bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron spectroscopy imaging cytochemistry : Use of boronated-protein A
Authors: Bendayan M. , Barth R.F. , Gingras D. , Londoño I. , Robinson P.T. , Alam F. , Adams D.M. , Mattiazzi L.
Published: June 2020

The ultrastructural organization of the cell and the functional properties of its components have reached a high level of knowledge due to the development of electron microscopy and related cytochemical techniques. Colloidal gold particles having a size down to 3 to 5 nm provide the best resolution in cytochemistry. In order to improve this, one must look at tracers detectable at the molecular and atomic levels.

Recent advances in analytical electron microscopy, including electron energy loss spectroscopy (EELS) and electron spectroscopy imaging (ESI) provide much higher levels of resolution. By choosing the appropriate edge of energy loss, ESI allows for the simultaneous detection and ultrastructural distribution of specific elements in ultrathin tissue sections, with a spatial resolution of 3 to 5 Å. We have recently tested this approach for the detection of an element, boron, introduced in the tissue as a cytochemical marker. Because of the advantages of protein A as a second reagent in indirect immunocytochemistry, we investigated the potential of boronated protein A (B-pA) for the detection of antigenic sites in tissue sections.
Immunogold labelling of neutral endopeptidase 24.11 (enkephalinase), on human neutrophils and neutrophil cytoplasts
Authors: Kriho V. , Wagner B. , Erdos E.G. , Becker R.P.
Published: June 2020

We have documented the presence of neutral endopeptidase 24.11 (NEP), on the surface of human neutrophils (PMN) and PMN cytoplasts. Cytoplasts are whole cell preparations which contain cytomatrix, but lack internal membranes and organelles ,such as nuclei and lysosomal granules. These structures have been extracted mechanically, leaving the plasma membrane “outside-out” topology intact. Cytoplasts are very useful in correlative studies of cell surface structure and function. Biochemically, the membrane component of cytoplasts is predominantly plasma membrane; structurally, chemical activity may be localized to domains of the intact cell surface. NEP is a membrane-bound metalloendopeptidase present in human PMN' s. We have marked NEP on the plasma membranes of PMNs and PMN cytoplasts via pre-embedding iramunocytochemistry. We used scanning electron microscopy (SEM) with backscattered electron imaging (BEI) to visualize Au labelled anti-NEP on the surface of a large number of cells. Transmission electron microscopy (TEM) was used to confirm the presence of the enzyme on PMN's and PMN cytoplasts.

Suspensions of PMN or PMN cytoplasts (2 x 106 cells/ml) were fixed for 8 min at room temp. in 0.25% glutaraldehyde in phosphate buffered saline (PBS) pH 7.2 rinsed in PBS, treated with 0.1% glycine in PBS for 10 rain and then incubated for 15 min in 5% normal goat serum (NGS) in 0.1% bovine serum albumin dissolved in PBS (BSA/PBS). Following this step, cells were incubated for 20 min in anti- NEP antibody, rinsed in BSA/PBS, incubated in goat anti-rabbit IgG coupled to 15nm colloidal Au particles (GARG15) for 1 h and again rinsed in PBS. Postfixation for 30 min in 2.5% glutaraldehyde and PBS rinsing followed. For SEM a drop of cell suspension was put on a polylysine- treated Formvar-carbon-coated Au grid and cells were allowed to settle and attach for 30 min. The grid was rinsed in water, dehydrated and critical point dried. Cells were coated with carbon before viewing by SEM. For TEM, following immunolabelling, cells were post-fixed in OsO4, rinsed, dehydrated and embedded in Epon for sectioning.
Microtubule number increases locally within regions of axonal compression
Authors: Price R.L. , Lasek R.J. , Katz M.J.
Published: June 2020

Along the length of myelinated axons, stereotyped variations in the interaction with the enveloping Schwann cell produce different amounts of compressive force on the axon and its internal elements. Comparisons of histologically distinct axonal regions indicate that compression of the axon by the Schwann cell increases in the following order: internodal regions of compact myelin < Schmidt-Lanterman (S-L) clefts < nodes of Ranvier. Quantitative morphometry has shown that regions of greater Schwann cell compression are associated with regions of smaller axon diameter and greater neurofilament (NF) packing density. By locally decreasing axon diameter and increasing NF density, Schwann cell compression (especially at the node of Ranvier) may slow the transport of those axonal structures - such as microtubules (MTs) - that move by a polymer sliding mechanism. Under this hypothesis, a local slowing of MT transport should produce a local increase in MT number, and here we have studied whether such a local increase in MT number can be quantitatively documented.

A twenty-eight day old chicken was perfused by cardiac puncture with 4 1 of fixative (4% paraformaldehyde, 2% glutaraldehyde, 0.005% CaCl2, in 0.1M cacodylate buffer, pH 7.4). The somatic motor branch of the oculomotor nerve was removed (noting the proximo-distal orientation) and fixed overnight at 4°C, followed by a buffer rinse in 0.1M cacodylate buffer at room temperature and postfixation in OSO4. The nerve was then dehydrated in a graded ethanol series and embedded in PolyBed/812. Starting at the proximal end of the nerve segment, l00nm sections were cut at consecutive 2-3 um intervals over a 225 um length of nerve, stained with an LKB Ultrostainer, and photographed on a JEOL 100CX microscope. We selected two axons with normal morphology, in which the cytoskeletal polymer profiles were consistently cross-sectioned and in which nodes were located approximately 120 um distal to the first section taken. All sections from these 2 axons were photographed, and MTs were counted on 58,000X micrographs.
A serial section analysis of rabbit foliate taste buds using the high voltage electron microscope
Authors: Royer S.M. , Kinnamon J. C.
Published: June 2020

We have examined the foliate taste buds of the rabbit using a combination of serial section analysis and computer-assisted three-dimensional (3-D) reconstructions. Previous investigators of rabbit foliate taste buds have classified taste cells into types I, II, and III, basal cells, and peripheral cells. Based on the presence of synaptic contacts with nerve processes, only the type III cells have been considered to be taste receptor cells. Our goals were the elucidation of morphological details of the various cell types and the characterization and quantification of synaptic contacts between taste receptor cells and intragemmal nerve fibers.

