bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of N-Vinyl pyrrolidone grafting into silicone rubber
Authors: Lee R. , Greer R.
Published: June 2020

Since 1960, hydrogels have been utilized in a large number of biomedical products. The poor mechanical strength of hydrogels necessitates the use of a stronger support material in order to provide structural reinforcement. Methods of surface preparation include radiation grafting. Parameters of importance for grafting N-vinyl pyrrolidone (NVP) into silicone rubber include the choice of solvent, monomer concentration, radiation dose and use of selective inhibitors. Post irradiation extraction of homopolymer and monomer from the silicone rubber substrate after grafting is an important step. NVP grafted polymers can be stained by many dyes for depth of penetration studies.

Fourteen cm sections of non-reinforced Silastic® (silicone rubber, Dow Corning) tubing, 0.078″x0.125″, were boiled for three 1 h periods in aqueous 2.0% sodium bicarbonate solution. Prior to irradiation, monomer solutions used to fill tubes were bubbled with nitrogen for at least 30 min. Several monomer/solvent formulations (100% NVP; 80% NVP/20% methanol; and 60% NVP/40% methanol, and gamma irradiation doses (50, 150, 200 and 250 krad) were used.) After irradiation, samples were flushed with either acetone/methanol (50:50 v/o) or ethanol/water (50:50 v/o) , followed by three 30 min agitated washes in the same type of solution. To test the effect of methanol concentration on rim thickness measurements, 0.5 cm lengths of cleaned Silastic , acetone/methanol extracted Silastic®, and grafted tubing of 100% NVP and 80% NVP/20% methanol formulations were immersed in 0.1% acid fuchsin stains in 10, 50, and 100% methanol for 48 h. After rinsing and drying, cross sections were cut, and the penetration depth of the stain (i.e., graft depth) was measured.
Localization of two structural wall proteins in an insect spermatophore by immunoelectron microscopy
Authors: Schuette J. L. , Happ G. M.
Published: June 2020

Spermatophores are acellular protein sacs which package sperm for transport. They are highly ordered structures derived from secretions that self-assemble in extra- cellular spaces. The mealworm spermatophore emerges from the male in an unexpanded state and consists of a multilayered fibrous outer wall, an organized inner core and a lumen containing semen. The spermatophore expands inside the female as its core is extruded and molded into new wall material. We are interested in the assembly and maturation of the spermatophore and have developed monoclonal antibodies which recognize epitopes of two different wall proteins, each secreted from a distinct cell type. We have employed immunoelectron microscopy (ABC Technique) to determine the location of these proteins in the wall and to examine their fate during the expansion process.

Spermatophores were collected from copulating male mealworms (Tenebrio molitor). Some were allowed to fully expand. Samples were fixed in 1% formaldehyde and 0.5% glutaraldehyde in 0.1M phosphate buffer, pH = 7.2, for 2 h; dehydrated through alcohol and embedded in Epon.
Effect of depolarization by potassium proprionate on synaptic vesicle diameter and distribution of the snake motor endplate
Authors: Hendricks Gregory , Coniglio Lisa , Marshall Ian , Searl Tim , Parsons Rodney
Published: June 2020

The morphological effects of elevated potassium (K+) at the neuromuscular junction of garter snake twitch muscle fibers were examined in vitro. Elevated K+ depolarizes the motor neuron terminals and causes quantal release of acetylcholine. It has been shown in amphibians that prolonged (30-60 min) depolarization with high K+ depletes terminals of synaptic vesicles ; but causes no change in the average diameter of the vesicles. In the snake, however, we have shown previously that miniature endplate currents (MEPCs) are present at high frequency even after 1-2 hours of exposure to high K+ . This indicates that in the snake, synaptic vesicles are not totally depleted by depolarization with high K+. In this study, we examined the effect of depolarization with high K+ on the distribution and diameter of synaptic vesicles.

Intercostal muscle preparations were exposed for 15 minutes to Ringer's + solution containing either a control (1.2mM) or high (70mM) concentration of K+ Preparations were fixed immediately in 1% glutaraldehyde for 15 minutes and washed with Millonig's buffer. The whole mount muscle preparations were then fixed for 1 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2; rinsed three times in fresh buffer and post-fixed in 2% OsO4 in the same buffer for 30 min, rinsed in fresh buffer and dehydrated in a graded ethanol series to 100% and then transferred into a 2% solution of uranyl acetate for 20 min to en-block stain the whole mounts. The preps were then transferred back to 100% ethanol to complete the dehydration and embedded in a mixture of Embed 812/Araldite 502 (Hard) between two microscope slides that had been precoated with Formen- Trennmittel Liquid Release Agent and polymerized overnight at 70°C.

The whole mounts were then removed from the microscope slides and examined on a compound light microscope where the neuromuscular junctions on the twitch muscle fibers could be identified, marked and cut out of the preps. These pieces of the whole mounts were then remounted into a slot on the end of a pre-cast Embed-Araldite block with a drop of fresh resin and polymerized.
A technique for protecting delicate specimens during processing
Authors: Mount R.J. , Harrison R.V.
Published: June 2020

The sensory end organ of the ear, the organ of Corti, rests on a thin basilar membrane which lies between the bone of the central modiolus and the bony wall of the cochlea. In vivo, the organ of Corti is protected by the bony wall which totally surrounds it. In order to examine the sensory epithelium by scanning electron microscopy it is necessary to dissect away the protective bone and expose the region of interest (Fig. 1). This leaves the fragile organ of Corti susceptible to physical damage during subsequent handling. In our laboratory cochlear specimens, after dissection, are routinely prepared by the O-T- O-T-O technique, critical point dried and then lightly sputter coated with gold. This processing involves considerable specimen handling including several hours on a rotator during which the organ of Corti is at risk of being physically damaged. The following procedure uses low cost, readily available materials to hold the specimen during processing ,preventing physical damage while allowing an unhindered exchange of fluids.

