bluuebunny/crossref_metadata_2025 huggingface dataset:
Three‐dimensional fine structure of the biliary tract: Scanning electron microscopy of biliary casts
Authors: Yamamoto Kazuhide , Itoshima Tatsuya , Tsuji Takao , Murakami Takuro
Published: February 2005

The three‐dimensional structure of the biliary tract was studied by scanning electron microscopy (SEM) of biliary casts. The replica of the biliary tract was successfully prepared by retrograde injection of low viscosity resin into the common bile duct. Bile canaliculi are intricate networks in which hexagonal and pentagonal meshworks are interconnected. Each hexagonal or pentagonal meshwork is on a plane, but adjoining meshworks are on different planes. Bile canalicular networks connect with bile ductules at the periphery of the portal tract. The intrahepatic bile duct showed considerable interspecies variation. The human bile duct has plexiform side branches and periductal sacculi, which are most numerous near the liver hilum and fewest in the smaller portal tracts. The hilar plexus and sacculi are present on opposite sides of the bile duct. The plexus formed at the bifurcation of the bile ducts exhibits a plane. Periductal sacculi were also observed in the monkey and pig bile ducts, particularly the latter, while rat bile ducts possess a peculiar portal bile ductular plexus situated between the portal tract and the surrounding liver parenchyma. No such structures were observed in either the dog or rabbit bile ducts. SEM of the biliary casts showed that the biliary tract was not a simple draining tube but had additional structures, such as periductal sacculi and plexiform side branches. These structures, together with the peribiliary vascular plexus, may be implicated in the modification of bile.
Scanning electron microscopic study of immunogold‐labeled human leukocytes
Authors: Helinski Ernest H. , Bootsma George H. , McGroarty Roy J. , Ovak Geraldine M. , De Harven Etienne , Pauly John L.
Published: February 2005

For many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein is a simple, efficient, inexpensive, reproducible, and safe procedure using Peldri II, a proprietary fluorocarbon compound that is solid at room temperature and a liquid above 25°C, as a sublimation dehydrant for processing specimens for SEM. The utility of Peldri II was demonstrated in studies using leukocytes from the blood of healthy donors and patients with leukemia as well as from long‐term lymphoblastoid cell lines. The application of the proposed Peldri II procedure was further documented in SEM studies in which the expression and distribution of the interleukin‐2 receptor (IL‐2R) on leukocyte surface membranes was imaged using colloidal gold‐labeled antibodies (i.e., immunogold). When compared with current SEM preparation procedures using CPD, Peldri II is a useful alternative that is thought to offer several important advantages.
Preparation of cross‐sectional specimens of ceramic thermal barrier coatings for transmission electron microscopy
Authors: Unal O. , Heuer A. H. , Mitchell T. E.
Published: February 2005

During the microstructural examination of ceramic thermal barrier coatings by transmission electron microscopy (TEM), initial efforts for the preparation of cross‐sectional thin foils from interface regions by conventional means were mostly failures. Delamination of the Y2O3‐stabilized ZrO2 ceramic coating from the nickel‐base alloy substrate sometimes occurred during fine polishing at around 80 μm thickness but mostly occurred during dimpling. Because of this sensitivity, special techniques for mechanical handling were developed so that ion milling could give thin enough regions of the metal‐ceramic interface. TEM showed convincingly that the highly fragile nature of the coatings is in fact due to the extensive porosity at the interface developed as a result of heat treatment.
Small colloidal gold conjugated to fab fragments or to immunoglobulin g as high‐resolution labels for electron microscopy: A technical overview
Authors: Baschong W. , Wrigley N. G.
Published: February 2005

Fab‐colloidal gold labelling in conjunction with negative staining and high‐resolution electron microscopy was used for targeting single protein units in regular arrays. These were bacteriophage T4 polyheads with Fab‐Au2.5, and a specific antibody binding site on the haemagglutinin polypeptide of influenza virus with Fab‐Au3, Fab‐Au2.5, and Fab‐Au1‐2. For the latter, IgG‐Au3 was also used. Experimental details are summarized to provide generally applicable methods for the preparation of small gold colloids Fab‐Au and of labelling.

The putative mechanism of protein‐gold complex formation and adsorption to preferred sites on Fab and IgG, most probably to sulphur‐rich regions, is discussed. The influence of pH during complex formation was found to be of minor importance in the samples investigated. Reported experimental details and our own experiences suggest that the importance of a protein's pI relative to its optimum gold complexing pH critically depends on the nature of the protein in question rather than being of general importance for protein‐gold complex stability.
Preparation of bone for high‐voltage electron microscopic radioautography
Authors: Pylypas Susan P. , Schurath Rodica M. , Johnson Roger B.
Published: February 2005