Adult white rabbits were perfused with a fixative containing 2.5% glutaraldehyde and 1% paraformaldehyde in 0.05 M cacodylate buffer at pH 7.3. The foliate papillae were excised fixed overnight in fresh fixative at 4°C, rinsed in buffer, postfixed with buffered 1% OsO4, washed, and stained en bloc in 5% uranyl acetate in 70% ethanol for 48 hours. The specimens were then dehydrated and embedded according to standard procedures. Serial 0.25 μm sections of the embedded tissue were cut with a Diatome Semi diamond knife using a Reichert Ultracut E ultramicrotome. Sections were mounted on Formvar-coated slot grids and poststained in triple lead stain and alcoholic uranyl acetate. After carbon coating, the sections were examined with a JEOL JEM-1000 high voltage electron microscope (HVEM) operated at 1 meV. Contours of interest were traced from micrographs onto a Summagraphics digitizing tablet and entered into an IBM-compatible PC, using programs developed by the authors.
Immunolocalization of the intracellular sites of accumulation of mutant influenza hemagglutinins by Electron Microscopy
Authors: McCammon K. , Segal M. , Sambrook J. , Gething M. J. , McDowall A.
Published: June 2020

The hemagglutinin (HA) of influenza virus has been used as a model system to study the biosynthesis and intracellular transport of integral membrane proteins in mammalian cells. To investigate the role of protein structure in facilitating transport along the secretory pathway, we have examined the expression in monkey CV-1 cells of a large number of mutant HA molecules. The majority of the HA mutants do not progress along the secretory pathway and accumulate in the endoplasmic reticulum (ER), and we have shown that assembly of newly-synthesized HA monomers into correctly folded trimeric structures is required for transport of the protein to the Golgi apparatus. By contrast, only one HA mutant has beegn characterized whose transport is blocked at a post-Golgi stage of the pathway and thus little is known about the factors involved in the sorting of the HA molecule from the Golgi apparatus to the plasma membrane (PM). In this study we are using electron microscopy to precisely define the intracellular site of accumulation of two mutant HAs whose transport is blocked at different stages of the secretory pathway. In mutant HAJS67, a cysteine residue (cys67) involved in a key disulfide bond has been substituted by a serine residue. In mutant HA164, the 10 amino acid cytoplasmic tail of the wild-type HA has been replaced by a non-homologous sequence of 16 amino acids. Biochemical and immunof1uoresence analyses have indicated that HAJS67 molecules remain in the ER compartment while HA164 is largely confined to a post-Golgi compartment, possibly the trans Golgi network (TGN).
Comparative fine structure of whole cells by High Voltage Electron Microscopy
Authors: Porter Keith R. , Anderson Karen , Peachey Lee D.
Published: June 2020

When fish scales are isolated and treated with collagenase and hyaluronidase, populations of five different kinds of cells are obtained. These can be maintained in vitro for several days and even encouraged to proliferate by enriching the culture medium. As a model for studying comparative fine structure, the system is ideal. For example the isolated cells can be cultured directly on formvar- and carbon-coated gold grids. In preparation for microscopy they are all exposed to precisely the same conditions and receive identical treatment. It is important also that they are not embedded and sectioned but are on the grids as whole cells with organelles etc. in functional relation to one another and to the cytoplasmic matrix. Such differences as are shown by the cells can be regarded as native to the various types.

These differences can be dramatic and extend from compact and thick iridophores to thinly spread epidermal units. It is essential for their successful study to use a high energy beam that can penetrate even the thickest cells or parts of cells. In this instance we have used a Zeiss 10 operated at 100 kV; a JEOL 400 kV (IVEM) and a JEOL 1000 kV (HVEM). As expected, penetration and resolution improve as the voltage is increased (Figs. 1-4) and so also does the information content of the micrographs. The cytoplasmic matrix heretofor poorly resolved and difficult to interpret, is seen as a 3D lattice of thin (50A) strands (microtrabeculae) that contains other components of the cytoskeleton as well as polysomes. The lattice varies strikingly in different kinds of cells found in these preparations. For example, in fibroblasts the cytomatrix is dense compared with the matrix in cytoplasm of similar depth in epidermal cells. In fibroblasts the microtrabeculae are shorter and thicker and the intertrabecular spaces are smaller. In a comparative examination of the cytoskeletons, it is possible to follow microtubules in a single cell from begining to end, a feat that is impossible in thin sections. All of these features of the matrix are under study using morphometric techniques to quantitate the differences. The structural dimensions of the lattice suggest it may be the intracellular equivalent of “plectin” isolated and characterized by Wiche and colleagues.
Hexamethyldisilazane: A modified procedure
Authors: Laliberté Verdon , Hayes L.L. , Stanulis-Praeger B.M.
Published: June 2020

