bluuebunny/crossref_metadata_2025 huggingface dataset:
Digital video and laser microscopic studies of developmental events in living neurons and nervous systems
Authors: Smith S. , Forscher P. , Cooper M. , Waxman A.
Published: June 2020

Motility and structural change are extremely important aspects of nervous system development. We are working to refine digital video and laser confocal light microscopic methods for visualization of neuronal motility both in situ and in vitro. Our efforts are oriented especially toward the collection of digitally-enhanced time-lapse movies, for which purpose we especially favor the use of the optical memory disc video recorder. It is our feeling that such dynamic observations are yielding insights not obtainable from examination of fixed specimens. Work to be discussed includes video microscopic studies of the basic motility mechanisms of neuronal growth cones in vitro and laser confocal observations of mammalian CNS growth cone motility and cell proliferation and migration events in slices of embryonic rat cortex.

As cell markers for the laser confocal studies, we have used a variety of fluorescent compunds including the lipid stain Dil(C18-3) and the Ca indicator Fluo-3. The latter compound is of special interest in revealing the cytosolic calcium transients associated with various forms of cell signalling as well as making cell structure and motility visible. We have found the use of slow-scan, single-sweep laser image acquisition especially valuable in the collection images from living, moving cells, as this mode circumvents the blurring of moving specimen details associated with the prolonged integration times of low-light-level video data acquisition and multi-sweep averaging in laser scanning systems.
Tissue ferritin concentration in breast carcinoma: A possible marker of proliferative index (A cytosol and Electron Microscopic study)
Authors: Elliott Robert L. , Ledford Lindsay , Werneke Debra , Elliott Mary C.
Published: June 2020

Elevation of serum ferritin in carcinoma of the breast is well known and levels suggest the severity of the disease. Weinstein et al. found that malignant tissue had six times the ferritin concentration as did benign breast tissue. Malignancies with the highest ferritin concentration were histologically more anaplastic suggesting that the major site of the increased ferritin was the malignant epithelium. They postulated that ferritin may be a marker of neoplasia.

To confirm and expand on this study and increase our knowledge on the role of transferrin receptors and iron metabolism in breast cancer, we measured tissue ferritin in cytosol extracts in 44 mammary carcinomas and 11 benign breast tissues. All specimens were examined ultrastructurally. Ferritin concentrations in malignant specimens ranged from 192-12,308 ng/mcp while in benign tissue it ranged from 33-296 ng/mcp. Electron microscopy showed abundant ferritin deposits in malignant epithelium while deposits were sparse in benign epithelium and connective tissue stroma.
Deficiency of leukocyte adhesion molecules
Authors: Hawkins H.K. , Smith C. W. , Anderson D. C.
Published: June 2020

Study of patients congenitally deficient in expression of the CD11/CD18 dimer has clarified the fundamental role of these transmembrane glycoproteins of the integrin family in multiple functions of polymorphonuclear leukocytes (PMNL). Deficient expression is associated with severe deficiency of PMNL aggregation, adhesion to protein-coated surfaces and endothelial monolayers, locomotion, chemotaxis, phagocytosis and bacterial cell killing. Severely affected patients develop leukocytosis and die in infancy of overwhelming infection. A patient we studied at autopsy died from septicemia and pneumonia. There was striking lack of any acute inflammatory response near sites of necrosis and infection. In the lungs, in contrast, a distinct but deficient acute focal inflammatory response was observed.

We used colloidal gold immunolabeling to study the subcellular localization of the CD11/CD18 adhesion molecules in human PMNL and their expression and movement on cell surfaces after stimulation with the chemotactic peptide formyl-met-leu-phe (fMLP). Human PMNL in pyrogen-free PBS were fixed for lh in 1% glutaraldehyde, dehydrated to 70% ethanol and embedded in LR White resin at 55°C. A polyclonal rabbit antiserum recognizing CD18 and absorbed with CD18-deficient PMNL was used to label sites of storage in thin sections, using 5-nm goat-anti-rabbit-IgG gold. We compared sites labeling for CD18, lactoferrin and gelatinase in adjacent thin sections. For surface labeling, cells were fixed in 1% glutaraldehyde for lh, exposed to OKM1 monoclonal antibody, washed and labeled using 36-nm rabbit-anti-mouse-IgG-gold. Controls for both studies included substitution of other or nonspecific primary and secondary antibody. Cells were attached to polylysine-coated coverslips, critical-point dried in CO2, carbon coated and examined by scanning EM using backscattered electron imaging. Other cells were exposed to f(ab’)2 fragments of OKMl and to f (ab’)2-rabbit-anti-mouse prior to fixation, followed by labeling with 36-nm goat-anti-rabbit-gold, or exposed to f(ab’)2-OKMl conjugated to 36-nm gold particles prior to fixation.
Correlative light and electron microscopic cytochemical demonstration of biodegradation of polyester implants
Authors: Hanker J. S. , Yates P. , Anderson E. , Giammara B.
Published: June 2020