Following fixation, the cochlea is dehydrated to 70% ethanol then dissected under ethanol to prevent air drying. The holder is prepared by punching a hole in the flexible snap cap of a Wheaton vial with a paper hole punch. A small amount of two component epoxy putty is well mixed then pushed through the hole in the cap. The putty on the inner cap is formed into a “cup” to hold the specimen (Fig. 2), the putty on the outside is smoothed into a “button” to give good attachment even when the cap is flexed during handling (Fig. 3). The cap is submerged in the 70% ethanol, the bone at the base of the cochlea is seated into the cup and the sides of the cup squeezed with forceps to grip it (Fig.4). Several types of epoxy putty have been tried, most are either soluble in ethanol to some degree or do not set in ethanol. The only putty we find successful is “DUROtm MASTERMENDtm Epoxy Extra Strength Ribbon” (Loctite Corp., Cleveland, Ohio), this is a blue and yellow ribbon which is kneaded to form a green putty, it is available at many hardware stores.
CFM holey films
Authors: Olson R. A.
Published: June 2020

Chloroform-formvar-mica (CFM) can be used to make holey films in the simplest and most reliable procedure described to date. A wide variety of approaches have been devised to produce thin membranes with holes in them. Hayat (1978) provides an excellent historical overview of these perforated film techniques. They range from complex and cumbersome to relatively simple, but even these simpler techniques require either special solutions or incorporate time-dependent steps which may yield variable results. The CFM technique suffers from neither of these drawbacks; the steps are outlined below.

Procedure:

l. Dip a freshly cleaved mica strip into a 0.25% solution of formvar in chloroform and withdraw it at a slow, even rate.
2. Wick off excess solution, allow to dry for about one minute, and trim the formvar coated lower edges of the strip with scissors.
3. Score the formvar membrane with a razor blade just below the “high water mark” and float it onto distilled water.
Changes in the plastid ultrastructure during greening in cultured soybean cells
Authors: Gillott M.A. , Erdös G. , Buetow D. E.
Published: June 2020

Mesophyll cells isolated from soybeans (Glycine max, L. Merr. var. Corsoy) can be grown photoautotrophically in suspension culture. The SB-P cell line can be bleached by maintaining them in total darkness in sucrose supplemented media for several weeks, and will regain photosynthetic competency when returned to the light. This system is ideally suited for the study of gene regulation and the biochemical and ultrastructural changes which occur during the greening process.

Cells were fixed for electron microscopy after 8 weeks of growth in total darkness and at intervals of 1 h to 12 d after transfer to the light. Chlorophyll measurements were determined for each sample. For transmission electron microscopy, the cells were fixed for 1-2 h in 4% glutaraldehyde in 0.1M Pipes buffer, pH 7.4, washed in the same buffer, then postfixed for 1 h in 1% OsO4 in Pipes, pH 6.8. Following a graded ethanol dehydration series the cells were transferred into propylene oxide and embedded in Epon. Sections were stained with uranyl acetate and observed on a JEOL 100C TEM operated at 80 kV.
A Scanning Electron Microscope study of the pecan phylloxera, Phylloxera devastatrix, with observations on emergence
Authors: Hall M.J. , Clawson R. C. , Saunders C. , Shelton R. R.
Published: June 2020

The pecan phylloxera, Phylloxera devastatrix Pergande, is a small, cecidogenic insect found throughout the native range of the pecan tree. Infestations of P. devastatrix have been reported to cause reductions in nut quality and quantity, premature defoliation, and shoot dieback. Galls formed by P. devastatrix are an alternate host for the larvae of the hickory shuckworm, Cydia caryana Fitch. Little information exists on the biology and ecology of this insect. The objective of this study was to examine the emergence patterns of P. devastatrix on different pecan cultivars to determine if variability in bud-break between cultivars effected emergence. This study also provided an opportunity to study gall formation and the life cycle in detail.

P. devastatrix overwinter as an egg within the body of the sexual female (Figs. 1 & 2). Upon hatching, the stem mother moves to the bud and begins feeding (Figs. 3 & 4). As the stem mother feeds, a gall begins to form around the insect (Fig. 3). Eggs deposited within the gall by the mature stem mother (Fig. 6) hatch into apterous and alate females (Fig. 7). Eggs produced by these females hatch into sexual males and females (Fig. 8 & 9). After mating, an egg is produced within the body of the female. The female next seeks a place on the tree to overwinter, thus completing the cycle. Stoetzel described the development of the stem mother and apterous and alate females as hemimetabolous (incomplete metamorphosis), and that of the sexual males and females as holometabolous (complete metamorphosis). Gal1s of p. devastatrix are classified as cover galls. The upwalling of the plant tissue around the insect, characteristic of this type of gall, can be clearly seen in the SEM photographs (Figs. 3 & 5).
Quantitative analysis of post-embedding immunogold Electron Microscopy with differently sized labelling gold particles
Authors: Gu J. , D'Andrea M.
Published: June 2020

The post-embedding immunogold staining technique is a powerful tool in localizing tissue antigens at the ultrastructural level. The highly electron dense colloidal gold particles are easily visible under electron microscope and the differently sized gold particles allow a simultaneous demonstration of more than one tissue antigens in the same grid. The sizes of the commercially available labelling gold particles range from 1 nm to 40 nm. It is generally believed that the smaller the gold particles the better the detecting sensitivity of the technique. However, no quantitative data in this regard is available. We carried out an experiment to assess the detecting sensitivities of differently sized labelling gold particles in the post-embedding electron microscopic indirect immunogold staining method using atrial natriuretic peptide (ANP) in rat atria as a model.