Scanning electron microscopy is a powerful tool in the study of cell contact in vitro, and has shown, specifically, that fibroblasts under conditions of growth restriction increase cell contact by filopodia. Accurate quantitation of surface features like filopodia, however, depends particularly upon artifact-free drying procedures. Critical point drying (CPD) of fibroblast monolayers all too often causes cell flattening, shrinkage, loss of surface detail and cracking
(Fig. 1). Newer methods utilizing hexamethyldisilazane (HMDS) and uranyl acetate (UA) preserve fibroblast contour and surface architecture better than CPD, but solvent choice in the preparation of UA is of critical importance. UA in 70% ethanol results in the formation of precipitate (Fig. 2) whether the specimens are in the cold (4°C) or at room temperature, overnight or for periods less than 1 hour regardless of filtering. It occurs when incubation is carried out in
the dark and when it is followed by copious rinses in 70% ethanol.
Cellulase-gold labelling of cellulose in forages
Authors: Woodward Janet H.
Published: June 2020

Current fiber analyses provide information on the chemical and structural composition of the fiber as a whole. However, these techniques do not offer insight into the compositional and structural differences between specific fiber tissues which may affect digestibility. The purpose of this study was to: (1) ultrastructurally localize one cell wall constituent, cellulose, in four forages; and (2) apply this technique as a tool for the evaluation of biodegradation in forages.

Insoluble fibers were prepared using a modified AOAC Total Dietary Fiber Technique. Two millimeter sections cut from leaflets of alfalfa (Medicago sativa L.) and 1-76 1espedeza [Sericea cuneata (Dumont)] and leaves of coastal bermudagrass (Cynodon dactylon L. Pers.) and orchardgrass (Dactylis glomerata L.) were incubated stepwise with: (1) α-amylase, pH 6.0 for ½ h at 100°C; (2) protease, pH 7.5 for ½ h at 60°C; (3) amyloglucosidase, pH 4.5 for ½ h at 60°C, and (4) 1% (w/v) cellulase in 0.05 M sodium acetate buffer, pH 5.0 for 24 h at 37°C.
Single molecule optical diffraction: A tool for understanding the structure of triple stranded left-hand helical cellulose
Authors: Ruben George C. , Krakow William
Published: June 2020

Tobacco primary cell wall and normal bacterial Acetobacter xylinum cellulose formation produced a 36.8±3Å triple-stranded left-hand helical microfibril in freeze-dried Pt-C replicas and in negatively stained preparations for TEM. As three submicrofibril strands exit the wall of Axylinum , they twist together to form a left-hand helical microfibril. This process is driven by the left-hand helical structure of the submicrofibril and by cellulose synthesis. That is, as the submicrofibril is elongating at the wall, it is also being left-hand twisted and twisted together with two other submicrofibrils. The submicrofibril appears to have the dimensions of a nine (l-4)-ß-D-glucan parallel chain crystalline unit whose long, 23Å, and short, 19Å, diagonals form major and minor left-handed axial surface ridges every 36Å.The computer generated optical diffraction of this model and its corresponding image have been compared. The submicrofibril model was used to construct a microfibril model. This model and corresponding microfibril images have also been optically diffracted and compared

In this paper we compare two less complex microfibril models. The first model (Fig. 1a) is constructed with cylindrical submicrofibrils. The second model (Fig. 2a) is also constructed with three submicrofibrils but with a single 23 Å diagonal, projecting from a rounded cross section and left-hand helically twisted, with a 36Å repeat, similar to the original model (45°±10° crossover angle). The submicrofibrils cross the microfibril axis at roughly a 45°±10° angle, the same crossover angle observed in microflbril TEM images. These models were constructed so that the maximum diameter of the submicrofibrils was 23Å and the overall microfibril diameters were similar to Pt-C coated image diameters of ∼50Å and not the actual diameter of 36.5Å. The methods for computing optical diffraction patterns have been published before.
Comparative ultrastructural study of lymphocystis disease virus and frog virus 3 by means of enzymatic digestions
Authors: Heppell J. , Berthiaume L.
Published: June 2020

Fish lymphocystis disease is a common viral infection reported in over a hundred teleost species. The causative agent, Lymphocystis disease virus (LDV), has been classified in the Iridoviridae family. Though it has an icosahedral shape, its capsid is complex and still not very well characterized. However, several studies have been reported on the ultrastructure of Frog virus 3 (FV3), another iridovirus, showing outside protein units with an underlying lipidic layer, surrounding the viral core. In order to verify whether or not the structure of LDV particles correspond to that of FV3 , a comparative electron microscopic study was undertaken.

LDV strain Leetown (ATCC VR-342) was propagated on BF-2 cells, and FV3 strain Granoff (ATCC VR-567) on FHM cells. Viral particles, concentrated and suspended in 0.1M Tris buffer (pH appropriate for the enzyme used), were adsorbed on formvar-coated grids prior to digestion with proteases (pronase E or proteinase K, 20 U/ml) and/or phospholipases (A2 or C, 15 U/ml) at 37°C for various incubation periods. Particles were negatively stained with 2% (w/v) phosphotungstic acid pH 6.0 before examination with a Philips 300 transmission electron microscope (TEM) at 80 kV.
Vaccinia-lassa recombinant produces lassa-like inclusions
Authors: Goldsmith C. S. , Morrison H. G. , Auperin D. D. , Whitfield S. G. , Palmer E. L.
Published: June 2020

Lassa virus, a member of the Arenaviridae, is the etiologic agent of Lassa fever. The virus is endemic in widespread areas of West Africa, particularly Nigeria, Liberia, and Sierra Leone. Rough estimates indicate that there are upwards of 300,000 human infections annually, resulting in approximately 5000 fatalities. A safe and effective vaccine for Lassa fever is not yet available. Recent studies in our laboratories have focused on the development of recombinant vaccinia viruses that express the nucleoprotein and envelope glycoproteins of Lassa virus as potential vaccine candidates. Tissue culture cells infected with various recombinant viruses were examined by thin-section TEH for any morphological alterations.