There are situations in which an implant material is required only temporarily, e.g., when it is needed as a scaffold to support a nondegradable material until the latter can be incorporated by the body; or when it is required for the uniform release of a therapeutic agent over a long period of time. Biodegradable organic polyesters such as poly(glycolic acid), poly(lactic acid) or their copolymers are of particular interest for such purposes because they undergo hydrolysis (Fig. 1) to form glycolic or lactic acids which are metabolites normal to the body. Recent studies were performed on the use of polyglactin 910 mesh (VicrylR, a lactic-glycolic acid polymer) as a conduit (sleeve) to guide the reconnection of regenerating fibers of the proximal stump of rat sciatic nerve, across an llmm gap, with fibers in the distal stump of the interrupted nerve. For experimental set-up and procedure see reference 3.Gross examination of the repaired sciatic nerves at 1 month showed complete reconnection of the stumps but the VicrylRmesh was not apparent.When cryostat or paraffin sections were made of repaired nerve there was some disruption of structure due to movement of the polyester filaments through the tissue during the sectioning process. This could be avoided by embedding the specimens in epoxy resin and making semi-thin sections with a glass knife. Staining of the sections with either toluidine blue or the PATS reaction (a light and electron microscopy variation of the Keriodic acidSchiff PAS, reaction depositing silver, 5) revealed numerous VicrylRfilaments in different degradation stages (Figs. 2-4). The toluidine blue stained filaments were reminiscent of earlier results on methylene blue-stained muscle sections that contained cross-sections of polyglactin 910 braided sutures.
Alterations in surface substructure and degranulation of subapical cytoplasmic granules by mezereinu (MZ) in toad urinary bladder epithelia
Authors: Mia A. J. , Oakfoid L. X. , Yorio T.
Published: June 2020

The increase in transepithelial water flow induced by antidiuretic hormone (ADH) occurs through the ADH V2 receptor, and includes the stimulation of adenylcyclase, an increase in cAMP synthesis and the activation of protein kinase A. These biochemical events in amphibian urinary bladder tissues are reported to be accompanied by apical membrane transformations including the induction of numerous microvilli, and an increase in apical surface area as a result of incorporation of water channels. Amphibian epithelia also contain an ADH V1 receptor coupled to phosphoinositide metabolism and inositol phosphate release. Recently, it was demonstrated that mezerein (MZ), a non-phorbol activator of protein kinase C (PKC)increased transepithelial water flow when added to mucosal surface. The magnitude of water transport was less and occurred over a longer period of time than compared to ADH-stimulated tissues (7,and also Table 1). However, there is little or no information available on ultrastructural studies linking the events due to the ADH V1 receptor and osmotic water flow. Presently, we report on morphological and cytological observations which suggests that the ADH V1 receptor cascade may play a role in the insertion of water channels into the apical membrane possibly through the activation of PKC. The present study utilizes mezerein (MZ), a non-phorbol activator of protein kinase C (PKC) to examine the effect of MZ on epithelial cytomorphology.
Septate-like junction of neoplastic cells in myxopapillary ependymoma of the human filum terminale
Authors: Ho Khang-Loon
Published: June 2020

Since the first description of septate desmosomes by Wood the term septate junction has been used to describe various junctional structures where in transverse section the intercellular space is partitioned by more or less regularly spaced septa. While septate junction is a common intercellular feature of invertebrate epithelium, vertebrates do not possess septate junctions. There have been occasional reports in the literature of septate-like structures in vertebrate tissues, mainly in nervous tissue and between epithelial cells.

In an ultrastructural study of three cases of myxopapi1lary ependymoma of the fi1um terminale, structures similar to septate junction were observed in two cases. They were circumferential bands around the finger-like processes of neoplastic cells wherein slightly widened 30-40 nm intercel1ular spaces (as compared to 15-20 nm in non-junctional region) were transversed at regular intervals by parallel septa resulting in characteristic ladder-like appearance (Fig.1,2). The electron-dense septa, 30-40 nm in cross-length and 20-30 nm in width, were arranged in a periodicity of 40-50 nm. The septa connected to the outer leaflets of the apposing cytoplasmic membranes which appeared undisrupted. Most junctions were short the longest one contained 15 septa in a length of 1.4 um. Similar septa in the same intervals were also noted between the cytoplasmic membrane and the investing basement membrane(Fig.3). Junctional complexes such as zonula adherens and gap junction were often present in the vicinity(Fig.4). Septate-like junctions have not been described in previous studies of myxopapi11ary ependymoma nor in other human brain tumors. Similar junctions have not been observed in the normal conus medullaris nor fi1um terminale. They may represent a specific intercel 1ular feature of myxopapi11ary ependymoma.
Localization of Bacillus thuringiensis Kurstaki toxin binding sites by means of BT toxin gold probes
Authors: Armbruster B. L. , della-Cioppa G. R.
Published: June 2020

Strains of the bacterium Baci11us thuringiensis (Bt) produce toxins which, when ingested by susceptible larvae, function as a microbial insecticide. In addition to this broad spectrum activity against lepidopteran and dipteran larvae, the toxin has no effect on humans, animals and beneficial insects. As a corollary to our effort to genetically engineer Bt toxin production in major crop plants, we have studied the basic toxin-membrane interactions in the midgut region of Manduca sexta (tobacco hornworm) larvae by means of Bt toxin coated colloidal gold probes.

