bluuebunny/crossref_metadata_2025 huggingface dataset:
Microtubules in plant cell division
Authors: Wick S. M.
Published: June 2020

Immunofluorescence microscopy has proven to be a valuable accompaniment to electron microscopy for study of the cytoskeleton of plant cells. Whereas electron microscopy provides greater resolution and details of the spatial relationships of the cytoskeleton to other cellular components, fluorescence visualization makes it possible to see the three-dimensional organization of cytoskeletal elements without laborious reconstruction of views from serial sections. An area in which immunofluorescence microscopy has been useful is the investigation of how plant cells organize and position the various microtubule arrays that are utilized during mitosis, cytokinesis and cell expansion phases. One of the earliest indications of an impending division in a meristematic plant cell is the formation of a preprophase band of microtubules in the cell cortex, at the site where the new wall will be placed at the subsequent cytokinesis. At its later stages, the band is narrower than when first identifiable. In most cells, preprophase band microtubules have the same general orientation as the preceding interphase microtubules, and so preprophase band formation here could, in theory, be achieved by lateral bundling of microtubules.

Cells in which the division site and the preprophase band that marks it are not oriented parallel to interphase microtubules are found in stomatal complexes of grass leaves . Fig. 1 illustrates the arrangement of two such cell types: the guard mother cell, which divides lengthwise to form two guard cells, side-by-side, and the subsidiary mother cell, which undergoes a very asymmetric division to produce one of the pair of lens-shaped subsidiary cells that flank the guard cells. Interphase and preprophase arrangements of microtubules for each cell type are diagrammed in Figs. 2-4. In order to examine how these cell types achieve the reorientation of microtubules that is necessary to progress from interphase to preprophase, sheets of epidermis containing actively dividing stomatal complex cells were examined with immunofluorescence microscopy using antibodies to tubulin. Thin epidermal slices of leaves were fixed and glued down to a slide, whereupon cell walls were enzymatically weakened so that unwanted cell layers could be removed . Because waves of division pass along grass leaves, cells of the same type in a given file tend to be at similar stages, which facilitates deduction of the developmental pattern.
Involvement of plasma membrane complexes in cellulose deposition in desmids
Authors: Giddings T. H.
Published: June 2020

Deposition of cellulose microfibrils in oriented patterns in plant cell walls is believed to be one of the mechanisms by which cell shape is controlled. In some desmids, such as Closterium. circumferentially oriented microfibrils appear to help generate an elongated spindle shaped cell. In contrast, the primary cell wall of Micrasterias contains an apparently random meshwork of microfibrils, yet an elaborate multi-lobed semicell is formed. Freeze-etch electron microscopy of unfixed, ultrarapidly frozen desmids was undertaken to identify plasma membrane and cytoskeletal elements that may participate in the deposition and orientation of cellulose microfibrils.

Micrasterias denticulata and aClosterium sp. were grown under a light/dark cycle to induce partial synchronization of cell division. Groups of several cells at the desired stage of semicell regeneration were loaded between two modified Balzers gold planchets coated with a slurry of yeast. A slot grid spacer prevented the cells from being crushed. Specimens were frozen in a propane jet freezing device designed by Gilkey and Staehelin. Replicas were produced in a Balzers BA 360 freeze-etch device and examined in a JEOL 100 CX electron microscope.
Ultrastructural characteristics of sporidia of Ustilago
Authors: Hackney Patricia N.
Published: June 2020

Ustilago hordei and Ustilago violacea are yeast-like basidiomycete pathogens ofHordeum vulgare and Silene alba respectively. The mating type system in both species of Ustilago is bipolar, with alleles, A,a, (U.hordei) and a1, a2 (U.violacea) at a single locus. Haploid sporidia maintain the asexual phase by budding, while the sexual phase is initiated by conjugation tube formation between the mating types during budding and conjugation.

For observation of budding, sporidia were prepared by culturing the four types on YEG (yeast extract glucose) broth for 24 hours. After centrifugation at 5000g cells were either left unmated or mated in a1/a2,A/a combinations. The sporidia were then mixed 1:1 with 4% agar and the resulting 1mm cubes fixed in 8% gluteraldehyde and post fixed in osmium tetroxide. After dehydration and embedding cubes were thin sectioned with a LKB ultratome and photographed in a Zeiss 9s transmission electron microscope or in an AE1 electron microscope of MK11 1MEV at the High Voltage Electron Microscopy Center of the University of Wisconsin-Madison.
Hair cell changes after cationic stimulation of corti's organ
Authors: Fermin Cesar D. , Zenner Hans-Peter
Published: June 2020

Contraction of outer and inner hair cells (OHC&IHC) in the Organ of Corti (OC) of the inner ear is necessary for sound transduction. Getting at HC in vivo preparations is difficult. Thus, isolated HCs have been used to study OHC properties. Even though viability has been shown in isolated (iOHC) preparations by good responses to current and cationic stimulation, the contribution of adjoining cells can not be explained with iOHC preparations. This study was undertaken to examine changes in the OHC after expossure of the OHC to high concentrations of potassium (K) and sodium (Na), by carefully immersing the OC in either artifical endolymph or perilymph. After K and Na exposure, OCs were fixed with 3% glutaraldehyde, post-fixed in osmium, separated into base, middle and apex and embedded in Araldite™. One μm thick sections were prepared for analysis with the light and E.M. Cross sectional areas were measured with Bioquant™ software.