Tissue culture cells were infected with wild-type Lassa virus or with recombinant vaccinia viruses expressing either the Lassa virus nucleoprotein (NP). the envelope glycoproteins (GPI,GP2), or NP, GPI, and GP2 in combination. Cells were fixed in 2.5% glutaraldehyde in 0.2M cacodylate buffer, postfixed in buffered 1% osmium tetroxide, and en bloc stained with 4% uranyl acetate. The samples were dehydrated and embedded in Polysciences Epon-substitute and Araldite. Sections were stained with uranyl acetate and lead citrate.
Observation of microtubule-like structures and crystalline array in Azotobacter vinelandii
Authors: Dobbins M. S. , Socolofsky M. D.
Published: June 2020

Many studies have been conducted on microtubules in eukaryotes, including protozoans, but few have dealt with similar structures in bacteria. Microtubule-1ike structures (MT) have been observed in Proteus mirabilis, Azotobacter vinelandii, and some bacterial L-forms . The function of these MT in prokaryotes is as yet undetermined. Microtubules in eukaryotes are involved in cytoskeleton support, cel1 motility, molecule transport, and the separation of chromosomes during mitosis . It has been suggested that A. vinelandii possesses forty chromosomes per cell , so this investigation speculates on the possibility these MT are involved in some form of chromosomal separation. While observing these structures a crystal lattice was noticed which has not been observed in the aforementioned prokaryotes.

Cells were fixed for 10-20 min in 2% glutaraldehyde/0.1M sodium cacodylate (pH 7.2), washed with buffer, post-fixed with 1% OsO4, and stained with uranyl acetate. Samples were thin-sectioned, collected on carbon-coated parlodion- reinforced grids, and post-stained with lead citrate. Other preparations involved whole or lysed cells negatively stained with 2% phosphotungstic acid. All specimens exhibiting MT or the crystalline array were photographed in stereo pair.
Immunocytochemical localization of p65 with one-nanometer gold particles following silver development
Authors: Burry Richard W. , Hayes Diane M.
Published: June 2020

Electron microscopic (EM) immunocytochemistry localization of the neuron specific protein p65 could show which organelles contain this antigen. Antibodies (Ab) labeled with horseradish peroxidase (HRP) followed by chromogen development show a broad diffuse label distribution within cells and restricting identification of organelles. Particulate label (e.g. 10 nm colloidal gold) is highly desirable but not practical because penetration into cells requires destroying the plasma membrane. We report pre-embedding immunocytochemistry with a particulate marker, 1 nm gold, that will pass through membranes treated with saponin, a mild detergent.

Cell cultures of the rat cerebellum were fixed in buffered 4% paraformaldehyde and 0.1% glutaraldehyde (Glut.). The buffer for all incubations and rinses was phosphate buffered saline with: 1% calf serum, 0.2% saponin, 0.1% gelatin, 50 mM glycine 1 mg/ml bovine serum albumin, and (not in the HRP labeled cultures) 0.02% sodium azide. The monoclonal #48 to p65 was used with three label systems: HRP, 1 nm avidin gold with IntenSE M development, and 1 nm avidin gold with Danscher development.
Cellular and structural diversity of Campylobacter pylori
Authors: Crooker A. R. , Myers M. C. , Kraft W. G.
Published: June 2020

Campylobacter pylori is a recently recognized microaerophilic, gram-negative bacterium found in the upper gastrointestinal tract of humans. The role of this organism in the pathogenesis of gastritis and ulceration is currently under intense investigation. The rapidly growing literature on C. pylori describes the bacterium as spiral shaped; however, oval and coccal forms are occasionally noted. We report here on the cellular and structural diversity of this organism, including a new, pleomorphic form.

Two Peruvian clinical isolates of C. pylori and the type strain NCTC 11638 were grown for up to 120 h in liquid media. Thin sections and negatively-stained whole mounts were prepared for electron microscopy. Chemical fixation was performed in suspension with 2% cacodylate-buffered glutaraldehyde followed by 2% OsO4 In the same buffer, then uranyl acetate en bloc. Specimens were dehydrated in acetone and embedded in Spurr's resin. Ultrathin silver sections were stained with uranyl acetate and Reynolds’ lead citrate. Briefly prefixed bacteria were negatively-stained with 1% PTA on SiO/Forravar-coated 300 mesh grids.
MDX mouse myopathy I: Presence or absence of sarcolemmal lesions in tibialis anterior muscles from mice of different ages
Authors: Geissinger H.D. , Rhodes L.D.
Published: June 2020

Since the ‘mdx’ mouse appears to have the same basic defect as sufferers of human Duchenne Muscular Dystrophy (DMD), much recent interest in this possible animal model for the human disease has been generated. Perforations in the sarcolemma have been reported recently in the necrotic tibialis anterior (TA) of 35-days-old and the extensor digitorum longus muscles of 39-days-old ‘mdx’ mice. It is the purpose of this communication to find out if these lesions occur not only in necrotic, but also in unaffected, or in centronucleated fibers of the TA of mice which are younger than 35, or older than 39 days.