This paper presents a reliable technique for the preparation of radioautographic specimens for study by high‐voltage electron microscopy. Tissues are fixed by ventricular perfusion of aldehydes, sectioned, collected on slides, and coated with nuclear track emulsion using a coating machine. Sections are developed after 17 weeks' exposure in either undiluted Microdol‐X or diluted D‐19 developer, fixed in 25% sodium thiosulfate, and stained through the emulsion with uranyl acetate in absolute methanol and aqueous lead citrate. The technique produces sections of even thickness and staining density with negligible numbers of background grains and non‐specific labeling. In this report, the efficacy of this technique for the study of the periosteal surface of demineralized alveolar bone specimens is demonstrated.
Improved cryoprotection and freeze‐substitution of embryonic quail retina: A tem study on ultrastructural preservation
Authors: Meissner Dieter H. , Schwarz Heinz
Published: February 2005

Conditions for cryofixation and freeze‐substitution crucial to the ultrastructural preservation of embryonic quail retina were improved. As freeze‐substitution makes gentle dehydration and chemical fixation of tissue possible, the suitability of different cryoprotectants were tested in the preceding cryofixation. Additionally, different conditions for chemical prefixation were studied. In cryofixation, all of the “classic” cryoprotectants caused more or less severe tissue destruction. Only dimethylformamide (DMF) and–with certain reservations–dimethylsulfoxide (DMSO) yielded improved structure preservation. Perfusion fixation with a mixture of formaldehyde/glutaraldehyde (FA/GA) was superior to GA alone. In comparison to conventional fixation and dehydration methods, freeze‐substitution yielded better ultrastructural preservation of the embryos with fewer artifacts.
Development of spiral ganglion cells in mammalian cochlea
Authors: Romand M.‐R. , Romand R.
Published: February 2005

The development of the spiral ganglion in the cat, the rat, and the mouse was studied by electron microscopy, from fetal stages in the cat and from birth in the rodent. In the earliest stages, a single population of ganglion cells is present. Immature spiral ganglion neurons possess small perisomatic processes that seem to disappear with development, before the myelination ganglion cells are surrounded by one or two layers of Schwann cell processes. With maturation, the Schwann process increases in number around the perikaryon and its processes, which leads to the onset of myelination. The onset of myelination of the cell body processes is asynchronous. The perikaryon may be delayed in myelination by several days. Moreover, ganglion neurons from a given region of the cochlea do not myelinate simultaneously. The differentiation of two types of fibers in the intraganglionic spiral bundle and the first appearance of TII neurons occurs around birth in the cat and a few days after birth for the rat and the mouse. The distinction of TII cells is possible due to characteristic accumulation of neurofilamentous structures in the cytoplasm.
Freeze‐fracture and immunomorphological analysis of spiral ganglion cells
Authors: Anniko Matti , Thornell Lars‐Eric , Virtanen Ismo , Ramaekers Frans C. S.
Published: February 2005

Freeze‐fracture analysis of adult spiral ganglion cells of CBA/CBA mice revealed two types of membrane specializations. Most cells (type I) had a smooth surface and were surrounded by Schwann cells. Type II spiral ganglion cells showed numerous membrane specializations with well‐delineated indentations similar to those previously found on hair cells adjacent to afferent and efferent nerve endings. Immunomorphological analysis (using well‐defined monoclonal antibodies directed against different subclasses of intermediate filament proteins) revealed a unique co‐expression of neurofilaments, vimentin and cytokeratins in spiral ganglion cells of 8‐to 22‐week human fetuses.
Comparison of demyelination and neural degeneration in spiral and Scarpa's ganglia of C57BL/6 mice
Authors: Cohen Glenn M. , Park Janie C. , Grasso Joseph S.
Published: February 2005

Spiral and Scarpa's (vestibular) ganglia of female C57BL/6 mice, ranging in age from 1.5 to 32 months, were examined by light and electron microscopy. The spiral ganglia of C57BL/6 mice undergo sweeping neuronal losses. Based on cytological characteristics that repeat themselves in both young and old mice, we divided perikaryal demyelination and degeneration of spiral ganglia into four arbitary stages: (1) incipient demyelination, myelin sheaths begin to loosen and unravel; (2) contact, the partially demyelinated perikarya abut as the extracellular matrix disappear; (3) clumping, clusters of naked perikarya clump together, cytoplasmic processes surround the exterior of the clump; and (4) resorption, the clumps gradually disintegrate, leaving at completion large, fluid‐filled spaces and few cellular remnants. In Scarpa's ganglia, a small number of neurons demyelinate and clump, but none degenerate or undergo resorption.
Synaptic morphology of inner and outer hair cells of the human organ of Corti
Authors: Nadol Joseph B.
Published: February 2005

Innervations of inner and outer hair cells of the organ of Corti of the human cochlea were studied by serial section electron microscopy. At the base of inner hair cells, presumed afferent fibers were of varying size and demonstrated synaptic specialization consisting of a presynaptic body, vesicles, and asymmetrical synaptic membrane specialization. Two types of neurons, vesiculated presumably efferent and nonvesiculated presumably afferent, synapsed at the base of outer hair cells. The synaptic specialization of afferent fibers included presynaptic body, vesicles, and asymmetrical membrane thickening, whereas efferent synapses demonstrated presynaptic vesicles and a subsynaptic cisterna. Some presumably afferent nerve terminals formed a reciprocal synapse with outer hair cells in both the human and the chimpanzee. Such a synaptic relationship demonstrated morphologic specialization consistent with both hair cell‐to‐neuron and neuron‐to‐hair cell transmission between the same outer hair cell and nerve terminal. The innervation density of inner and outer hair cells and the comparative anatomy of the afferent and efferent innervation are discussed.
Serial reconstruction of inner hair cell afferent innervation using semithick sections
Authors: Siegel Jonathan H.
Published: February 2005