Potassium and sodium both cause isolated guinea pig OHC to contract. In vivo high K concentration may cause uncontrolled and sustained contractions that could contribute to Meniere's disease. The behavior of OHC in the vivo setting might be very different from that of iOHC. We show here changes of the cell cytosol and cisterns caused by K and Na to OHC in situs. The table below shows results from cross sectional area measurements of OHC from OC that were exposed to either K or Na. As one would expect, from the anatomical arrangement of the OC, OHC#l that are supported by rigid tissue would probably be displaced (move) less than those OHC located away from the pillar. Surprisingly, cells in the middle turn of the cochlea changed their surface areas more than those at either end of the cochlea. Moreover, changes in surface area do not seem to differ between K and Na treated OCs.
Mass determination of the nuclear pore complex and its sub-complexesl by high-resolution Scanning Transmission Electron Microscopy (Stem)
Authors: Reichelt R. , Holzenburg A. , Engel A. , Aebi U.
Published: June 2020

The nuclear pore complex (NPC) is an elaborate membrane-bound molecular machine residing in the nuclear envelope of all eukaryotic cells and acting as a passageway for molecular transport between the nucleus and the cytoplasm. Complementary to transmission electron microscopic studies of negatively stained and frozen hydrated NPCs yielding detailed information about their overall size, shape and substructure, the high-resolution scanning transmission electron microscope (STEM) was used to measure the mass, to map the radial mass distribution, and to evaluate the rotational symmetry of unstained native NPCs and of several distinct sub-complexes. “Native” NPCs were prepared for STEM as follows: the nucleus of Xenopus laevis oocytes was placed on a 4 nm carbon film supported by a holey film mounted on a copper mesh grid, and its nuclear envelope ruptured and spread out using fine glass needles. The grid was washed with low salt buffer to remove macroscopic contaminants, and excess liquid was withdrawn with filter paper. Next, tobacco mosaic virus (TMV; serves as internal mass standard) was co-adsorbed, before the grid was washed for 10 sec each on 3 drops of bi-destilled H2O. To disintegrate the NPC into distinct sub-complexes, nuclei were placed on a 4-nm thick carbon film supported by a thick holey carbon foil on a copper grid, ruptured and spread as above, and then detergent extracted. After washing, the samples were frozen in liquid N2, transferred to a VG HB-5 STEM and freeze-dried at 153 K. Low-dose annular dark field (AD) images (50 to 300 e/nm2) were acquired digitally, corrected for nonlinearities in the AD signal due to high mass thickness, and stored on tape for further processing.

Native NPCs, with and without plug, are displayed in Fig. 1. As shown in Fig. 2, two types of rings can be distinguished after detergent extraction: bright rings (“outer” rings; thick arrow) and dimmer rings (“inner” rings; thin arrow). In the same preparations particle “pairs” consisting of a ring (Fig. 3, thin arrow) and a plug-spoke complex (Fig. 3, thick arrow) can often be depicted. From these data averaged and 8-fold symmetrized mass maps of the native NPC with plug (Fig. 4; ˜50 NPCs) and without plug (Fig. 5; ˜50 NPCs), the outer ring (Fig. 6; ˜50 rings) and the inner ring (Fig. 7; ˜15 rings) were computed.
Corresponding radial mass distributions are displayed at the same scale for comparison (Figs. 8 to 11). The average masses of the different structures are summarized in Table 1 together with a tentative model. These measurements bring speculations about the mass of the NPC to an end which have stated values ranging from 20 MDa to over 100 MDa, and they suggest that the NPC could easily be made of the order of 100 different polypeptides.
Colder specimens give a sharper impression: Prospects for higher resolution Electron Microscopy at liquid helium temperature
Authors: Lamvik M.K.
Published: June 2020

Designers of electron microscopes started putting liquid helium into their plans with the hopes of improving instrumental resolution. Liquid helium can be used to cool specimen stages within conventional lenses, but it can also be used to make some materials superconducting. Once started, current flows continuously in a superconducting coil because it faces negligible electrical resistance. Superconducting lenses were introduced in electron microscopy to take advantage of the high magnetic field strengths that could be generated as well as the stability of the persistent current in the superconducting coils. As time passed, it became clear that electronic regulation of power supplies could produce more than adequate current regulation to supercede the need for the persistent current maintained in a superconducting lens. Meanwhile, the realization grew that low temperatures might give another kind of advantage to electron microscopy of radiation-sensitive materials. The very low temperature might help protect sensitive specimens from damage.

Several methods are available for detecting damage to specimens in electron microscopes. The loss of mass from specimens is one sign of radiation damage. A mass loss measurement with a liquid helium cooled stage seemed to show a significant reduction in mass loss; a different measurement that included a superconducting lens also indicated reduced damage. Another indicator of damage is the rate of fading of diffraction patterns of ordered specimens, and in this case it has been established that liquid helium temperature causes a moderate reduction of damage.
Three-Dimensional reconstructions of ’light’ and ’intermediate’ capsids of equine herpes virus
Authors: Baker T. S. , Newcomb W. W. , Booy F. P. , Brown J. C. , Steven A. C.
Published: June 2020

Equine herpes virus type 1 (EHV-1) belongs to an extensive family of large, genetically complex, and medically important animal viruses. The virion consists of an icosahedral nucleocapsid (T=16) separated from the viral envelope by a proteinaceous tegument layer. Assembly occurs in the nucleus of infected cells where capsids assemble, are packaged with DNA, then bud through the nuclear membrane. Two morphological species of EHV-1 capsids have been distinguished: “lights” which are abortive particles incapable of packaging DNA and “intermediates” which are precursors in the assembly of mature virions. Purified “intermediates” contain an additional proteinP22 (46 kDa), which is not present in “lights” and accounts for ˜10% of the total particle mass. In order to characterize the capsid structures of the two particle types and explore differences between them, we have applied three-dimensional reconstruction techniques to electron micrographs of frozen-hydrated specimens.