ANP is a newly discovered cardiac hormone with 28 amino acids. In the heart it is located exclusively in the specific electron dense granules in the cytoplasm of atrial cardiocytes. The high concentration and the well defined location of this tissue antigen makes it an ideal model to evaluate the detecting sensitivities of immunostaining techniques.
Biological photocathodes: Complexes of chlorophylls and hemes examined by photoelectron microscopy
Authors: Griffith O.H. , Habliston D.L. , Birrell G.B. , Skoczylas W.P. , Hedberg K.K.
Published: June 2020

Chromophore binding proteins are important in cell bioenergetics. They are, however, difficult to distinguish by electron microscopy because the atomic composition and hence scattering properties are not very different from those of other proteins. Photoemission is one relatively unexplored property of chromophores that offers a solution to this problem. Essentially all biological specimens exposed to radiation of sufficiently high energy, typically in the ultraviolet region of the spectrum, emit electrons (photoelectrons). The photoelectrons are accelerated and imaged in a photoelectron microscope as diagrammed in Fig. 1. Since the photoelectrons originate at or near the sample surface, photoelectron microscopy is essentially a surface technique. Up to now biological applications of photoelectron imaging have been restricted by the relatively low magnifications attainable due to the low emissivity of biological specimens. The goal of this study is to convert the biological surface into a sensitive photocathode while retaining the differences in photoemission of the various components, such as the chromophores, which provide important functional information in the biological preparations.

We find that the photoemission of biomolecules can be greatly increased by exposure to a very thin layer of cesium (approximately 1 nm thick). The free cesium evaporates, leaving behind cesium atoms that interact with the surface components. One mechanism is evidently electron transfer from cesium to the chromophores, resulting in charge transfer complexes (ion radical salts). Other mechanisms are almost certainly also involved. An example of the dramatic improvement in photoemission, and hence magnifications attainable, is shown in Fig. 2.
Signs of nerve regeneration following pancreas transplantation in an insulin-dependent diabetic with neuropathy
Authors: Beggs J.L. , Johnson P.C. , Olafsen A.G. , Cleary C. , Watkins C.J. , Targovnik J. , Koep L.
Published: June 2020

Nerve disease is a common complication of diabetes mellitus. Hyperglycemia directly or indirectly causes structural damage and functional impairment of nerve fibers. Despite conventional therapy, there continues to be a substantial incidence of diabetic complications. Development of complications is thought to be due to abnormal fluctuations in blood glucose levels. The number of pancreas transplants performed for the treatment of diabetic complications has increased dramatically in the last few years. Unlike conventional therapy, functional pancreas grafts provide normal fasting blood glucose levels, near-normal glucose tolerance, and normal levels of glycosylated hemoglobin. In this report, we address the following question: will functional pancreas grafts prevent or reverse the structural nerve damage caused by diabetes mellitus?

A 53 y/o man developed insulin-dependent diabetes at age 35. Nephropathy, retinopathy, and neuropathy developed despite conventional insulin therapy. To treat these complications, pancreas segment transplant was performed. The donor was his identical twin. The recipient was treated with low-dose immunosuppressive drugs (azathioprine and prednisone). He has remained euglycemic for 3 years and requires neither insulin therapy nor oral hypoglycemic agents. Clinical examinations during the 3 years post-transplantation have revealed progressive improvement in vision, nerve function and a stabilization in renal function.
MDX mouse myopathy II: Degenerative changes in centronucleated fibers from mice of different ages
Authors: Geissinger H.D. , Rhodes P.A.
Published: June 2020

Ultrastructural features of centronucleated (regenerating) fibers of ‘mdx’ mice have been reported in three different papers and in all three, different features of these fibers were described in some detail. Thus, in one paper “abortive attempts at regeneration” in very young fibers, in another early degeneration of regenerating fibers and in the third paper “healthy” regenerating fibers, as well as degenerating myotubes were shown. We show the probable pathogenesis of degeneration of centronucleated fibers in ‘mdx’ mice.

METHODS: Tibialis anterior muscles from 22-, 25-, 41-, 61- and 99-days-old C57BL/10ScSn/MDX mice were pinned on corkboard in a relaxed state, prefixed for 30 minutes in glutaraldehyde followed by routine processing for TEM.

RESULTS: In a 41-days-old ‘mdx’ mouse a fiber which had apparently fully regenerated from a previous episode of necrosis is shown in FIG. 1. Since there are no pathological changes in this fiber, it will serve as a control. The nucleus has a fairly prominent nucleolus and there are no lesions in the
myofibrils and the rest of the sarcoplasm.
Postmortem changes of the gill lamellae of fish
Authors: Liu K. C.
Published: June 2020

The fine structure of the gill lamellae of fish has been reported for several species. All of them consisted the same types of cells and showed the same characteristics. The structural change of the gill lamellae under natural condition, after death was not reported. This report is to present a study on that event.