The afferent innervation pattern of inner hair cells in the apex of the guinea pig cochlea was studied using serial reconstruction of semithick (0.25–μm) sections and high‐voltage electron microscopy (HVEM). This thickness produced a good compromise between the ability to resolve details of the synaptic contacts between the hair cells and sensory neurons and the number of sections required to reconstruct the nerve terminals within the receptor organ. The use of a goniometer allowed the sections to be tilted to angles optimum for viewing either the synaptic membrane specializations or the presynaptic bodies. Reasonably good images of 0.25‐μm sections could be obtained using a conventional 120‐keV microscope, but the images produced by the HVEM were clearly superior. The sensory nerve terminals and hair cells were reconstructed using a microcomputer‐based computer‐aided‐design system. Nerve terminals with complex shapes could be successfully rendered as surface models viewed as stereo pairs. The advantages and limitations of the techniques used are discussed.
Immunoelectron microscopic localization of neurotransmitters in the cochlea
Authors: Eybalin Michel , Altschuler Richard A.
Published: February 2005

This paper presents the works and methods of our respective laboratories using electron microscopic immunocytochemistry to identify and localize cochlear neurotransmitters. Antibodies to various prospective neurotransmitters and associated enzymes have been used to study the ultrastructural localization of several candidates for olivocochlear efferent neurotransmitters previously suggested by light microscopic immunocytochemistry. Antibodies against enkephalins label lateral olivocochlear efferent fibers. Antibodies against choline acetyltransferase (ChAT) (an enzyme marker for acetylcholine) label a major population of both lateral and medial efferent fibers and terminals, whereas antibodies to γ‐aminobutyric acid (GABA) label what might be a small subpopulation of both the lateral and medial efferent systems. The GABA‐like immunostained medial efferent fibers are preferentially located in the upper turns of the guinea pig cochlea, particularly the third turn. Immunoelectron microscopy shows that neither GABA nor ChAT immunolabels all medial efferent terminals, regardless of cochlear turn. All the different types of immunolabeled efferent terminals have been observed to make characteristic synaptic contacts; lateral efferent terminals on afferent dendrites and medial efferent terminals on outer hair cells and occasionally on type II afferent dendrites. Other types of contacts involving GABA‐like, and sometimes met‐enkephalin‐like, immunostained fibers are occasionally seen particularly in the upper turns of the cochlea. Immunoelectron microscopic results suggest that both medial and lateral efferent systems might be further subdivided on the basis of differences in neurotransmitters. Future trends of immunocytochemical research on cochlear neurotransmitters are proposed, particularly colocalization studies, which show a complex pattern of coexistence of neurotransmitters in the lateral efferent system.
Autoradiographic studies of selective amino acid uptake by neural and nonneural elements in the gerbil cochlea
Authors: Schwartz I. R. , Ryan A. F.
Published: February 2005

The cochlea is well suited for studies of the uptake properties of auditory neurons and nonneuronal supporting cells. Probe concentrations of radioisotopically labeled amino acids, including putative neurotransmitters and their precursors, breakdown products, and blockers, can be introduced via the natural, fluid‐filled channels of the inner ear. Uptake patterns can be mapped at cellular and intracellular levels using light and electron microscopic autoradiographic methods. The procedures for introduction of label, fixation, plastic embedment, and light and electron microscopic autoradiography are described with special reference to the cochlea. Labeling patterns observed with over 20 amino acids are summarized for hair cells, spiral ganglion neurons, efferents, and nonneural elements of the stria vascularis, limbus, and modiolus. Limitations on the interpretation of results and their implications for the general usefulness of the methods are discussed.
Synaptic circuitry identified by intracellular labeling with horseradish peroxidase
Authors: Hamos James E.
Published: February 2005

During the past two decades new techniques have been developed to directly test the dogma that neuronal structure is correlated with neuronal function. In the earliest experiments, Procion yellow was injected into neurons after they had been characterized physiologically; these neurons were then viewed through the light microscope. Recent advances in the method generally employ horseradish peroxidase as the dye which is injected since it diffuses quite readily throughout the injected neuron and produces a stable reaction product for both light and electron microscopic studies. This review explores the utility of examining synaptic circuitry after physiologically recording from axons or neurons and then injecting horseradish peroxidase into them. As a model system, we studied the cat lateral geniculate nucleus and investigated, at the electron microscopic level, the synaptic contribution to this nucleus from retinogeniculate axons, from interneurons, and from the axon collaterals of neurons that project to visual cortex.
Scanning image detection (SID) system for conventional transmission electron microscope (CTEM) images
Authors: Tanji Takayoshi , Tomita Masahiro , Kobayashi Hiroyuki
Published: February 2005