The Kentucky A strain of EHV-1 was propagated in L-929 cells, and the two types of capsids were obtained as separate fractions from Renografin-76 density gradients. Neither fraction contained any significant amount of DNA. Cryo-electron microscopy of capsids on carbon film substrates was performed as described earlier. Micrographs were recorded at a nominal magnification of x36,000 at 2-3μm underfocus and digitized with a 50/μn step size (˜1.38nm sampling at the specimen). Three-dimensional reconstructions of the two particle types were computed to 4.5nm resolution using modified “common-lines” procedures. Images of 37 “light” and 39 “intermediate” particles were separately combined to compute three-dimensional density distributions.
Morphological polarity of intercalating deep mesodermal cells in the organzer ofXenopus laevisgastrulae
Authors: Keller Ray , Shih John , Wilson Paul
Published: June 2020

The dorsal lip of the blastopore constitutes the “organizer” of the amphibian body plan, both in terms of its tissue interactions and its morphogenetic movements of convergence and extension during gastrulation. This tissue autonomously narrows (converges) and lengthens (extends) during early development, functioning prominently in the morphogenetic movements of both gastrulation and neurulationXenopus laevis. High resolution time-lapse recording of cell behavior in cultured explants and cell labelling studies have shown that the movements of convergence and extension are produced by radial intercalation of cells, in which several layers rearrange to produce fewer layers of greater area, and by mediolateral intercalation of cells, in which several rows of cells rearrange to produce a narrower, longer array. By labelling individual cells with the fluorescent compound, DiI, and making low light level recordings, we found that cells of the notochord intercalate mediolaterally using polarized protrusive activity at their internal medial or lateral ends. Thus polarized protrusive activity appears to play a major role in mediolateral cell intercalation after the boundary between the notochord and somites forms in the late gastrula stage.

We examine further the morphology of the deep mesodermal cells with scanning electron microscopy at earlier stages, to search for morphological manifestations of a similar polarity of protrusive activity. The dorsal deep mesodermal cells of early gastrulae were exposed by rapidly pulling the. epithelial endoderm off the deep cells with forceps, in Danilchik's solution, and fixing the embryo within 15 seconds in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) as described previously. The dorsal deep cells adjacent to the epithelium are elongate and aligned parallel to one another and to the circumference of the glastopore (Fig. 1). The medial and lateral ends bear broad, lamelliform protrusions (large pointers, Fig. 1, 2) whereas the anterior and posterior ends bear numerous small filiform protrusions (small pointers, Fig. 1, 2). This characteristic morphology is found only in the dorsal marginal zone, which undergoes convergence and extension by mediolateral intercalation.
New approaches to the diagnosis of childhood leukemias
Authors: Mierau G.W.
Published: June 2020

Electron microscopy probably is the single most precise method for classifying acute leukemias. However, because it is a relatively expensive technique it must be used very selectively. Ultrastructural studies are of genuine value and fully cost-justifiable in circumstances where: (1) routine cytochemical studies produce conflicting, confusing, or equivocal results; (2) insufficient or aberrant leukemic cell differentiation causes uncertainty; or (3) an unusual or uncommon diagnosis is under consideration. Such cases comprise only about five percent of the total. Establishing an accurate specific diagnosis in the problem case generally will require the use of one or more “special” ultrastructural techniques.

The electron microscopist is faced with a challenging set of practical problems when presented with a specimen from a patient suspected of having leukemia, especially when that patient is a child — options are to be preserved, costs minimized, the sample very small, the time element critical, and so on. This presentation will attempt to demonstrate how we deal with these problems. The methodological details for the techniques used in our laboratory have been presented elsewhere.
Correlation of Intracellular Calcium Measurements Using X-Ray Microanalysis and Fluorescence Microscopy Techniques
Authors: Hagler H.K.
Published: June 2020

It is my thought that calcium plays a major role in the development of irreversible cellular injury in the myocardium.

There are two basic forms of calcium within the cell, the active, ionized calcium and the inactive form which may be bound to cell proteins or held in storage sites within the cells.

The use of energy dispersive x-ray microanalysis in an analytical electron microscope permits the localization and measurement of the total elemental content of subcellular regions of cells. These measurements generally require that the cells be cryofixed , cryosectioned, cryotransfered and freeze dried in the electron microscope. The Hall method of continuum normalization is then used to convert the x-ray intensity measurements into dry weight concentrations.

Because the cells have to be cryofixed, it is not possible to follow the development of elemental changes within a single cell over time, thus many cells have to be frozen at different time intervals to measure time dependent changes of irreversible injury and cell death. Thus, selected time points are identified and sampled for x-ray microanalysis.
Electron Microscopic observations of the aquatic caterpillar Acentropus niveus (Olivier)
Authors: Green I.S. , Smith D. L , Boylen C. W.
Published: June 2020

Acentropus niveus is of special interest because it is one of a few species
of Lepidoptera that is truly aquatic and because it feeds on Eurasian (Myriophyllum spicatum). M. spicatmn, an introduced aquatic plant, is considered a major nuisance in many lakes in North America and Europe. It has been speculated that A. niveus may act as a biological control agent (1). Although a number of ecological studies of this caterpillar have been done, none has included electron microscopy.