Gill filaments from culturedAnguilla japonica,Tilapia sp. andChanos chanos, of marketing size, both fresh and dead for different length of time were studied. The samples were fixed with 3% glutaraldehyde and 1% osmic acid, dehydrated with ethanol, and embedded with Epon and Araldite mixture. Thin section were stained with uranyl acetate and lead citrate. Observations were made with a JEOL 100 CX electron microscpe at 60 KV.

The gill lamella of all three species studied showed similar fine stuctural characteristics . They consisted a central raw of pillar cells which formed the blood lumen. A layer of epithelium covered the outer surface . There was a prominent layer of basal limina between those two types of cells (Fig. 1). The epithelium showed degeneration first. It disintegrated and ditached from the basal lamina, and formed debris.
Microtubule motors and other maps
Authors: Vallee Richard B.
Published: June 2020

Microtubules are involved in a number of forms of intracellular motility, including mitosis and bidirectional organelle transport. Purified microtubules from brain and other sources contain tubulin and a diversity of microtubule associated proteins (MAPs). Some of the high molecular weight MAPs - MAP 1A, 1B, 2A, and 2B - are long, fibrous molecules that serve as structural components of the cytamatrix. Three MAPs have recently been identified that show microtubule activated ATPase activity and produce force in association with microtubules. These proteins - kinesin, cytoplasmic dynein, and dynamin - are referred to as cytoplasmic motors. The latter two will be the subject of this talk.

Cytoplasmic dynein was first identified as one of the high molecular weight brain MAPs, MAP 1C. It was determined to be structurally equivalent to ciliary and flagellar dynein, and to produce force toward the minus ends of microtubules, opposite to kinesin.
Initial formation of glucan fibrils of the reverting protoplast in fission yeast, elucidated with a newly developed ultrahigh-resolution, low-voltage scanning electron microscope
Authors: Osumi Masako , Yamada Naoko , Taki Akiko , Naito Nobuko , Kobori Hiromi
Published: June 2020

The resolution of the SEM has progressed markedly by means of an “in-lens” type field emission (FE) SEM. However, its performance at low beam energy, where we could expect an improved resolving power to visualize the detail of the surface of the specimens, was insufficient. Therefore an improved ultrahigh resolution (UHR) FESEM, the S-900H, was developed jointly with Hitachi to give better resolution at low voltages (LV, below 5 kV) by improving the objective pole piece. This instrument, which we called model S-900 LV, allows visualization of an about 1-nm object at 2.5 kV and x 30,000. The instrument made it possible to observe uncoated biological specimens at a magnification of 200,000 in the LV from 2 to 3 kV. The present study was intended to elucidate the u1trastructure of a fission yeast protoplast in the early stage of its regeneration, especially the formation of glucan fibrils from secreted small particles.
Immunoprobe localization in mammalian embryos
Authors: Calarco Patricia G. , Siebert Margaret C.
Published: June 2020

Visualization of preimplantation mammalian embryos by electron microscopy is difficult due to the large size of the ircells, their relative lack of internal structure, and their highly hydrated cytoplasm. For example, the fertilized egg of the mouse is a single cell of approximately 75μ in diameter with little organized cytoskelet on and apaucity ofor ganelles such as endoplasmic reticulum (ER) and Golgi material. Thus, techniques that work well on tissues or cell lines are often not adaptable to embryos at either the LM or EM level.

Over several years we have perfected techniques for visualization of mammalian embryos by LM and TEM, SEM and for the pre-embedding localization of antigens. Post-embedding antigenlocalization in thin sections of mouse oocytes and embryos has presented a more difficult challenge and has been explored in LR White, LR Gold, soft EPON (after etching of sections), and Lowicryl K4M. To date, antigen localization has only been achieved in Lowicryl-embedded material, although even with polymerization at-40°C, the small ER vesicles characteristic of embryos are unrecognizable.
Immunoelectron microscopy provides a road-map for antigen processing and presentation
Authors: Guagliardi Lynne , Crumrine Debra , Brodsky Frances
Published: June 2020

Immunoelectron microscopy was used to identify the intracellular routes traveled by molecules involved in antigen processing and presentation by human B cell lines. To mimic the uptake of multivalent antigen, anti-immunoglobulin coupled to 15-nm colloidal gold beads was used to cross-link surface immunoglobulin and induce its internalization at 37°C. At various timepoints following internalization, cells were fixed in cacodylate buffer with 2% paraformaldehyde, postfixed briefly with OsO4 ferricyanide, and en-block stained with aqueous uranyl acetate. After brief dehydrations in 50% and 70% ethanol, cells were gradually infiltrated with L.R. White Resin (medium hardness) and polymerized at 50°C overnight. 60-nm sections were labelled with monoclonal antibodies and polyclonal antisera adsorbed to 2- or 5-nm colloidal gold to characterize the intracellular compartments containing immunoglobulin with respect to their pH, enzyme content, the presence of Class II histocompatibility molecules, the invariant chain, and markers for the endocytic pathway. Using 2 sizes of gold probes, we simultaneously mapped the distribution of 2 different molecules in the same immunoglobulin-containing compartment.
Immunocytochemistry after fast freeze fixation-freeze substitution: Advantages and limits
Authors: Nicolas Marie-Thérèse
Published: June 2020

An alternative to aqueous chemical fixation consists in immobilizing physically the specimen by freezing it as fast as possible without using any cryoprotectant. This Fast Freeze Fixation (FFF) followed by Freeze Substitution (FS) avoids osmotic artefacts due to the slow penetration of liquid chemical fixative. Associated with Immuno-Gold labeling (IGS), FFF-FS allows a more precise localization of antigens.