METHODS: TA from 22-, 25-, 41-, 61- and 99-days-old C57BL/10ScSn/MDX and C57BL/lOScSn control mice were pinned on corkboard in a relaxed state, prefixed for 30 minutes in 2.5% glutaraldehyde followed by routine processing for TEM. Appropriate micrographs were evaluated for a more detailed morphological analysis
of the sarcolemma (SL) and the basal lamina (BL).

RESULTS: It should be stated beforehand that in all muscles examined the BL appeared to be intact. In the muscles of a 25-days-old control mouse the SL appeared quite intact (FIG. 1). In contrast to this small perforations or large tears in the SL could be seen in otherwise unaffected muscles of 22- (FIG. 2), 25-
and 41-days-old ‘mdx’ mice, as well as in necrotic and regenerating fibers of mice
from these ages.
High-resolution SE-I SEM of HIV-infected cells
Authors: Apkarian R.P. , Pasquinelli G. , Cenacchi G. , Preda P. , Re M.C. , Becker R.P.
Published: June 2020

The characteristic TEM appearance of HIV is well known. Our studies describe SE-I high resolution (HR) SEM imaging of H9 cells derived from human T-cell leukemia which expresses CD4 and cultured CD4- human endothelial cells (EC) each infected separately with HIV. Both cell systems displayed 100 nm single or clumped roughly spherical viruses with the same characteristic morphology displayed by HIV in TEM studies. By SE-I HRSEM, HIV appears as rasberry shaped structures with surface knobs. These knob like projections may represent gp120 of the virus envelope. SE-I imaging provided information of viral envelope/host cell surface HIV association. Correlated by TEM observations, particulate dense bodies were associated with the RER of ECs. SE-I mode operation which eliminates the SE-III signal component, collects only the specimen specific SE-I and II signal components using an above the lens secondary electron detector. Specimens coated with an ultrathin fine grain chromium film provide a continuous coat with a mass average thickness and have a very low mobility on the sample surface thereby avoiding decorations.
Sperm migration into post partum cervical mucus: scanning microscopy study
Authors: Vigil P. , Morales P. , Uribe L. , Perez A. , Bustos-Obregón E
Published: June 2020

Breast-feeding is associated to the infertile period occurring post-partum. During this period there is arise in prolactin and FSH, with lowered levels of LH, progester one and estradiol, indicating ovarian in activity. There assumption of ovarian ciclic activity, with rising estradiol levels should be associated with a change in the pattern of cervical mucus secretion from an infertile type of mucus to a fertile type. The objective of the present study was to evaluate the score of the cervical mucus, according to WHO parameters, present in amenorrhoeic women under exclusive breast feeding at 30, 60, 90, 120, 150 and 180 days post partum. In addition, sperm migration into the mucus was evaluated using the slide contact test method according to WHO (1987). Scanning electron microscope studies were done in order to stablish the ultrastructural characteristic as of post partum mucus and it with control peri-ovulatory cervical mucus. Cervical mucussamples from 54 women were collected at the time intervals described before. Six samples from normal lycycling women were used as control. Twenty-one (39%) of the 54 post partum cervical mucus samples gave a positive result in the slide contact test.
SEM and TEM observations of sperm development in the testis of the freshwater fish Oreochromis mossambicus
Authors: Cornish Daryl A. , Smit George L.
Published: June 2020

Oreochromis mossambicus is currently receiving much attention as a candidater species for aquaculture programs within Southern Africa. This has stimulated interest in its breeding cycle as well as the morphological characteristics of the gonads. Limited information is available on SEM and TEM observations of the male gonads. It is known that the testis of O. mossambicus is a paired, intra-abdominal structure of the lobular type, although further details of its characteristics are not known. Current investigations have shown that spermatids reach full maturity some two months after the female becomes gravid. Throughout the year, the testes contain spermatids at various stages of development although spermiogenesis appears to be maximal during November when spawning occurs. This paper describes the morphological and ultrastructural characteristics of the testes and spermatids.

Specimens of this fish were collected at Syferkuil Dam, 8 km north- west of the University of the North over a twelve month period, sacrificed and the testes excised.
The ultrastructure of the developing sperm tail middle piece in the skink Eumeces latices (Lacertilia: Scincidae)
Authors: Okia Nathan O.
Published: June 2020

The typical reptilian sperm has only a head and tail without the neck. However, the proximal centriole is capped by a neck cylinder, which also encloses the connecting piece and the base of the axoneme. Much variation in the ultrastructure of reptilian sperm tails is found in the connecting piece, neck cylinder and middle piece. yhe current study was aimed at elucidating the ultrastructure of the area between the nucleus and the annulus in the skink Eumeces laticeps, a squamate lizard.

Broad-headed skinks Eumeces laticeps were sacrificed between January and May, during the period of spermatogenesis. Testes were fixed in cold Karnofsky’s fixative, pH 7.4, postfixed in 1% OsO4 at room temperature, dehydrated in alcohols and acetone, embedded in Spurr’s epoxy mixture and examined with a Philips 300 EM.