A. niveus larvae were collected from recently established milfoil beds in Lake George, New York. Individuals were fixed either overnight in 2% glutaraldehyde in 0.1M Na-cacodylate buffer, pH 7.2, or for several months in 70% ethyl alcohol. Glutaraldehyde fixation was followed by rinsing three times in 0,1M Na-cacodylate buffer for 15 minutes each, dehydration in a graded ethanol series, critical-point drying, and sputter-coating with gold. The ethanol-fixed larvae were dehydrated, critical-point dried, and gold coated. All were examined with an Etec Autoscan SEM operated at 20 KV.
Ultrastructure of the digestive tube of the black mollie
Authors: Caceci Thomas , Hrubec Terry C.
Published: June 2020

The black mollie (Poecilia spp.) is a teleost fish found in Central American estuaries, and taxonomically related to the carps and goldfish. The common strains sold in pet stores are hybrids of P. mexicana and P. sphenops. The ultrastructure of its digestivetract has not previously been described.

As with other Cyprinidontiforms, the gut is anatomically simple, consisting of a relatively undifferentiated tube from esophagus to anus. The tube is lined with a simple columnar epithelium and has an external wall composed of two layers of smooth muscle. A serosal investment covers this external muscular tunic. Six intestines were examined by scanning and transmission EM. The fish were anesthetized by chilling and opened on the left side. The gut was removed, unwound, and fixed intact in a 3.0% glutar-aldehyde/ 2.0% formaldehyde/ 2.5% picric acid mixture at RT and buffered with cacodylate at pH 7.4. After primary fixation, each intestine was arbitrarily divided into a cranial, middle, and caudal segment, and four specimens were post-fixed in osmium tetroxide, dehydrated, and embedded for TEM examination. Two specimens for SEM examination were washed, treated with 1.0% tannic acid, then post-fixed in osmium tetroxide, dehydrated, and critical-point dried through CO2.
Ultrastructure of lymphatic endothelium
Authors: Singh A. , Ezeasor D.N. , Sidhu N.K.
Published: June 2020

Recently, the lymphatics in the caprine hemal nodes have been described. The present study describes some ultrastructural features of the endothelium in the lymphatics of the goat hemal nodes.

Hemal nodes located along the aorta, were collected from 15 clinically healthy, adult, West African dwarf goats. The tissues were fixed by either immersion or perfusion methods. For immersion fixation, the nodes were excised, and immersed temporarily in cold 2.5% glutaraldehyde in 0. IM cacodylate buffer, pH 7.3. After 5 min, the nodes were diced into 1-2 mm cubes and further fixed for 2 h in the same fixative. The par-aortic nodes were fixed by vascular perfusion. The two batches of tissues were post-fixed in 2% osmium tetroxide in cacodylate buffer, and further processing was done by conventional methods for transmission electron microscopy. The thin sections were examined and photographed in a
Hitachi H-7000 electron microscope.

The lymphatic wall comprised a continuous layer of fenestrated endothelial cells. The nuclei were oval and contained
heterochromatin. The cytoplasm exhibited fenestrations that were closed by a membrane, and vesicular compartments bound on luminal and/or abluminal aspects by fenestrated membranes(Fig. 1). occasionally lymphocytes(Fig. 2) or erthyrocytes, most probably intransit, occupied these compartments. In material fixed by immersion, most of the vesicles contained flocculent material similar to that contained in the lumen(Fig. 3).
Capillary endothelial morphometric profiles in fast and slow twitch muscle: alterations in response to anesthesia and electrical stimulation
Authors: Manfredi T.G. , Edington D.W.
Published: June 2020

Recent investigations focusing on transcapillary exchange have correlated physiologic and ultrastructural findings in an effort to understand mechanisms associated with endothelial function. The vascular endothelium is permeable to water and macromolecules such as lipoproteins and plasma proteins. Plasmalemmal vesicles inside the endothelium are involved with the transport of substances. These vesicles can fuse on both endothelial fronts and they can form transendothelial channels by way of vesicular fusion. These ultrastructural alterations can increase the capillary endothelial surface area by a factor of 0.5-2.0. Recent studies suggest that endothelial permeability is modulated by the fusion of vesicles into clusters which may act as transendothelial channels. The density of vesicles differ from one tissue to another. Vesicle numbers are under physiologic control and have been reported to increase in lung capillaries in response to edema and hydrostatic pressure. Physiology and cell biology must be integrated in an effort to understand the complexity of endothelial function. Therefore, the purpose of this study was to compare the morphometric profiles of capillary endothelium response to anesthesia and electrical stimulation in two muscles of the rat hind limb, the capillary enriched slow-twitch soleus, and the low oxidative fast-twitch plantaris.
The structure of patch-clamped membranes in high voltage Electron Microscopy
Authors: Ruknudin A. , Song M.J. , Auerbach A. , Sachs F.
Published: June 2020

The study of single ion channel kinetics in cell electrophysiology has been made possible by the introduction of patch-clamp techniques. Recordings can be made from a patch of membrane either attached to the cell or excised into controlled solutions. Though this technique has been widely used for more than a decade, the structure of the membrane patch and its associated cytoplasmic elements are not known except for recent work done in this laboratory . We have improved this technique to visualize membrane patches using high voltage electron microscope and also identified one class of channels in the patch by immunocytochemistry.