Using the bioluminescent bacteria Vibrio harveyi, a comparison of IGS with an antibody directed against its luciferase (enzyme of the luminescent reaction) has been done after liquid chemical fixation versus FFFFS. This later technique, beside an expected improvement of the ultrastructure always shows a better preservation of antigenicity and a lower background. In the case of FFF-FS technique (Figure 3):

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labeling in acrylic resin (LRWhite) is 2 to 4 fold more intense than in epoxy resin (Epon),

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but the ultrastructure is always better in Epon.

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but the ultrastructure is always better in Epon.

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The addition of fixatives in the substitution medium, results in a decrease of labeling which is more important in the case of a mixture of fixatives than with osmium tetroxide alone; with one exception: the substitution with glutaraldehyde which produces a dramatic increase in the density of the labeling but also, at the same time, a swelling of the cells of about 30%.
Modifications of sperm proteins leading to acquisition of fertilizing ability
Authors: Shalgi R. , Jones R. , Phillips D.M.
Published: June 2020

Mammalian spermatozoa acquire their fertilizing ability following release from the seminiferous tubules, as they transverse the epididymis, the female tract and the egg investments. We have examined changes occurring in the distribution of antigens of rat spermatozoa during epididymal maturation and after ligation of the caput epididymis by using immunocytochemistry at the light microscopy and at the electron microscopy (EM) levels.

Spermatozoa were collected from the test is and different segments of the epididymis. To obtain aging of sperm in vivo, male rats were operated and one side of the epididymis was ligated at the caput and corpus junction. Spermatozoa were washed, and divided into 4 tubes, and treated as follows: 1. no treatment; 2. permeabilization by freeze thawing; 3. Trypsin; 4. NP-40;. Sperm samples were washed and the nincubated for 30 min with monoclonal antibodies. Monoclonal antibodies were raised in mice against antigens on cauda sperm of rats.
Ultrastructural observations of the ciliated cyst in the human uterine tube epithelium
Authors: Hagiwara Haruo , Shibasaki Susumu , Ohwada Nobuo
Published: June 2020

The ciliated cysts have been identified as a common manner of development of ciliated cells through light microscopic studies, however recent ultrastructural studies have Reported that they should be regarded as an expression of atypical ciliogenesis. In the present study we attempt to elucidate the detailed structure of the ciliated cysts and their significance in ciliogenesis in the human uterine tube epithelium.

Human uterine tubes were obtained from 20 women who underwent total hysterectomies due to gynecological diseases. The ampullar segments were cut into small pieces, and fixed in 2.5% glutaraldehyde-2% paraformaldehyde (1/2 Kamovsky) in a 0.1M phosphate buffer solution (pH 7.4). These specimens were postfixed in a 1% Osmium tetroxide solution (Millonig), dehydrated with a graded series of ethanol and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined in a transmission electron microscope.

The ciliated cysts were detected as a round or oval intracytoplasmic cavity provided with many ciliary apparatuses and microvilli (Fig. 1).
Reflection Confocal Laser Microscopy in Neurobiology: Peroxidase and Golgi Preparations
Authors: Turner James N. , Szarowski Donald H. , Deitch Jeffrey S. , Smith Karen L. , Swann John W.
Published: June 2020

One of the first applications of confocal microscopy was reflection imaging of unstained neurons. However, most applications employ either Golgi (silver impregnation) or peroxidase staining. Golgi preparations have been imaged by tandem scanning, but both need optimization for confocal laser scanned microscopy (CLSM) due to low signal levels and internal reflections. The imaging wavelength is critical. Specimen absorption of the argon ion laser lines is an order of magnitude larger than the HeNe red line. We compare the imaging of Golgi and peroxidase stained neurons using both lasers.

Rat hippocampal neurons were filled with biocytin, fixed in 4% paraformaldehyde, incubated in avidin-HRP with Triton and reacted with DAB with or without Ni. Specimens were imaged in permount using a Bio-Rad MRC 500 and Olympus BH-2.
Using a TEM Electron Energy Loss Spectrometer to Eliminate Offensive Carbon “halos” from Platinum Replicas of Quick-Frozen, Deep-Etched Biological Samples
Authors: Heuser John , Probst Wolfgang
Published: June 2020

A significant improvement in image quality can be achieved, when imaging deepetch replicas via TEM, by using an electron energy loss spectrometer in a rather unorthodox manner. Microscopes equipped with true imaging spectrometers, such as the Zeiss EM 902, permit the viewer to subtract the offensive carbon “background halo” which is characteristic of all deep-etch replicas. Heretofore, this “halo” has been an unavoidable consequence of the need to “back” or physically support the otherwise extremely delicate platinum replica. In fact, much thicker carbon supports would be desirable, since fragmentation of platinum replicas during their cleaning represents the single greatest impediment to successful use of the deep-etch technique. Until now, such thick carbon has created hopeless “halos” and excessive blurring of replicas in the TEM. Amazingly, such imaging problems can be circumvented, regardless of the thickness of the carbon “backing”, by “dialing out” the carbon signal from the TEM image!
Ultrastructural Characteristics of Glioblastoma Multiforme
Authors: Sundaram A. , Bharati Arumugam Sarasa , Arumugam S. , Rajalakshmi Y.
Published: June 2020

The ultrastructural studies of Glioblastoma multiforme was carried out from tissue obtained from human intracranial neoplasms obtained in Madras Institute of Neurology.