A new image detection system has been developed to display transmission electron microscope (TEM) images on a CRT without a video camera system. Deflection coils placed in both the upper space of an objective lens and in the lower space of the first intermediate lens scan a small electron probe simultaneously. The electrical signal acquired through an improved scintillator and a photomultiplier is synchronized with the scanning signal and displayed in a similar fashion to a conventional scanning TEM (STEM) instrument. A preliminary system using a 100 kV conventional TEM (CTEM) equipped with a hairpin‐type electron gun, produced an image with a spatial resolution of 1 nm.
On high‐resolution transmission electron microscopy in an unstable magnetic environment
Authors: Luzzi David E.
Published: February 2005

Owing to the particular environment of the JEM4000EX HREM at the University of Pennsylvania, it became necessary to control the magnetic environment around the microscope. The unstable magnetic environment included two components, a slowly wandering vertical DC magnetic field and an AC magnetic field. The effects of these fields on the microscope performance were to introduce an uncertainty in the objective lens defocus value over a series of images and to reduce the attainable resolution of the microscope, respectively. A solution is presented in which these fields are stably reduced well within the limits of sensitivity of the JEOL, Ltd. JEM4000EX for high‐resolution imaging. This solution is based on a feedback loop using a pseudo‐Helmholtz coil to generate a well‐defined vertical magnetic field.
Lectin binding patterns to plasmalemmal glycoconjugates of goblet cells undergoing differentiation in vitro
Authors: Frisch Emily B. , Phillips Thomas E.
Published: February 2005

The plasmalemmal glycoconjugates of the HT29‐18N2 (N2) cell line were characterized on cells grown as (1) undifferentiated multilayers in glucose‐containing culture media and (2) monolayers of columnar cells acquiring the goblet cell phenotype in glucose‐free media. Lectins were unable to bind sheets of detached N2 cells in the absence of fixation. Following fixation with aldehydes, a dramatic unmasking of lectin binding sites was seen. When fixed monolayers were stained prior to embedding, biotinylated lectins, visualized by the avidin‐biotin‐complexed peroxidase technique, were more efficient than collodial gold‐coupled lectins. Lectin binding sites could also be detected by using collodial gold‐coupled lectins to stain monolayers embedded in LR White, Lowicryl K4M, and Lowicryl HM20. The binding of 5 lectins (wheat germ, Dolichos bifluros, peanut, soybean, and Ulex europeus) was found to be independent of the stage of differentiation; “pre‐differentiated” columnar cells which had prominent microvilli and no or few mucous secretory granules had identical staining patterns as well‐differentiated goblet cells with large numbers of secretory granules. Ricinus communis I was the only lectin whose binding was influenced by the stage of differentiation; it intensely labeled undifferentiated multilayers of N2 cells but only weakly labeled basolateral membranes of differentiated monolayers. Canavalia ensiformas (ConA) caused a moderate and even labeling of both apical and basolateral membranes of fixed monolayers stained prior to embedding, but post‐embedding labeling revealed heavy labeling along the lateral margins of all columnar cells and weak to moderate binding along the apical and basal cell surface.
A Stopped‐flow/rapid‐freezing machine with millisecond time resolution to prepare intermediates in biochemical reactions for electron microscopy
Authors: Pollard Thomas D. , Maupin Pamela , Sinard John , Huxley Hugh E.
Published: February 2005

We have developed an instrument capable of freezing transient intermediates in rapid biochemical reactions for subsequent freeze‐fracturing, replication, and viewing by transmission electron microscopy. The machine combines a rapid mixing unit similar to one widely used in chemical kinetics (Johnson, 1986) with a propane jet freezing unit previously used to prepare static samples for freeze‐fracturing (Gilkey and Staehelin, 1986). The key element in the system is a unique thin‐walled flow cell of copper that allows for injection and aging of the sample, followed by rapid freezing. During freeze‐fracturing, a tangential cut is made along the wall of the flow cell to expose the sample for etching and replication. The dead time required for mixing and injection of the reactants into the flow cell is less than 5 ms. Electronic controls allow one to specify, on a millisecond time scale, any time above 5 ms between initiation of the reaction and quenching by rapid freezing.
Ultrastructural aspects of oogenesis and oocyte growth in fish and amphibians
Authors: Wallace Robin A. , Selman Kelly
Published: February 2005