The procedure for preserving biological structure in a glass patch pipette is basically same as described earlier as “dry mounting technique” . Briefly, after making a patch, the pipette is removed from the bath and the tip is freeze-fixed in liquid propane. A slight negative pressure is applied to the pipette while freeze-fixing in order to stretch the shape of the patch. Once fixed, the tip is not exposed to temperatures higher than-126° C. A 0.5 cm bit of the pipette tip is broken off, stored in IN2, and then freeze dried between -126° to -80° C under high vaccum (10-6Torr).
Ultrastructural evaluation of hippocampus in rhesus monkeys after intravenous delta-9-tetrahydrocannabinol (THC): 90-day study
Authors: Andrews A.M. , Scallet A.C. , Uemura E. , Ali S.F. , Bailey J.W. , Paule M.G. , Brown R.M. , Slikker Wm.
Published: June 2020

A previous study of two rhesus monkeys exposed either to intravenous delta-9- tetrahydrocannabinol (THC) or marijuana smoke via inhalation presented ultra- structural evidence of increased synaptic width (Harper et al., 1977). Studies with rats have shown decreased synaptic density and decreased dendritic branching in the hippocampus following withdrawal after 90 days of administration of THC, the major psychoactive ingredient of marijuana (Scallet et al., 1987).

Our preliminary study in monkeys was to assess the risk, of potential neuropatho- logical alterations produced by intravenous THC for 90 days in rhesus monkeys to prepare for a longer term study of inhalation exposure.

Juvenile male rhesus monkeys, 2-3 years of age and weighing 2.5-3.5 kg were used. 1.0 or 0.1 mg/kg THC or vehicle (4-5 animals per group) were administered intravenously once daily for 90 days. The monkeys were sacrificed the day after completion of treatment. Specimens from 14 rhesus monkeys, perfused with mixed aldehydes as described by Harper et al., 1977, were taken for transmission electron microscopy (TEM) of the mid-body of the hippocampus (Demeter et al., 1985) as well as certain other brain regions. Samples were post-fixed in 4% glutaraldehyde, followed by osmium tetroxide and embedded in Epon-Araldite resin, lμ sections were stained with methylene blue and azure II and examined with the light microscope to select the CA3 region for thin sectioning. The CA3 region was identified by reference to the blades of the dentate gyrus and the presence of mossy fibers. Thin sections (l00nm) were stained with uranyl acetate and lead citrate then examined at 60kV in a Philips 201 TEM.
Spindle shaped bodies in foveolar cones of the baboon retina
Authors: Krebs W. , Krebs I.
Published: June 2020

Various inclusion bodies occur in vertebrate retinal photoreceptor cells. Most of them are membrane bound and associated with phagocytosis or they are age related residual bodies. We found an additional inclusion body in foveal cone cells of the baboon (Papio anubis) retina.

The eyes of a 15 year old baboon were fixed by immersion in cacodylate buffered glutaraldehyde (2%)/formaldehyde (2%) as described in detail elsewhere . Pieces of retina from various locations, including the fovea, were embedded in epoxy resin such that radial or tangential sections could be cut.

Spindle shaped inclusion bodies were found in the cytoplasm of only foveal cones. They were abundant in the inner segments, close to the external limiting membrane (Fig. 1). But they also occurred in the outer fibers, the perikarya, and the inner fibers (Henle’s fibers) of the cone cells. The bodies were between 0.5 and 2 μm long. Their central diameter was 0.2 to 0. 3 μm. They always were oriented parallel to the long axis of the cone cells. In longitudinal
sections (Figs. 2,3) they seemed to have a fibrous skeleton that, in cross sections, turned out to consist of plate-like (Fig.4) and tubular profiles (Fig. 5).
Scanning Electron Microscopic study on chicken pituitary cells
Authors: Tai S. , Velasquez C.
Published: June 2020

The cytology and histology of the vertebrate pituitary glands have been studied extensively under light microscopy and transmission electron microscopy. These informations form the basis for the generally accepted concept that the specific cell types synthesize, store and release their particular hormones. These hormonal products are stored in membrane-bound secretion granules in the cytoplasm.

Tanaka developed the 0 smium-DMS 0-0 smium (ODO) method which can remove the excess cytoplasmic matrices from the frozen-fractured surface of pre-fixed cells and revealed intracellular structures of many kind s of cells from liver, retina, etc. In this work, we have used the ODO method and Field Emission SEM to obtain 3-dimensional images of the fine structure of pituitary cells, as a new approach to have a better idea about the intracellular organization of the hormone secretory cells .

Chicken pituitary glands were fixed in 0.5% glutaraldehyde in 0.2M phosphate buffer(pH=7.2) for ½ h; rinsed with the same buffer; postfixed in 0.5% OsO4 for another ½h. After been rinsed throughly, specimens were passed through 25% and 50% dimethyl sulfoxide(DMSO) solution for 30 minutes each.
Localization of actin in boar spermatozoa using myosin S1-colloidal gold
Authors: Bozzola J. J. , Peterson R. N. , Hunt W. P.
Published: June 2020

Actin has been reported to be present in the sperm cells of a number of mammalian species. The location and form of actin (monomeric or polymeric) is variable from species to species with monomeric or G-actin being the predominant type. The cytoskeleton is thought to be involved in maintaining the structural/chemical specialization of the plasma and acrosomal membranes of mammalian sperm cells, since actin has been localized in various regions. Cytochalasin D, a specific inhibitor of actin polymerization, inhibits fertilization of hamster eggs, indicating that F-actin may play an important role in the fertilization process. Since the exact form and location of actin has not been resolved, we used the myosin SI subfragment coupled to colloidal gold to localize F-actin in boar caput and cauda epididymal as well as ejaculated sperm cells.