In spermatids undergoing chromatin condensation, dense material appears in the post-nuclear cytoplasm (Fig. 1). Some of this, developing close to the spermatid membrane, might be associated with the developing annulus (Fig. 1 AN), but most of the dense material which concentrates arround the proximal centriole (Figs 1,2,3,5), gives rise to the conical capitulum, and the connecting piece.
Structure of DNA and Nucleosome Observed by Ultrahigh-Resolution SEM
Authors: Inaga Sumire , Osatake Hitoshi , lino Akihiro , Tanaka Keiichi
Published: June 2020

So far, the ultrastructure of DNA strand and nucleosome had been observed mainly by transmission electron microscopy with some techniques (thin-sectioning, spreading method, replica method and so on). Among them, the freeze-etching replica method gave high magnified images of DNA double helix (Ruben et al., 1989). Further, scanning tunneling microscopy also elucidated the images of major and minor grooves in a helical DNA duplex. Though scanning electron microscopy (SEM) was also applied for observing chromatin structures, it had been difficult to observe clearly such small materials. Because, the resolution of SEM was too poor to investigate such fine structures. The obstruction of resolution, however, was overcome by the development of an ultrahigh resolution SEM (UHS-T1, Tanaka et al., 1985). Using the SEM, we could successfully observed naked DNA strands and nucleosomes of chicken erythrocyte nuclei without any metal-coating.

Preparations were made by the microspreading procedure basically according to the method of Seki et al.
Structural organization of kinetoplast DNA and its compactization in vitro
Authors: Martinkina Larissa , Novikova Elena , Kolesnikov Alexander , Vengerov Yuri
Published: June 2020

Studies on compactizatlon and decompactizatlon of the genome are of great importance for elucidation of structural mechanisms taking part in the regulation of gene activity. Kine to plast DNA (kpDNA) is a convenient model for studies of compactizatlon processes. Leishmania gymnodactyli kpDNA represents a gigantic association of catenated circular molecules of two types: 0.3μm minicircles and 10μm maxicircles. Fig. 1 demonstrates that kpDNA prepared by protein film method is a network of linked circular DNA molecules shaped as a shallow cupor a basket. Part of minicircles exists in relaxed form, part-in a supertwisted. Electron microscopic studies of Leishmania gymnodactyli cells by Miller's technique showed that kpDNA looks like irregularly out line dagglomerations of crossed or sometimes branched rod-like structures 100 to 200nm long and 20nm thick (Fig.2). They seemtore present compacted minicircles. On the electron microscopic preparation these accumulations are lying separately from nuclear chromatin agglomerations and are often torn out together with the flagellum at the basis of which the kinetoplast is located.
Immuno-gold post-embedding labeling for electron microscopy: Technical procedures and potential limitations
Authors: Bendayan Moise
Published: June 2020

The successful post-embedding immunolabeling of tissue sections requires several essential steps.

1) The use of an highly specific and well characterized antibody.

2) The use of an efficient probe that will bind the antibody molecule.

3) The use of tissue sections prepared for microscopy which will combine good preservation of the cellular structures and retention of protein antigenicity.

The characterization of the antibodies and their monospecificity are prerequisite conditions that will determine the final quality of the results.

Several immunoprobes are available for the labeling of antigenic sites. The use of protein A or protein G, secondary antibody molecules tagged with colloidal gold particles or the biotin-avidin gold system, have been able to generate very good and reliable results of similar value. The question on the size of the gold particles remains a decision that should be taken according to the results expected. Labeling of large cellular structures and their visualization at low magnification require relatively large gold particles (10 to 15 nm).
Use of a Biodegradable Neuroprosthesis Containing Laminin for Avulsive Peripheral Nerve Injury Results in Early Endoneurial and Perineurial Collagen Type III Formation
Authors: Giammara B. , Anderson E. , Yates P. , Zuniga J. , Hanker J.
Published: June 2020

The direct anastomosis of nerve stumps is most desirable after peripheral nerve transection. There are many avulsive nerve injuries, however, where a nerve segment is destroyed or must be removed -- and the proximal and distal stumps are separated by a gap requiring bridging. Earlier studies showed that a biodegradable polyester mesh (polyglactin 910, Vicryl™) sleeve can act as a conduit and guide regenerating axons into the distal stump across an 11 mm gap created in rat sciatic nerve. This is facilitated by filling the sleeve with Matrigel™, an extracellular basement membrane gel containing 80% laminin, smaller amounts of collagen type IV, heparan sulfate proteoglycan and entaction. The avulsed sciatic was reconnected in animals examined after 30 and 45 days and sensation and leg movement were much improved.

The PATS variation of the PAS reaction can be used to demonstrate all glycomacromolecules containing alpha-diglycol groups. Although it also stains basement membrane or type IV collagen brown, it stains type III collagen or reticulin intensely black.
Myelinated Nerve Fixation Techniques: Problems and Comparative Morphology
Authors: Van Over James E. , Fassel Theresa A. , Hauser Christopher C. , Matloub Hani S.
Published: June 2020

The morphology of peripheral nerve trauma is necessary to understand the resulting alterations in nerve function. Specific traumas, such as Vibration Syndrome, directly effect nerve function. Earily changes in myelinated axons closely resemble artifact damage due to preperations for electron microscopy. If the correlation between structure and function in traumatic conditions is to be studied, it is imperative to establish a fixation technique that yields consistently good results. It is also important to characterize fixation artifacts and correlate these changes to traumatic damages.