Oogenesis, the early events of primary oocyte growth (meiotic arrest, synapsis, ribosomal gene duplication), and folliculogenesis can be seen to particular advantage in the germinal ridge of the syngnathan ovary. After budding off the germinal ridge (a compartment of the luminal epithelium), nascent follicles then enter into a linear array of developing follicles within which temporal and stage‐specific events can be correlated with spatial distribution. Prominent features of the later phase of primary oocyte growth include intense transcriptional activity and the formation and subsequent dispersal of the Balbiani vitelline body (mitochondrial cloud) concomitant with an increase in cytoplasmic organelles and volume. Further oocyte growth is characterized by a period of cortical alveolus (in teleosts) or cortical granule (in anurans) formation, in which Golgi elements play a predominant role, and finally vitellogenesis. The latter process, which is responsible for the preponderance of oocyte growth, includes the hepatic synthesis and secretion of vitellogenin (VTG), the uptake of VTG from the bloodstream into the oocyte by receptor‐mediated endocytosis, and the transport of VTG via endosomes and multivesicular bodies to forming yolk platelets. In the process, VTG is proteolytically cleaved into the yolk proteins, which assume either a monoclinic (in cyclostomes) or orthorhombic (in teleosts and amphibians) crystalline array. Other structures associated with the growing oocyte are also briefly discussed, including nuage, the vitelline envelope, intercellular junctions between the oocyte and overlying follicle cells, pigment, intramitochondrial crystals in ranidae, and annulate lamellae.
Transmission electron microscopic X‐ray quantitative analysis of human dentin at 200 kV accelerating voltage
Authors: Yonehara Katsuhisa , Shinohara Minoru , Kanaya Koichi
Published: February 2005

Qualitative and quantitative x‐ray energy dispersive spectroscopy is now used successfully to analyze many features and processes in inorganic samples. When applied to inorganic samples, however, the results are often less satisfactory due to problems of preparation of organic samples, difficulty of measuring x‐rays from organic samples, damage of the sample by the electron beam, and other practical problems.

In the present study we used a high voltage transmission electron microscope equipped with an energy dispersive x‐ray spectrometer to examine accurate quantitative standardless analysis of thin sections of an organic sample, human dentin.

Based on our experiments we found the important parameters for quantitative analysis were sample thickness and appropriate choice of model sample. Further, we show that the method of Cliff and Lorimer can be used with biological samples at 200 kV, and we show that quantitative analysis of human dentin can be carried out at 200 kV. Finally, we show that areas of human dentin can be differentiated by their morphological characteristics and x‐ray analyses obtained in the transmission electron microscope.
Modified embedding procedure for microtomy of large particle zeolites
Authors: Ulan J. G. , Schooley C. , Gronsky R.
Published: February 2005

A method is described to strengthen the binding of organic resin to inorganic zeolite, allowing large particles to be microtomed. For FeZSM‐5 aggregates the particle size limit increased from 3 μm to greater than 20 μm in diameter by application of this method. This technique can be applied to a variety of oxide powder samples, extending the utility of microtomy as a materials science tool.
Perspectives on golgi apparatus form and function
Authors: Mollenhauer Hilton H. , Morré D. James
Published: February 2005

In 1898, Camillio Golgi reported a new cellular constituent with the form of an extensive intracellular network (the apparato reticolare interno), which now bears his name. However, the history of Golgi's apparatus is replete with controversy regarding its reality, what components of the cell should be included under its aegis, and what terminology should be used when referring to it. Electron microscopy has resolved many of these controversies and it is appropriate that this volume emphasize that aspect of Golgi apparatus discovery.

The principal structural component of the Golgi apparatus is the stack of cisternae, or dictyosome. As determined both biochemically and at the level of electron microscopy, the dictyosome is a highly ordered and polarized structure. The maintenance of order within the stack is thought to result from either intercisternal bonding constituents, or filamentous structures (or both) that bridge the space between adjacent cisternae.

Mechanisms proposed for movement of membrane and product into and out of the dictyosome (i.e., the Golgi apparatus stack) include a serial mode which functions exclusively by the formation, displacement, and loss of cisternae from the stack, and a parallel mode which functions exclusively by the movement of membrane, product, or precursor molecules directly into the peripheral edges of the cisternae. In the parallel mode, all cisternae can be accessed either singly or simultaneously, at least in theory, at any position within the stack. It is probable that both the serial and the parallel modes function concomitantly and need not be mutually exclusive.

Finally, the peripheral tubules of the cisternae represent a major membranous constituent of the cell with potentially unique functions. These tubules interconnect cisternae of adjacent stacks and may represent the major site of receptors for the shuttle (i.e., parallel) type of transfer among cisternae. Peripheral tubules as extensions of the cisternal lumina into the cytoplasm presumably have other functions, but these, like the tubules themselves, have only rarely been accommodated into functional models of Golgi apparatus dynamics in secretion or membrane flow.
Cytochemical characteristics of the golgi apparatus
Authors: Pavelka Margit , Ellinger Adolf
Published: February 2005

Lectinocytochemistry provides a useful tool for localizing subcompartments of the complex reticular apparatus of Golgi. The technique is based on interactions of lectins with glycoconjugates present in the limiting membranes and luminal spaces of Golgi elements. Application of a series of lectins of different sugar specificities permits a differentiation between Golgi subcompartments containing glycoconjugates with different oligosaccharide side chains. These may be (a) different glycoconjugates or (b) glycoconjugates at different stages during synthesis or repair of their glycans. The lectinocytochemical studies with mannose‐, glucose‐, N‐acetyl‐glucosamine‐, N‐acetylgalactosamine‐, galactose‐, fucose‐, and sialic acid‐recognizing lectins revealed predominating patterns that labeled distinct, i.e., cis, medial, trans, and transmost, regions of the Golgi apparatus. A further refinement could be achieved by differential lectin‐inhibition that enables a dissection of lectin binding reactions on the basis of their binding affinities. High‐affinity binding reactions showed that subcompartments are not necessarily confined to one single Golgi subregion and may change their position from one to another subregion. Some of the patterns observed may be interpreted in relation to certain steps during synthesis and modifications of glycans.
Secretory pathways in animal cells: With emphasis on pancreatic acinar cells
Authors: Beaudoin Adrien R. , Grondin Gilles
Published: February 2005