The sperm cells were washed in an F-actin stabilizing buffer (pH 7.0) consisting of 5 mM Na2HP04, 5 mM NaH2P04, 135 mM NaCl, 5 mM MgCl2, 2 mM EGTA, 0.005% PMSF, 0.005% PTAME, and 1 mg/ml soybean trypsin inhibitor.
Enhancement of the fine structure of Pneumocystis carinii using potassium ferrocyanide and tannic acid
Authors: Goheen M.P. , Blumershine P. , Hull M.T. , Bartlett M.S. , Smith J.W.
Published: June 2020

Pneumocystis carinii (PC) is an obligate parasite found in the lungs of many vertebrates including man. A recent upsurge of interest in this organism is due to its role in producing severe pneumonia in over two thirds of individuals with acquired immunodeficiency syndrome (AIDS). Many studies have been published of the ultra- structure of PC in steroid treated rats or immunocompromised patients. Several laboratories have reported growing PC in culture, but few ultrastructural studies have resulted. Additionally, PC from culture or vertebrates has been difficult to fix in a fashion which would preserve the fine structural detail. This study reports that post-fixation with tannic acid-osmium and ferrocyanide-osmium mixtures greatly improves the ultrastructural morphology of PC from culture.

Human embryonic lung fibroblasts were cultured in minimal essential medium with 10% fetal calf serum. The cultures were inoculated with viable trophozoites obtained from steroid treated rat lung and incubated in 5% O2 10% CO2, balance N2 at 35°C for seven days. At this time three aliquots of tissue culture fluid were centrifuged to obtain pellets of PC. The supernatants were decanted and replaced with Karnovsky's fixative, and the pellets allowed to fix for two hours at 20°C. Following a rinse in cacodylate buffer two of the specimens were post-fixed in 1% buffered osmium, and the third specimen was immersed in a mixture of 1.5% OSO4 and 2.5%
Freeze substitution fixation of fungal reproductive structures and spores
Authors: Mims C. W. , Richardson E. A.
Published: June 2020

The advantages of freeze substitution fixation over conventional chemical fixation for preservation of ultrastructural details in fungi have been discussed by various authors. As most ascomycetes, basidiomycetes and deuteromycetes do not fix well using conventional chemical fixation protocols, freeze substitution has attracted the attention of many individuals interested in fungal ultrastructure. Thus far most workers using this technique on fungi have concentrated on thin walled somatic hyphae. However, in our laboratory we have experimented with the use of freeze substitution on a variety of fungal reproductive structures and spores with promising results.Here we present data on freeze substituted samples of sporangia of the zygomyceteUmbellopsis vinacea, basidia ofExobasidium camelliaevar.gracilis, developing teliospores of the smutSporisorium sorghi, germinating teliospores of the rust Gymnosporangium clavipes, germinating conidia of the deuteromyceteCercosporidium personatum,and developing ascospores ofAscodesmis nigricans.

Spores ofG. clavipesandC. personatumwere deposited on moist pieces of sterile dialysis membrane where they hydrated and germinated. Asci ofA. nigricansdeveloped on pieces of dialysis membrane lying on nutrient agar plates.U. vinaceawas cultured on small pieces of agar-coated wire. In the plant pathogensE. camelliaevar.gracilisandS. sorghi, a razor blade was used to remove smal1 pieces of infected host issue. All samples were plunged directly into liquid propane and processed for study according to Hoch.l Samples on dialysis membrane were flat embedded. Serial thin sections were cut using a diamond knife, collected on slot grids, and allowed to dry down onto Formvar coated aluminum racks. Sections were post stained with uranyl acetate and lead citrate.
Preservation of embryo ultrastructure by high-pressure freezing and freeze substitution
Authors: McDonald Kent , Morphew Mary K.
Published: June 2020

Ultrarapid freezing is a well-established technique for the optimal preservation of cellular ultrastructure. The size of the sample which can be well-frozen is determined by the physics of heat transfer and under normal atmospheric pressure the upper size limit of material without cryoprotectant is about 40 um. However, if one can freeze the sample under high pressure (optimum about 2100 bars) then the maximum sample diameter can be around 0.6 mm. Moor and Riehle provided the theoretical basis and design for the high pressure freezer which has evolved into the commercial instrument (the Balzers HPM 010) now available from Balzers Corporation, Hudson, NH. We have used the Balzers HPM 010 plus the specimen holders designed by Craig et al. for the results presented here.

The materials selected for this study include the early embryonic stages of Drosophila melanogaster, Strongylocentrotus purpuratus, Caenorhabditis elegans and oocytes of Xenopus laevis, Samples were mixed with a cryoprotectant (either a yeast paste made up with 10% methanol or 1-hexadecane) and loaded into the specimen holders, making sure that no air spaces remained. The time between loading and freezing was typically less than a minute to minimize prefreezing damage. Tests with S. purpuratus showed that brief exposure to the cryoprotectants had little or no effect on embryo viability.
Localization of acid phosphatase in rice calli
Authors: Huang Y. F. , Chen Y. R.
Published: June 2020

Acid phosphatase is an important hydrolytic enzyme and widely distributed in plants. The role of acid phosphatase in plant tissues has been suggested involving in mobilization of nutrient reserves, sugar transport, differentiation of plastid, cell autolysis and senescence. In culture, the differentiating calli show the changes in isozyme patterns and activities, and anatomical localization. In this study, subcellular localization of acid phosphatase in regenerating callis is emphasized.

