the cytoplasm and, in some cases, the aggregation of few gold particles suggested the presence of packed DNA fibrils. Various control experiments confirmed the specificity of the labeling pattern obtained. Lactoferrin‐gold complex appears to be a valuable probe for the intracellular demonstration of DNA molecules in double‐fixed and Epon‐embedded tissues.
Observation of GaAs—AlxGa1 — x as heterostructures and quantum‐well‐wire structures using backscattered electron image
Authors: Hirayama Yoshiro , Okamoto Hiroshi
Published: February 2005

As a microscale tool for observing GaAs‐Alx Ga1–xAs heterostructures, backscattered electron (BE) images in the scanning electron microscope (SEM) were compared with conventional secondary electron (SE) images. BE images were found to be more sensitive to compositional differences between GaAs and AlxGa1–xAs and less sensitive to surface roughness. BE images have a spatial resolution of 10 nm or better. This method enables the nondestructive observation of ultrafine lateral periodic structures, such as quantum‐well‐wire (QWW) structures, fabricated by compositional disordering technology using focused Ga ion‐beam (Ga‐FIB) implantation into GaAs‐AlxGa1–xAs material.
Use of membrane filters and osmium tetroxide etching in the preparation of sperm for scanning electron microscopy
Authors: Van Der Horst Gerhard , Kitchin Robert M. , Curry Patrick T. , Atherton Robert W.
Published: February 2005

A new method was developed which is suitable for the preparation of mammalian sperm for scanning electron microscopy under either laboratory or field conditions. Samples of ejaculates from humans, two ferret species, and epididymal sperm from the African elephant were diluted in Millonig phosphate buffer and then fixed in glutaraldehyde solution. A small sample of the fixed sperm suspension was diluted in the same buffer, withdrawn with a syringe, and injected very slowly onto either a cellulose acetate or a polycarbonate membrane filter. This step was essential to concentrate the dilute sperm samples. During the various dilution steps most of the granular prostatic secretions were lost. However, a protein‐like sheath, which remained attached to most sperm, obscured the surface features and had to be removed for SEM studies. It was removed by prolonged fixation/etching in 1% osmium tetroxide. Membrane filters containing sperm on their surfaces then were dehydrated, dried by the critical point drying method, and sputter coated with gold. Polycarbonate filters were superior to cellulose acetate filters in producing a flat and homogeneous background.
Application of ion‐beam etching techniques to the fine structure of biological specimens as examined with a field emission SEM at low voltage
Authors: Yonehara Katsuhisa , Baba Norio , Kanaya Koichi
Published: February 2005

The term “etching,” in electron microscopy, refers to the removal of specimen surface layers and includes chemical, electrolytic, and ion‐beam methods. The ion‐beam etching process is used to remove layers of a target material by bombarding it with ionized gas molecules. Recently, the method has been applied to the field of biological specimens; however, the practical procedures for such organic materials have not been developed. In the present study, we used an apparatus in which a beam of argon ions is collimated and focused by electrostatic lenses onto an appropriate target. We demonstrated the optimum conditions to observe biological specimens that were treated with osmium tetroxide and tannic acid. The specimens were examined uncoated at low accelerating voltage using a field emission scanning electron microscope.

According to our experiments, when a biological specimen was observed under high‐resolution conditions at over 50,000x magnification, the optimum condition of ion‐beam etching consisted of an accelerating voltage of E = 1 keV and an ion‐beam dose of It = 360 ∼ 400 μA. min, depending on parts of the specimens. In order to decrease overetching, we had to choose factors such as E = 1 ∼ 2 keV and It = 500 μA. min.
Three‐dimensional morphometrical study of dendritic spines of the granule cell in the rat dentate gyrus with HVEM stereo images
Authors: Hama Kiyoshi , Arii Tatsuo , Kosaka Toshio
Published: February 2005

Number, length, and diameters of dendritic spines of the granule cell in the dorsal leaf of the rat dentate gyrus were measured by using high‐voltage electron microscope stereo images of 5‐m̈m‐thick Golgi preparations with the aid of a three‐dimensional image analyzer system.

Spine densities of 2.02 ± 0.28, 2.28 ± 0.33, and 3.36± 0.35 per 1 μm at distal, middle, and proximal portions of the dendrite were obtained. These values were about 1.6‐fold of the previous light microscopical report.

Mean three‐dimensional spine length were 1.244 ± 0.506 μm, 1.262 ± 0.563 μm, and 1.254 ± 0.584 μm at distal, middle, and proximal portions, respectively, which were about 1.4 times longer than those measured in two dimensions.

By using measured morphometrical parameters of spines such as lengths, diameters, and population densities, total spine surface areas of 2.401 μm2, 2.806 μm2, and 4.180 μm2 per 1 μm of the dendrite at distal, middle, and proximal portions, respectively, were obtained. The total surface area of dendrite was about doubled by the addition of the spines at each dendritic portion.

The advantageous features and the problems of the present method are discussed.
Lysosomal movements during heterophagy and autophagy: With special reference to nematolysosome and wrapping lysosome
Authors: Sakai Masahiro , Araki Nobukazu , Ogawa Kazuo
Published: February 2005

Recent studies on lysosomal movements during heterophagy and autophagy performed in our laboratory for the past several years were reviewed; methods for the investigation of lysosomes and the cytoskeleton in these studies mainly involved electron microscopic cytochemistry.

Lysosomal movements during heterophagy were observed in cultured rat alveolar macrophages taking up horseradish peroxidase (HRP) and rat peroxidase‐antiperoxidase (PAP) by fluid‐phase pinocytosis and adsorptive pinocytosis, respectively. A characteristic lysosomal change which was induced by the pinocytosis was the appearance of long, threadlike lysosomes (nematolysosomes) in the cytoplasm. The effects of actin filament destabilizer and antimicrotubular drug on lysosomal changes revealed that the appearance of nematolysosomes was dependent on the presence of both actin filaments and microtubules. The close morphological relationship between lysosomes and cytoskeletal elements, such as actin filaments and microtubules in the alveolar macrophages, supports the participation of the cytoskeletal system in the regulatory mechanism of lysosomal movements.

In the study of the lysosomal wrapping mechanism (LWM), which is one type of lysosomal movement that occurs during autophagy, it was found that the occurrence of LWM was dependent on energy—namely, the supply of ATP—and on the presence of actin filaments. However, deconstruction of microtubules induced or favored the occurrence of LWM. It is conceivable that the LWM is also related to the cytoskeletal system.

We conclude that intracellular dynamics of lysosomes during heterophagy and autophagy are largely a consequence of complicated modulation by the cytoskeletal system.
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

In 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

Cytoskeletons 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

Single 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

A 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.
Symmetry analysis
Authors: Tanaka M.
Published: February 2005

The crystal point‐ and space‐group determination method using convergent‐beam electron diffraction (CBED) is reviewed. Crystal symmetry elements that can be observed by CBED are discussed.
Measurement of lattice parameter and strain using convergent beam electron diffraction
Authors: Randle Valerie , Barker Ian , Ralph Brian
Published: February 2005

A 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

The 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

Particle 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

Molecular 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

Understanding 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

Kupffer 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

A 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

We 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

Cholinergic 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

GABA 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

A 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

We 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.