Studies over the past three decades have clearly established the existence of at least two distinct pathways for the intracellular transport and release of secretory proteins by animal cells. These have been identified as the regulated and constitutive pathways. Many observations have indicated that in certain cells, such as those of the exocrine pancreas and parotid glands at least, these pathways coexist in the same cells. Although the general scheme of protein transport within these pathways is well established, many fundamental aspects of intracellular transport remain to be unraveled. How are proteins transported through the endoplasmic reticulum? How are the transitional vesicles formed and what are the underlying mechanisms involved in their fusion with the cis‐Golgi cisterna? Even the general mode of transfer through the Golgi stack is debated: Is there a diffusion through the stack by flow through intercisternal tubules and openings or is there a vesicle transfer system where membrane quanta hop from one cisterna to the other? What is the fate of secretory proteins in the trans‐Golgi area and by what mechanisms is a fraction of newly synthesized molecules of a given secretory protein released spontaneously while the majority of such nascent molecules are diverted into a secretory granule compartment? In this review, we have examined these and other aspects of intracellular transport of secretory proteins using pancreatic acinar cells as our reference model and we present some evidence to support the existence of a paragranular pathway of secretion associated with secretory granule maturation.
Localization of glycosylation sites in the golgi apparatus using immunolabeling and cytochemistry
Authors: Roth Jurgen
Published: February 2005

This review summarizes data on the distribution of certain glycosylation steps in the Golgi apparatus as revealed by immunolabeling and lectin techniques. The methodical basis for such investigations was provided by the introduction of the colloidal gold marker system for immunolabeling and the development of new means of tissue processing such as the low‐temperature embedding technique using Lowicryl K4M. The application of these techniques together with highly specific antibodies has provided much of the basis for our current understanding of the Golgi apparatus in functional terms. Thus, in many cell types, three Golgi apparatus compartments can be distinguished, whereas in others no such functional subdivision is evident. Investigations on sialyltransferase distribution have also provided direct evidence that GERL is structurally and functionally part of the Golgi apparatus.
Imaging friction tracks at diamond surfaces using reflection electron microscopy
Authors: Wang Z. L.
Published: February 2005

Reflection electron microscopy (REM) is applied to image the structure of polished natural diamond (001) surfaces (of 5 × 4 mm size) after friction experiments under a pressure below the critical value. Friction tracks marked by a diamond needle after a single pass movement under a pressure of 13 GPa can be seen in REM images and show non‐uniform contrast. The surface shows relatively dark image contrast at the light contacted area, which is possibly due to the structural modification at the top atomic layer. The high local contacting pressure pushes part of the needle into the surface which causes fracture, resulting in the formation of grooves at the surface. It is possible to have plastic deformation in this process, but no evidence has been found for the presence of cracking. The observations support the adhesion frictional mechanism rather than the micro‐cleavage model.
Fertilization‐Induced changes in the vitelline envelope of echinoderm and amphibian eggs: Self‐assembly of an extracellular matrix
Authors: Larabell Carolyn , Chandler Douglas E.
Published: February 2005

The surface of the unfertilized sea urchin egg is covered by the vitelline layer (VL), a fibrous extracellular matrix that contains receptors for sperm. At fertilization, cortical granule exocytosis releases enzymes and structural proteins that cause the VL to elevate and become remodelled into the mechanically and chemically tough fertilization envelope. This envelope prevents further penetration of sperm and protects the embryo during early development. A thicker, more complex vitelline envelope surrounds the Xenopus laevis egg. This fibrous coat is also restructured at fertilization to produce an impenetrable barrier to sperm. The biochemical steps that occur during self‐assembly of these fertilization envelopes are reviewed, and the ultrastructural changes that occur, as seen in platinum replicas of quick‐frozen, deep‐etched, and rotaryshadowed eggs, are illustrated.
Suitability of different silver enhancement methods applied to 1 nm colloidal gold particles: An immunoelectron microscopic study
Authors: Stierhof York‐Dieter , Humbel Bruno M. , Schwarz Heinz
Published: February 2005

In order to exploit the recently introduced 1 nm gold colloids in routine electron microscopic labeling experiments, an efficient enhancement step for a better visualization of this small marker is a prerequisite. Efficiency and reproducibility of enhancement as well as growth homogeneity of gold particles were evaluated for three different silver intensifying solutions: silver lactate/hydroquinone/gum arabic (Danscher, 1981), Ilford L4/Metol (Bienz et al., 1986), and the commercially available IntenSE M silver enhancement kit (Janssen Pharmaceutica). The best results were obtained by using the silver lactate/hydroquinone/gum arabic mixture. The quality of enhancement of the IntenSE M kit was considerably increased by the addition of the protective colloid gum arabic.
Influence of calcium and amino acids on the osmium impregnation of the endoplasmic reticulum
Authors: Thiery G. , Bergeron M.
Published: February 2005