Sterized rice seeds(Oryza sativaTainong 67) were grown on modified MS medium containing 2 ppm 2,4-D. After 2 weeks, calli 2 to 3 mm in size were transfered on differentiation medium containing 1 ppm NAA and 2 ppm kinetin. Different stages of calli were collected and cut into thin slice with razor blade. Slices were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer(pH 7.0) for 2h and then washed, overnight . Specimens were incubated in Gomori solution containing 3mM p- nitrophenyl phosphate as substrate at 37°C for 90 min, postfixed with 1% OsO4for lh, dehydrated in ethanol series and embedded in Spurr's resin. Control experiments were done as follows: l), incubation medium without substrate; 2),incubation medium without lead nitrate; 3), incubation medium containing 0.01% NaF. Sections were doubly stained with uranyl acetate and lead citrate, and finally observed under Hitachi H-600 at 75 KV.
Electron Microscopic study of two strains of bovine viral diarrhea virus (BVDV)
Authors: Alain R. , Cardin S. , Berthiaume L. , Lecomte J.
Published: June 2020

Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle throughout the world, causing a wide range of clinical syndromes. Virion is spherical, 40-70 nm in diameter, with an envelope tightly applied to a spherical nucleocapsid 25-35 nm in diameter with icosahedral symmetry (figure 1 and 2) (Matthews, 1979) . It is presently included in the Togavlridae family, genus Pneumovirus but Collett et al. (1988) have proposed to group this genus in the Flaviviridae family. In the present study, two strains of BVDV were compared by electron microcospy ultrathin sections: one cytopathogenic, NADL strain, and the other non-cytopathogenic, NY-1 strain. These were Inoculated on Madney-Darby Bovine Kidney (MDBK) cells to a multiplicity of infection (m.o.i) between 3,0 and 5,0. Control and virus infected cells were fixed with glutaraldehyde (2,5% in cacodylate buffer) at 24 , 48, 72 and 96 h following incubation at 37°C. Cells were postfixed with osmium tetroxide and embedded in Vestopal.

The NADL-CP strain infected cells showed vacuolization of the endoplasmic reticulum, ribosomal activity in the cytoplasm and appearance of virus particles 48h after infection (figure 3). At 72h, an expensive vacuolization was apparent (figure 4). Maximum viral production was seen 96 h after infection (figure 5). From 24 to 72 h post infection, the NY-l-NCP strain of BVD infected cells showed normal vacuolization like in control cells. They produced weak vacuolization with few particles, not apparent before 96h after infection (figure 6). Viral particles of 45-50 nm in endoplasmic reticulum vacuoles were in general homogenous in size while those in smooth membranes, probably of cellular origin, heterogenous in size.
Immunogold labeling of phycobilisomes in a cyanobacterium
Authors: Zianni M. R.
Published: June 2020

Phycobilisomes are pigment-protein complexes that act as the major light- harvesting antenna, in addition to chlorophyll, for photosynthesis in red algae and Cyanobacteria.In vivo, phycobilisomes transfer their energy mainly to the photosystem II reaction center to which they are assumed to be attached. This assumption will be tested by the use of gold labeling of antibodies raised against photosystem II reaction center components to localize the reaction centers and to compare the location of the antibodies with that of the phycobili- somes. In Cyanobacteria phycobi1isomes are difficult to fix particularly under conditions required for immunocytochemistry. Here, two methods for efficient fixation of phycobi1isomes are described, together with immunocytochemical results to confirm the identity of the phycobilisomes.

For phycobilisome detection,Synechocvstissp. PCC 6803 cells were either freeze substituted or fixed in a high molarity buffer followed by cryomicrotomy. For freeze substitution the cells were collected, subjected to ultra-rapid freezing with a slammer device and a copper block at liquid helium temperature, placed in 5% acrolein in ethanol at -85°C for 4 days, and embedded in LR White. For cryo- sectioning the cells were fixed with 1% glutaraldehyde in 0.75M sodium potassium phosphate buffer, pH 7.4, for 2 h, enrobed in 10% gelatin, infused with 1.6M sucrose in 0.1M sodium phosphate buffer,pH 7.2, for 3 h, and frozen in liquid freon. Cryosections were prepared with an RMC CR2000 cryoultramicrotomy unit on an MT-6000 ultramicrotome. In both procedures 0.05% Carnation nonfat dried milk in 0.1M sodium phosphate buffer was used to block nonspecific binding of the anti serum. Sections were treated with rod-phycocyanin (phycobilin protein) anti - serum followed by protein A conjugated to lOnm gold particles. Subsequently the plastic sections were stained with uranyl acetate and lead citrate. The cryosections were subsequently treated with 1% OSO4, 1% tannic acid, uranyl acetate, and alkaline bismuth, dehydrated in ethanol, and embedded in LR White. Sections were observed in a Philips EM201.
Ultrastructural study of langerhans cells in a murine UV-induced tumor
Authors: Bucana C.D. , Song M.J. , Dunner K. , Sanchez R. , Kripke M.L.
Published: June 2020

Langerhans cells (LC) are dendritic cells of the macrophage-monocyte lineage that are found in the skin, other epithelial-1ined tissues and lymphoid tissues. LC are al so present in a wide variety of pathologic tissues. LC are characterized by the presence of a unique rod- or racquet-shaped pentalaminar organelle called the Langerhans cell granule. Ultrastructural and immunohistochemical analysis of several murine UV-induced tumors revealed the presence of infiltrating cells that are morphologically similar to LC in some tumors.