Light microscopy showed a highly cellular neoplasm with areas of sinusoidal necrosis palisaded by spongioblasts. The malignant cells exhibited hyperchromatism, pleomorphism, bizarre nuclei and there were marked endothelial proliferations. Mono nuclear cell aggregates are also seen.

Ultrastructurally Glioblastoma multiforme exhibited great variation in size and shape of cells. In the main, the cells were off moderate size with ovoid nuclei and fine chromatin distributed evenly throughout the nucleus. Nucleoli were prominent and here also the two distinct nuclear membranes could be easily be made out. Fine pores were seen in the nuclear membranece.

The cytoplasm was relatively lucent and showed various organelles such as mitochondria, Golgi apparatus and elements of the rought endoplasmic reticulum in moderate numbers. The endoplasmic reticulum was dilated in many regions and numerous electron dense bodies could also be seen.
Early Cellular Events in the Development of Atherosclerotic Lesions in a Model of Accelerated Atherosclerosis in the Rabbit
Authors: Beesley Julian E. , Hassall David , Jessup Eileen A. , Honey Andrew , Dopping-Hepenstal Trish J.C. , Martin John F. , Moncada Salvador
Published: June 2020

We have generated several of the characteristic features of human atherosclerotic lesions in the rabbit with in 14 days by the placement of a soft, silastic collar around the carotid artery. Preliminary microscopical studies show that the lesion in the region of the collar consists of proliferating smooth muscle cells, foam cells in animals maintained on a high cholesterol diet and that the endothelial cells appear intact. The histology of the contralateral arteries was unaffected by the placement of the collar. This report describes our observations of the early cellular changes in the collar region with particular regard to the presence of monocytes, macrophages and polymorphonuclear leukocytes.

One group of New Zealand White rabbits were fed normal laboratory diet (0.004%cholesterol, w/w) supplemented with 1.5% cholesterol, whilst the other group were maintained on normal laboratory diet. The rabbits were anaesthetised and both carotids exposed surgically. The collar was positioned on the left artery, and the right contra lateral artery served as the sham operated control. Animals were killed at several time points between 1hr and 56days after arterial manipulation.
Diethylnitrosamine-induced spongiosis hepatis in Medaka, Oryzias latipes
Authors: Norton William N. , Gardner Hank
Published: June 2020

Investigators representing such diverse disciplines as biochemistry, pathology, genetics and environmental toxicology currently employ fish models as routine and reliable bioassay testing systems for the detection of toxins and/or carcinogens in the environment, and more appropriately, as rapid indicators of carcinogenicity. The Japanese medaka, Oryzias latipes, has served as a popular fish model for carcinogenicity studies, many of which have included diethylnitrosamine (DENA), a site specific carcinogen for hepatic neoplasms. One of the more intriguing manifestations of nitrosamine exposure reported for both fish and rats is spongiosis hepatis, a hepatic lesion characterized by multilocular cyst-like complexes. Spongiosis hepatis has not been reported in medaka exposed as embryos to hepatic carcinogens and the single article indicating the presence of such a lesion in adult medaka is limited in cytological descriptions.
Histological evaluation of cochlear hair cell damage from noise-induced hearing loss in chinchillas
Authors: Harrison R.V. , Mount R.J. , White P. , Fukushima N.
Published: June 2020

In studies which attempt to define the influence of various factors on recovery of hair cell integrity after acoustic trauma, an experimental and a control ear which initially have equal degrees of damage are required. With in a group of animals receiving an identical level of acoustic trauma there is more symmetry between the ears of each individual, in respect to function, than between animals. Figure 1 illustrates this, left and right cochlear evoked potential (CAP) audiograms are shown for two chinchillas receiving identical trauma. For this reason the contralateral ear is used as control.

To compliment such functional evaluations we have devised a scoring system, based on the condition of hair cell stereocilia as revealed by scanning electron microscopy, which permits total stereociliar damage to be expressed numerically. This quantification permits correlation of the degree of structural pathology with functional changes. In this paper wereport experiments to verify the symmetry of stereociliar integrity between two ears, both for normal (non-exposed) animals and chinchillas in which each ear has received identical noise trauma.
Electron microscopy of simian immunodeficiency virus isolated from a sooty mangabey (Cercocebus Atys) in Liberia, West Africa
Authors: Munn Robert J. , Marx Preston , Brotman Betsy , Hanson Aloysius
Published: June 2020

Simian immunodeficiency virus (SIV) is a Lentivirus that exhibits both morphological and genomic similarities to HIV. SIV has been isolated from sooty mangabeys in several primate colonies, and has been shown to be about 80% related to HTV-2. Since HIV-2 and sooty mangabeys are both indigenous to West Africa, isolation of a virus from sooty mangabeys in West Africa would be of great interest in determining the origins of these two viruses. We report here thin section transmission electron microscopy of a lentivirus from a pet sooty mangabey living in Liberia, West Africa.

Peripheral blood mononuclear cells (PEMC) from a healthy pet sooty mangabey in Liberia were frozen in liquid nitrogen and shipped to the California Primate Research Center. The cells were thawed and stimulated for 72h with 0.5μg/ml Staphylococcal enterotoxin A in RPMI 1640 containing 10% fetal calf serum. 2×106PEMC were then co-cultivated with 2×106CEMxl74 cells and processed for EM.