Several techniques were compared in an attempt to illustrate the quality of morphology obtained from each fixation. Femoral nerve was microsurgically harvested from Sprague Dawley rats. The nerve was then cut into 1 mm segments and fixed by a variety of methods. The basic fixation was for 2.5 hr in 2.5% glutaraldehyde (GA), followed by 3 10-minute washes in 0.1 M cacodylate buffer pH 7.2, and 2.5 hr post-fixation in 1.0% OSO4 with an additional three washes in buffer.
Acoustic microscopy: A study of contrast mechanisms in fresh tissue
Authors: Hoagland Patricia A. , Scherba Gail , O'Brien William D.
Published: June 2020

There are two general classes of acoustic microscopes, viz., scanning acoustic microscope (SAM) and scanning laser acoustic microscope (SLAM). Both were developed independently in the early 1970’s. Operating at frequencies generally between 100 MHz and 1 GHz, the SAM’s resolution capability ranges between 1 and 15 μm whereas, the SLAM, which operates at frequencies between 10 MHz and 500 MHz, exhibits resolution capability in the 3 to 150 μm range. While the SAM tends to operate at ultrasonic frequencies greater than the SLAM, each acoustic microscope technology is distinct and complementary since resolution is not the only benchmark to judge system capability. The SLAM provides real-time (TV frame rate) image capability while the SAM yields temporally discrete images since it generates images at a slower rate. The SLAM has proven quantitative capabilities, able to yield attenuation coefficient and speed of specimens under study. The SAM’s quantitative capability has not been completely documented, particularly in terms of determining the specimen’s ultrasonic speed.
Study of Sporotrichosis by TEM and SEM
Authors: Li Zhen , Wang Jianha , Suen Zhengcheng , Cu Fei , Li Nan
Published: June 2020

Sporotrichosis is a common infective mycosis that is caused by Sporotrichum schenckii, a dimorphic fungus. The ultrastructures of primary skin in sporotrichosis in human tissue were observed under a transmission electron microscope (TME) and a scanning electron microscope (SEM). The results are reported below.

The primary skin lesions were obtained from patients with sporotrichosis by biopsy. TEM and SEM specimens were prepared by routine methods and refrigeration cutting. Under the TEM, the gap between epidermic kerationcytes was wider than normal and the amount of desmasome decreased. Infiltrations of Langerhans’s cells, granulocytes, lymphocytes, macrophage, and plasma cells were observed in the epidermis. Relicts of S. schenckii were found in the gap of stratum granulosum. A number of inflammatory cells infiltrated into the dermis. Spores of S. schenckii were phagocyted by histiocyte in a shallow layer of the dermis.
Atomic structural images of crystalline cholesterol monohydrate in human atheroma
Authors: Lee Ying-Shiung
Published: June 2020

To gain further insight regarding the micromechanisms of progression and regression of human atherosclerotic plaques the present study was designed for characterization of crystal lattices of cholesterol monohydrate, demonstration of cholesterol crystallization processes, and identification of crystal disorders of cholesterol monohydrate by means of high resolution electron microscopy (HREM).

Clusters of cholesterol clefts containing crystalline cholesterol monohydrate were obtained from atherosclerotic human aortas under a dissecting microscope at autopsy. Cholesterol clefts were milled to a powder with a porcelain pestle. The powdered samples without staining dispersed in distilled water were spread on the copper-grid and dried at room temperature. The air-dried specimens were directly examined by a Hitachi H-9000 high resolution electron microscope operated at 300 KV. High resolution lattice images were taken under an optimal defocus condition. Selected area images and electron diffraction patterns were simultaneously taken for the analysis of crystalline characteristics.
The ultrastructure of the molluscum contagiosum lesion
Authors: Burmeister Veronika , Shelley W.B.
Published: June 2020

Molluscum contagiosum is unique among the viral induced growths of the skin. Unlike other viral tumors, the molluscum contagiosum lesion is a mass made up of myriads of free, discrete virions. It is not a proliferating growth of epidermal keratinocytes such as one observes in the common wart. There is no better way to perceive the nature of this tumor mass than by scanning electron microscopy.

Skin lesions of molluscum contagiosum were excised from patients and immediately fixed in 2.5% glutaraldehyde. After fixation the specimens were rinsed in Millonig’s Phosphate buffer. The entire lesion was than cross-sectioned with a razor blade, postfixed in 1% OsO4, rinsed again in buffer, dehydrated in grading ETOH, critical point dried, positioned on an aluminum stub and gold-coated. Examination was made using a JEOL SEM (JSM-35).
Quantification of the nuclei of human bronchial epithelial cells from electron micrographs for radon risk analysis
Authors: Robbins Edith S. , Meyers Owen A. , Harley Naomi H.
Published: June 2020

Lung cancer in either smokers or in uranium miners exposed to radon occurs preferentially in the epithelium of the bronchi (airway generations 2 to 6). Accurate carcinogenesis risk/dose modeling requires quantitative data on normal bronchial epithelial parameters including: (a) the average number and volume densities (VD) of the nuclei of the possible target cell types and (b) the distances to the free surface of the nuclei of each type. Current dose models use data from an inadequate 1972 light microscopic study. Since smoking and radon effects may be synergistic, these parameters of the bronchi of smokers and non-smokers should be compared.