The aim of this study was to define further the interaction between osmium and organelle content in cells prefixed with glutaraldehyde. We have studied the reaction of osmium with divalent or trivalent cations (calcium, barium, zinc, aluminum, and iron) and various amino acids in the same conditions prevalent in histological techniques, in particular with Thiéry's technique of metal impregnation. Experiments were carried out in vitro in test tubes, on cellulose acetate discs, or with an immunodiffusion apparatus. Some experiments were also carried out with tissue extracts (kidney and intestine). Our studies suggest that calcium is in general essential for the formation of osmium black, but also that lysine is reactive even in the absence of calcium and that a few amino acids–such as tryptophan, ornithine, cysteine, and aspartic acid–are only slightly reactive in the absence of calcium. Other amino acids do not seem to participate in the endoplasmic reticulum osmium impregnation even in the presence of calcium ions. Our studies also suggest that osmium reactivity reflects calcium binding sites and not only calcium content.
Three‐Dimensional imaging of fertilization and early development
Authors: Holy Jon , Simerly Calvin , Paddock Stephen , Schatten Gerald
Published: February 2005

The field of biological microscopy has recently enjoyed major technical advances, exemplified by the development of field‐emission low‐voltage scanning electron microscopes and laser scanning confocal light microscopes. In addition, computer processing of microscopical data is revolutionizing the way morphological information is imaged. In this paper, we illustrate methods by which this new technology can be used to examine events in fertilization and early development in three dimensions. Different types of specimen preparation protocols, using both echinoderm and mammalian gametes and embryos, are evaluated for their ability to preserve accurately the threedimensional organization of these specimens for imaging by both low‐voltage scanning electron microscopy and laser scanning confocal light microscopy.
Confocal microscopy and three‐dimensional reconstruction of electrophysiologically identified neurons in thick brain slices
Authors: Turner J. N. , Swann J. W. , Szarowski D. H. , Smith K. L. , Marko M. , Leith A.
Published: February 2005

Three‐dimensional morphology and electrophysiology were correlated from individual neurons in a thick brain slice preparation. The hippocampal formation from immature and adult rats was cut transverse to the longitudinal axis into 500 μ Um‐thick slices which were maintained under physiologic conditions. Individual neurons were impaled and physiologically characterized using microelectrodes. Recordings were made from the soma and in some cases from a dendrite. The impaled neurons were filled through the microelectrode with the fluorescent dye lucifer yellow and imaged by confocal scanning laser microscopy using an analog preprocessor. As many as 180 optical sections were recorded as a function of depth through the slices. Images are presented as a series of optical sections, stereo pairs, or three‐dimensional reconstructions. Both stereo contouring and volume rendering methods were employed, and the reconstructions were viewed from any arbitrary perspective. Dendritic and axonal fields were separated from each other and displayed separately or as different pseudocolors. The three‐dimensional reconstructions provided perspectives that were difficult or impossible to appreciate by viewing the optical sections or conventionally formed stereo pairs.
The use of confocal microscopy and sterecon reconstructions in the analysis of sea urchin embryonic cell division
Authors: Summers Robert G. , Musial Cora E. , Cheng Ping‐Chin , Leith Ardean , Marko Michael
Published: February 2005

A laser scanning confocal microscope has been used to investigate the development of the sea urchin embryo. The samples were fixed in Carnoy's solution at various developmental stages, stained for DNA with the Feulgen reaction, and optically sectioned with a BioRad MRC‐500 confocal microscope. Computer‐generated stereographic projection images and a three‐dimensional contour tracing and reconstruction system were employed to investigate the cleavage pattern during the 6th cleavage division. Cell division is found to be asynchronous during the 6th cleavage, with macromere derivatives completing division first, followed by mesomeres, and finally by the outer quartet of micromeres (which begins division only after macromeres and mesomeres have completed their respective divisions). Sixth cleavage produces an embryo comprising 60 cells. Asynchronous division was also observed within individual tiers of blastomeres. Variations in the orientations of cell division axes within individual tiers of cells were also observed. The utility of computer‐graphics reconstruction techniques for both quantitative and qualitative developmental analysis are discussed.
Applications of confocal microscopy to the study of myelin development and neuron structure
Authors: Staugaitis Susan M. , Rowan Todd , Sanes Dan H. , Colman David R. , Smith P. R.
Published: February 2005

Confocal laser scanning microscopy has been used to study the localization of myelin basic proteins expressed in nonglial cells, and to probe the three‐dimensional structure of central auditory neurons in the lateral superior olive. The paper focuses on the techniques used to obtain the results. The key roles of confocal microscopy and computer image processing of the images obtained are emphasized as they relate to the discovery of essential structural information about these specimens.
Microfibril‐microvessel connections in the rabbit eye: Correlative confocal and electron microscopy
Authors: Korte Gary E. , Manche Edward E.
Published: February 2005