CEMxl74 cells co-cultivated with infected PBMC exhibited syncytial giant cells with patchy foamy cytoplasm (Fig 1). large numbers of virus particles were observed within these vacuoles (Fig 2).
Ultrastructural evidence for mast cell-macrophage interrelationships in the dura mater of the rat: Infrequent mast cell-nerve associations
Authors: Dimlich Ruth V.W.
Published: June 2020

Mast cells in the dura mater of the rat may play a role in cerebral pathologies including neurogenic inflammation (vasodilation; plasma extravasation) and headache pain . As has been suggested for other tissues, dural mast cells may exhibit a close spatial relationship to nerves. There has been no detailed ultrastructural description of mast cells in this tissue; therefore, the goals of this study were to provide this analysis and to determine the spatial relationship of mast cells to nerves and other components of the dura mater in the rat.

Four adult anesthetized male Wistar rats (290-400 g) were fixed by perfusion through the heart with 2% glutaraldehyde and 2.8% paraformaldehyde in a potassium phosphate buffer (pH 7.4) for 30 min. The head of each rat was removed and stored in fixative for a minimum of 24 h at which time the dural coverings were removed and dissected into samples that included the middle meningeal vasculature. Samples were routinely processed and flat embedded in LX 112. Thick (1 um) sections from a minimum of 3 blocks per rat were stained with toluidine blue (0.5% aqueous).
Oxidant damage and deterioration of the blood-brain barrier in optic neuritis: A correlative study in the guinea pig by MRI and TEM
Authors: Ann Ellis E. , Guy John R. , Fitzsimmons Jeffery R. , Beck Barbara L.
Published: June 2020

Optic neuritis, a demyelinating autoimmune disease, is usually the initial site of multiple sclerosis (MS). Experimental allergic encephalomyelitis (EAE), the experimental model for MS, is induced by injecting spinal cord emulsion in complete Freund’s Adjuvant into susceptible animals; clinical symptoms of ataxia and hind quarter paralysis develop 8 to 14 days later. Damage to the bloodbrain barrier (BBB) and free radical derived oxidants, including hydrogen peroxide (H2O2), have been implicated in the pathology of EAE and other inflammatory diseases. Cytochemical localization of H2O2 in the optic nerve head of animals with EAE corresponds to areas of extravasation of horseradish peroxidase (HRP) at later stages of EAE. Damage to the BBB is monitored clinically in human cases of MS by magnetic resonance imaging (MRI) of gadolinium-DTPA (Gd-DTPA), contrast agent, leakage. Preliminary studies of EAE with MRI at 4 days post antigen sensitization showed leakage of Gd-DTPA in the optic nerve; light and electron microscopy (TEM) of sections from the same nerve showed substantial inflammation from the optic nerve head to the optic chiasm.
Microfibrillar paracrystalline inclusion (MPI) within dorsal root ganglia (DRG) of humans and ferrets in cisplatinum toxicity
Authors: Dancea S. , Montpetit V.J.A. , Mikhael N. , Stewart D.
Published: June 2020

MPI consist of large aggregates of regularly arranged microfilaments (MF) occuring in the soma and proximal processes of neurons of humans and various animal species. These inclusions have been described in the sympathetic ganglia, the caudate, the thalamus and the substantia nigra but their occurence in the DRG has not yet been reported. In the present study, they were found in DRG of humans and ferrets treated with cisplatinum as well as in the controls.

On one micron epon sections stained with methylene blue, MPI appeared as bright blue inclusions of variable size, with an average diameter of 5 μm. At the ultrastructural level, their tendency to be located at the periphery of the cytons with a predilection for the axon hillocks (Fig. 1) and the proximal unmyelinated axons (PUMA) was confirmed (Fig. 2). They formed compact bundles of straight filaments measuring 5-6 nm in diameter. Within each individual bundle, the MF were organized in a highly parallel fashion with periodic intervals of 5-6 nm and regular transverse striations 5-7 nm apart (Fig. 3).
Transmission and Scanning Electron Microscopic study of rat brain tissue after exposure to enriched versus impoverished environments
Authors: Hicks Heraline E. , Stahl Jeanne M. , Mills Adell
Published: June 2020

Previous research has indicated that when rats are raised in enriched versus impoverisshed environments significant structural and chemical changes occur in the brain. Enriched environments usually include group housing in a large cage with many inanimate objects that are changed daily. The purpose of this study was to examine by electron microscopy, cellular morphology in rat brains after exposure to enriched versus impoverished environments.

Neonatal Long-Evans hooded rats were raised in either enriched or impoverished environments from day 10 to day 70. After 60 days of enrichment, the animals were given several behavioral tests, were euthanized, the brains were surgically removed, and the septum, hippocampus, and cortex were dissected free. For scanning electron microscopy (SEM), the tissue was fixed in 2.5% glutaraldehyde in Sorensen’s phosphate buffer for 24 hr, rinsed and then post-fixed in 2% osmium tetroxide for 1 hr, dehydrated through a graded ethanol series, critical point dried, mounted on specimen holders, sputter coated and examined. Other specimens were embedded in Epon 812, sectioned for transmission electron microscopy (TEM) stained with uranyl acetate followed by lead citrate and examined. SEM demonstrated that brain tissue from rats exposed to enriched environments had more and larger neuronal vessels extending through the tissue than brain tissue from impoverished animals (Fig. 1 & 2).
Further studies on the ultrastructure of the cochlea
Authors: Wang Zhixian , Zhu Pinjin , Sun Jianhe , Song Xuezheng
Published: June 2020

Hearing research is important not only for clinical, professional and military medicine, but also for toxicology, gerontology and genetics. Ultrastructure of the cochlea attracts much attention of electron microscopists, (1―3) but the research lags far behind that of the other parts of the organnism. On the basis of careful microdissection, technical improvment and accurate observation, we have got some new findings which have not been reported in the literature.