Murine UV-induced tumors were processed for routine histology by paraffin embedding and conventional TEM. Samples were screened for morphology of tumor cells. Tumors in which LC were identified ultrastructurally were also identified by an immunoperoxidase technique for the presence of S-100 protein.

Infiltrating cells that are positive for S-100 protein are found in some UV-induced tumors. These cells are not uniformly distributed in these tumors and their numbers varied significantly from region to region. They are found among tumor cells and their dendrites are observed between tumor cells. Ultrastructural analysis of these cells showed that they exhibit a more electron lucent cytoplasm than the surrounding
cells in the tissue and they contain neither desmosomes nor tonofilaments.
Ultrastructural morphology of cells transformed with temperature-sensitive mutant of Mo-MuSV
Authors: Al-Bagdadi F. , Singh B. , Arlinghaus R.B.
Published: June 2020

It is known that one of the abnormal behavior of cancer cells is changes in morphology. There is very little information in the literature on the morphological aspects of cell lines at the electron microscopic level transformed by viral mos gene (Culp, 1971; Hynes et al., 1978; Brown et al., 1981; Arlinghaus, 1985; Terasaki et al., 1986). NRK-6m2 is a cell line infected with a temperature sensitive mutant (tsll0) of Moloney murine sarcoma virus (Arlinghaus, 1985). The attempt in this study is to examine the ultrastructure of NRK-6m2 cells and relate the morphological changes to gag-mos induced cellular transformation.

NRK-6m2 cell cultures were maintained in McCoy's 5a medium supplemented with 15% fetal calf serum. Subconfuluent cells were washed twice in cold phosphate buffered saline and fixed in situ in 1% glutaraldehyde, scraped and centrifuged at 1500 g for 5 min at 4°C, post fixed in osmium tetroxided and processed for electron microscopic study. Silver sections were stained with uranyl acetate and lead citrate and examined on a Joel 1200 EX electron microscope.
Localization of heavy metals in the hepatopancreas of the terrestrial isopod, Oniscus asellus
Authors: Vernon G. , Greco R. , Witkus R.
Published: June 2020

Heavy metals have been identified in tissues of a variety of terrestrial isopods exposed to metal polluted sites in Europe. Heavy metals have little effect on some isopods indicating that perhaps the hepatopancreas functions as a detoxifying organ for the isopod by storing the metal. In some lead and copper contaminated rivers tolerant races of the isopod Asellus meridianus were found.

Specimens of Oniscus asellus were collected from a cadmium contaminated site adjacent to a marsh in Cold Spring, N. Y. and from an uncontaminated site in New Rochelle, N. Y. Isopods and leaf letter were transported to the lab where some isopods were prepared for scanning electron microscopy (SEM), and others were prepared for atomic absorption spectroscopy. For SEM the hepatopancreatic lobes were fixed in 3,5% glutaraldehyde in 0.1M sodium cacodylate buffer with 0.05% calcium chloride. Specimens were dehydrated in acetone and embedded in Araldite-DDSA. Five micron thick cross sections were deplasticized and gold coated - Sections were viewed and analyzed using an Hitachi S-510 SEM at 25kV with an Ortec 5000 EDX system.
Morphometric analysis of peroxisomes in hepatocytes of alcohol-fed rats
Authors: Dominguez D.C. , Ellzey J.T.
Published: June 2020

Peroxisomes which participate in 1ipid metabolism have been shown to be altered in several metabolic disorders and toxic conditions. In alcoholic liver disease, the single lesion most frequently found is lipid accumu1ation in hepatocytes. However, the mechanisms for this 1ipid accumu1ation are not clear. The occurrence of modifications of liver peroxisomes due to excess alcohol consumption has not been subjected to a controlled study. We utilized a combination of cytochemica1 and morphometrictechniques to study the size and number of liver peroxisomes in rats fed an alcohol-supplemented diet compared to those of matched-paired control animals.

Male Sprague-Daw1ey rats (400-500 g) received a liquid diet. The experimental group (N = 5/group) was fed a diet containing 30% ethanol-derived calories (EDC) and the control group was fed an isocaloric diet to 30% EDC. A pair feeding procedure was employed to control for caloric intake. Small pieces of liver randomly selected, were fixed in 2.3% -glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, incubated in a DAB medium and postfixed with. 2% aqueous osmium tetroxide. EM photographs were taken from sections of 3 tissue blocks from each sample (7,200X) with a Zeiss EM10-A (60 kV). With the use of a point counting method and a digital planimeter the volume density (Vv) and numerical density (Nv) were determined.
A high-resolution membrane probe
Authors: Hainfeld James F. , Lipka James J. , Elsie Quaite F.
Published: June 2020

Labeling the lipids of membranes for E.M. is frequently done by using radioactive phospholipids. Membrane dynamics can then be followed by autoradiography. Unfortunately, it takes approximately 6 months to expose the emulsion due to the low specific activity of the labels. Also, the resolution is limited to about 0.2 μm by this technique.

An alternative is to develop a phospholipid-like molecule that is electron dense and could be visualized directly. Several such molecules have been synthesized and one is shown in Fig. 1. They have a phosphotungstate “head” group that has a 4 (-) charge and is hydrophilic and an aliphatic “tail” of varying length that is hydrophobic. The tungstate cluster contains 11 tungsten atoms in the “Keggin” structure with a 1.0 nm diameter and has previously been studied in the electron microscope. It was shown to be almost completely resistant to beam induced mass loss.