The connections between elastic tissue and microvessels (arterioles, capillaries, and venules) in the rabbit eye were examined by light and electron microscopy. In particular, confocal scanning laser microscopy of tissue stained with orcein and examined by fluorescence using a rhodamine filter was correlated with electron microscopic observations. The goal was an analysis of the way in which elastic tissue of the uvea (i.e., choroid, ciliary body, and iris) and the optic nerve of the eye connect to the microvessels in these structures. Confocal microscopy revealed these connections advantageously and showed how they link the elastic tissue meshwork in the perivascular tissue spaces with the wall of the blood vessels. Electron microscopy showed that the connections consist of bundles of 10–12 nm diameter microfilaments that insert into vascular basement membranes. These connections may contribute to the vascular response to changes in blood pressure or intraocular pressure in the eye.
Immunoalkaline phosphatase with cerium‐based cytochemistry for double staining at the transmission electron microscopic level
Authors: Rowden Geoffrey , Dean Shirley
Published: February 2005

Immunoelectron microscopic localization of surface antigens on lymphocytes is possible using alkaline phosphatase combined with cerium‐based cytochemical methods. Distinctive electron‐dense deposits are easily identified at sites of antibody binding. Mouse splenocytes showing surface immunoglobulin localization and human peripheral blood lymphocytes stained for the MHC‐Class II antigen HLA‐DR illustrate the results. Double staining for murine Ia antigen by the alkaline phosphatase procedure, combined with immunogold labeling of antigens identified on dendritic cells, i.e., NLDC‐145, demonstrates the utility of the cerium cytochemical method.
Circular harmonic averaging of rotary‐shadowed and negatively stained creatine kinase macromolecules
Authors: Winkler H. , Gross H. , Schnyder T. , Kunath W.
Published: February 2005

The structure of mitochondrial creatine kinase is investigated by high‐resolution shadowing at very low temperature and conventional negative staining. The electron microscopic images are analyzed with circular harmonic averaging, a method suited for the processing of single molecules. The rotational alignment and averaging is performed with the circular harmonic components, which allows data compression and several steps of noise reduction to be carried out within the averaging procedure. In addition, the symmetry can be deduced. For the mitochondrial creatine kinase, a fourfold symmetry is found that is compatible with the biochemical and biophysical characterization of the molecule.
Loss of grain boundary segregant during ion milling
Authors: Kenik Edward A.
Published: February 2005

It is shown that material segregated to grain boundaries can be lost during ion milling. This specimen preparation artifact has been studied in the case of bismuth in copper and has also been observed for phosphorus in stainless steel. The loss is associated with specimen heating during ion milling and can be alleviated by good clamping and cooling of the specimen during milling. Specimen heating permits grain boundary diffusion of the segregating element to the specimen surfaces with subsequent loss of segregant from the specimen by evaporation or sputtering during ion milling. Loss of bismuth during in situ heating to 200–300°C is demonstrated. Therefore, care must be taken in specimen preparation for analytical electron microscopy measurement of such segregation. Similar effects may occur during ion milling of other materials, especially those where low thermal conductivity will result in high beam heating. In these cases, care must be taken to avoid loss of segregant during specimen preparation. Additional tests showed that no significant loss of segregant was observed during X‐ray microanalysis, even at nominal room temperature and probe currents five‐fold higher than that normally used for microanalysis.
Negative staining‐carbon film technique: New cellular and molecular applications
Authors: Robin Harris J.
Published: February 2005

Two techniques are presented which extend the original negative staining‐carbon film technique into new areas of cellular and molecular application. These relate (1) to the production of negatively stained specimens of single‐layer plasma membrane split from intact cells during the overall procedure that are negatively stained from the cytoplasmic face and (2) to the production of negatively stained specimens directly from glycerol‐containing protein solutions, membrane or viral suspensions. In both cases in vacuo drying onto mica from glycerol is performed, prior to deposition of a carbon film. (For the cellular technique, freshly cleaved mica is firstly rendered positively charged by immersion in Alcian blue.) This is followed by release of the carbon film plus adsorbed membrane or protein by floating onto water, with subsequent negative staining. Selected preliminary applications using human erythrocyte membrane and the high molecular weight (native) human erythrocyte tripeptidyl peptidase‐II complex are given and considered speculation as to the future application of the techniques is provided.
A fast image simulation technique for high resolution electron microscopy with multicomponent atomic species
Authors: Krakow William
Published: February 2005

A new method has been developed for simulating high resolution electron microscope images of weak phase objects via a digital television frame store system and fast Fourier transforms of a graphical representation of structures containing several atomic species. Here masks are constructed which consist of circular disk regions whose areas are proportional to the scattering power of the different atom types. These masks represent the object transmission function. The method extends the previous work on image computations of monatomic species objects using small point‐like model representations of atom positions. Several examples will be given to verify the relative scattering power from masks corresponding to different atomic species such as Y, Ba, Cu, and O, as well as the limitations of this method for representing objects. Image simulations, which are in agreement with experiments, will also be presented for superconducting oxide materials of the form YBa2Cu3O7 using the circular disk method.