We collected four cochleas from human corpses. Temporal bones dissected 1 h after death and cochleas perfused with fixatives 4 h after death were good enough in terms of preservation of fine structures. SEM:The apical surface of OHCs (Outer hair cells) and DTs (Deiters cells) is narrower than that of IPs (Inner pillar cells). The mosaic configuration of the reticular membrane is not typical. The stereocilia of IHCs (Inner hair cells) are not uniform and some kinocilia could be seen on the OHCs in adults. The epithelial surface of RM (Reissner’s membrane) is not smooth and no mesh could be seen on the mesothelial surface of RM. TEM.
Immunocytochemical localization of desmin and vimentin in chick embryonic myocardial cells
Authors: Sugi Yukiko
Published: June 2020

In cultured skeletal muscle cells of chick, one intermediate filament protein, vimentin, is primarily formed and then synthesis of desmin follows. Coexistence of vimentin and desmin has been immunocytochemically confirmed in chick embryonic skeletal musclecells. Immunofluorescent localization of vimentin and desmin has been described in developing myocardial cells of hamster. However, initial localization of desmin and vimentin in early embryonic heart has not been reported in detail. By quick-freeze deep-etch method a loose network of intermediate filaments was revealed to exist surrounding myofibrils. In this report, immunocytochemical localization of desmin and vimentin is visualized in early stages of chick embryonic my ocardium.

Chick embryos, Hamburger-Hamilton (H-H) stage 8 to hatch, and 1 day old postnatal chicks were used in this study. For immunofluorescence study, each embryo was fixed with 4% paraformaldehyde and embedded in Epon 812. De-epoxinized with sodium methoxide, semithin sections were stained with primary antibodies (rabbit anti-desmin antibody and anti-vimentin antibody)and secondary antibody (RITC conjugated goat-anti rabbit IgG).
The Z band in skinned fibers of a slow skeletal muscle
Authors: Goldstein Margaret A. , Schroeter John P.
Published: June 2020

We have shown that Z bands in rat soleus muscles exhibit two structural states. The small square (ss) form is seen in relaxed intact muscle and the basketweave (bw) form is seen in maximally activated muscle (tetanized and in rigor). The Z spacing measured in the bw form is 20% larger than that in the ss form. The dimensions of the two Z band lattice forms in these muscle states are independent of A band spacing. Skinned fibers have been used to assess various muscle properties and expansion of the A band has been observed in some cases. We have examined the effects of chemical skinning on Z band form and dimension and A band spacing in relaxed muscle.

Rat soleus muscles were dissected and tied to applicator sticks and removed to a solution of 1% Triton X-100 in lOmM PIPES buffer with 5mM EGTA and lOmM ATP overnight at 4°C, then rinsed with the PIPES buffer with EGTA and ATP only for 4 hours.
Electron microscopic analysis of the microtubular structure of HEp-2 cells
Authors: Kellermayer Miklós S.Z. , Henics Tamás , Szücs György , Pollack Gerald H.
Published: June 2020

The HEp-2 cell line was first established by Moore, et al. and today it serves as a widely used subject for a large variety of microbiological and cell-biological experiments. A tubular structure of unknown origin and function has been described first in virally infected, and later in uninfected HEp-2 cells. However, this tubular structure has not been further analyzed. We performed transmission (TEM) and whole cell mount electron microscopic studies of monolayer HEp-2 cells to morphologically describe the structure and relate it to other organelles of the cell.

For embedding, HEp-2 cells were grown on glass coverslips in Eagle's minimal essential medium. 24 hours after passage, three types of procedures were carried out: (1) The cells were fixed with 2.5% glutaraldehyde in 0.14 M Na-cacodylate buffer. (2) The cells were treated for 10 min. with Hank's solution containing 0.2% Brij-58 and 2.5% glutaraldehyde. Fixation was completed with 2.5% glutaraldehyde in 0.14 M Na-cacodylate. (3) Cells were treated with 0.2% Brij-58 in Hank’s solution for 10 min. Subsequently, they were fixed with 2.5% glutaraldehyde in 0.14 M Na-cacodylate.
Fine structure of the rectal papillae of the oriental fruuit fly,Dacus dorsalisHendel (Tephritidae, Diptera Insecta)
Authors: Wen-Yung Len , Lin Mei-Jung
Published: June 2020

Four cone-shaped rectal papillae locate at the anterior part of the rectum inDacus dorsalisfly. The circular base of the papilla protrudes into the haemolymph (Fig. 1,2) and the rest cone-shaped tip (Fig. 2) inserts in the rectal lumen. The base is surrounded with the cuticle (Fig. 5). The internal structure of the rectal papilla (Fig. 3) comprises of the cortex with the columnar epithelial cells and a rod-shaped medulla. Between them, there is the infundibular space and many trabeculae connect each other. Several tracheae insert into the papilla through the top of the medulla, then run into the cortical epithelium and locate in the intercellular space. The intercellular sinuses distribute in the posterior part of the rectal papilla.

The cortex of the base divides into about thirty segments. Between segments there is a radial cell (Fig. 4). Under the cuticle, the apical cell membrane of the cortical epithelium is folded into a regular border of leaflets (Fig. 5).