Human bronchial specimens (72) dissected by airway generation were obtained from non-involved areas of lungs/lobes removed for various pathologies (16 smokers, 8 non-smokers). Samples were fixed with 2% glutaraldehyde, 0.1 M cacodylate, 0.1M sucrose for routine TEM (graded ethanols, Epon embedding, lead and uranyl staining) and sections were examined on a JEOL 1200 EX at l,000x at 60 KV.
The Effect of Active Oxygen Species on the Cornea
Authors: Nguyen H. , Carubelli R. , Chace K.V. , Graham S.A. , Nordquist R. E.
Published: June 2020

Corneal ulceration (“corneal melting”) can occur following various types of insults to the eye, e.g., alkaline or thermal burns, ocular laceration, rheumatoid arthritis, eye infections and vitamin A deficiency. A common link in this multiplicity of etiologic factors is the probable participation of active oxygen species (i.e., hydrogen peroxide, singlet oxygen and free radicals such as superoxide anion and hydroxyl radical). These compounds could play an important role in the pathogenesis of corneal melting since they have been implicated in numerous types of tissue injury. Therefore, we decided to investigate in rabbit cornea the effects of hydrogen peroxide (H2O2) and superoxide anion () generated in situ. Oxidation of endogenous corneal glucose by glucose oxidase was utilized for the generation of H2O2. A cationic preparation of glucose oxidase Aspergillus niger was obtained by amidation of its free carboxylic groups. This modification enhances retention of the enzyme through ionic interaction with polyanionic corneal proteoglycans. Intrastromal injections of 30-60 ug of this enzyme cause corneal ulceration in the rabbit.
Anatomical Variations in the Ocular Vasculatue of Rabbits, Rats and Cats
Authors: Okubo H. , Koss M.C. , Nguyen H. , Lerner M.P. , Gherezghiher T. , Wen J. , Nordquist R.E.
Published: June 2020

Anatomical studies of the vasculature of the eye have been reported in a variety of animals. However, published reports of comparative studies are few in number and there are fewer yet published reports on the long posterior ciliary artery (LPCA). The present study focuses on the organization and structure of the LPCA and its branches in three different species. Our study of the vasculature of the eye was performed utilizing methacrylate vascular casting and observation by light microscopy and scanning electron microscopy. The study was designed to elucidate the anatomical formation of the long posterior ciliary artery and its branches and how this system differed in rabbits, cats and rats. Our findings showed that each species had a characteristic vascular pattern. In the rabbit eye, the LPCA supplied the anterior segment, the choroid, and was considered the only arterial supply of the uvea. In cat eyes, the choroid was supplied by many short posterior ciliary arteries (SPCAs) that arose from the ophthalmic artery.
Ultrastructural characteristics of experimental splenomegaly in portal hypertensive rats: An scanning electron microscopic study of the red pulp
Authors: Misawa T. , Hataba Y. , Baba N. , Kanaya K. , Sakurai K. , Suzuki T.
Published: June 2020

Because splenomegaly in humans is generally investigated at advanced stage, little is known about the relationship between the change in portal pressure and the pathological characteristics of splenomegaly or the pathological features of splenomegaly in the early stage. To clarify the relationship between the development of splenomegaly and portal hypertension, a morphological study on splenomegaly induced by portal hypertension was performed mainly by scanning electron microscopy (SEM) in liver cirrhotic rats.

Male Wistar rats, weighing 200 to 250 g, were used in this study. Liver cirrhosis was produced by intraperitoneal injections of 4% thioacetamide (TAA) in saline according to the previous report. Rats were divided into 5 groups (n=5 for each): groups 1 to 5 corresponding to 20, 40, 60, 80, and 100 injections of TAA, respectively. As a control for each experimental group, rats were injected with saline instead of TAA. The injection needles were placed in the portal vein trunks and abdominal aorta.
Atypical virus particles in glandular tissues from mice injected with a recombinant murine retrovirus encoding an avian V-MYC oncogene
Authors: Beatrice Murray A. , Schmidt Jörg , Becke Helma , Luz Arne
Published: June 2020

A murine retrovirus construct encoding an avian v-myc oncogene (gift from B. Vennström EMBL, Heidelberg, F.R.G.) was injected into new born female NMRI mice. All mice developed malignant thymic lymphoblastic lymphoma within four months. Four of 9 mice also developed adenocarcinomas of the pancreas. Tissues were fixed in buffered formalin and samples from the pancreas tumours, lymph nodes, normal pancreas, liver and salivary gland were embedded in epon and examined by transmission electron microscopy.

Large numbers of atypical virus-like particles were observed in all the samples from the pancreas tumours, normal pancreas and salivary gland, but only rare scattered particles in lymphomas. The pancreas tumours contained massive accumulations of particles in the intercellular spaces. In the normal pancreas, particles were preferentially located in pocketlike areas between the basal laminar and the surface of acinar cells (Fig.1), as well as in the narrow zone between adjoining acinar cells. Particles were occasionally seen budding from the cell surface.
Effect of ketotifen on the ultrastructure of the Erythrocytic stage of Plasmodium Yoelii in mice
Authors: Han-min Cao , xing-qing Pan
Published: June 2020

Since plasmodia have developed resistance to many of the drugs widely used as anti-malarials throughout the world, there is thus an urgency in the search for new types of anti-malarials (WHO technical report series 711; Liu et al. 1984). The search is going on worldwide. Several reports of the efficiency of ketotifen have been published in China. Base on biochemical analysis, Pan et al. reported that ketotifen showed strong anti-malarial effects on both a chloroquine sensitive strain of P. yoelii and a chloroquine resistant strain of P. berghei. In this paper we study the ultrastructural changes in plasmodia exposed to ketotifen.

Experimental mice were inoculated intraperitoneally with 1 × 10 P.yoelii infected red blood cells. After four days, the parasitemia reach approximately 20-30%. 20mg ketotifen base per kg body weight was given to the mice orally. Blood samples were collected at 1,2,3,6,12,24,36,and 48 hours after drug administration.