The midgut of 2nd instar M.sexta larvae was removed, incised along its length, inverted and incubated for minimal time in phosphate buffer. Bt gold probes were produced by incubating 15nm diameter colloidal gold in the purified 68kDa active toxin fragment. Dissected midgut was placed in drops of Bt toxin gold solution for 1, 4, 8, 16, and 32min. The samples were fixed for 3hr in 2% glutaraldehyde, post-fixed in 1 % osmium tetroxide, dehydrated in a graded ethanol series and embedded in Embed 812.
Quantitative immunogold localization of laminin and Type IV collagen in gastric mucosa
Authors: Smith Kathryn B. , Schmidt Karmen L.
Published: June 2020

Upon exposure to alcohol, necrotic lesions form in the gastric mucosa and surface epithelial cells detach leaving an exposed basal lamina. Since the basal lamina acts as a scaffold during restitution and repair in other organs, it may play a significant role in gastric tissue recovery, directing epithelial cell migration to the denuded surface. Type IV collagen and laminin are two of the major macromolecules which compose the basal lamina type IV collagen furnishes strength and flexibility, while laminin functions in cell adhesion and migration. However, despite the likely role of the basal lamina in gastric tissue restitution and repair, it has not been studied as a target of alcohol injury. The technique of TEM immunocytochemistry enabled us to quantify the response of the basal lamina to acute alcohol through examining ensuing changes in immunogold labelling (IG) to type IV collagen and laminin in rat gastric mucosa.

Female Sprague-Dawley rats weighing 180-200 g were fasted overnight. The next day 1 ml of 50%, 100% ethanol (50E, 100E) or saline (S) was administered via orogastric tube. After either 5 or 60 min, anesthetized animals were perfused with fixative. For IG studies, gastric tissues were fixed with 1% paraformaldehyde, which is known to preserve the antigenicity of basal lamina components. Since ultrastructural characteristics were not optimal with this fixative, however, parallel TEM studies were also executed using standard techniques. Tissues for IG studies were embedded in Lowicryl K4M at -30°C, sectioned, and incubated in anti-type IV collagen or anti-laminin followed by protein A-gold. Uranyl acetate-stained sections were viewed on a JEOL 1200EX TEM at 60kV. Micrographs from the interfoveolar, pit and gland isthmus regions of the gastric mucosa were examined at 30,000x, the area of lamina densa measured and number of gold particles per area counted. Results were analyzed using analysis of variance and Student-Newman-Keuls’ test for multiple comparisons (α= 0.05).
The binding of potassium to proteins and to nucleic acids best explains the potassium ion content of cells
Authors: Cameron I. , Haskin C. , Hunter K. , Smith N. K. R.
Published: June 2020

It is generally assumed that the Mg2+ dependent Na+, K+ ATPase “pump” located within the plasma membrane is the primary mechanism responsible for both the generation and the maintenance of the well known intra- to extracellular Na+ -K+ gradients in mammalian cells. This pump operates to pump out cellular Na+ and pump in K + . It is also generally assumed that Na+ and K+ in the cells are free in solution. If these two assumptions are true one would expect that the Na+ content of a post-mitotic tissue cell population should be negatively correlated to and should be a major predictor of K+ content; and that detergent (Brij 58) permeabilization (disruption of the plasma membrane) would allow free passage of Na+ and K+ down their concentration gradients. This would lead to the rapid loss of cellular K+ and to a rapid increase of cellular Na+. Our recent experiments (Cameron et al. 1988) showed that Brij permeabilized cells do not rapidly lose K+ as predicted by the assumption that K+ is free in the cell.

If, on the other hand, most of the cellular K+ is bound to proteins (the major source of total cellular sulfur) and to nucleic acids (the major source of total cellular phosphorus) then the S and P content should he positively correlated to and should be the major predictor of K+ content. Analysis of variation in ion and element content of the cytoplasm of many different cardiac myocytes (n = 90) of the ventricle wall by electron probe x-ray microanalysis in thin freeze-dried cryosections allowed a test of this hypothesis (Fig. 1 and Table 1). Multiple regression analysis showed both S and P content to be positively correlated to and the major predictor of cellular K+ content. In normal post-mitotic cardiac myocytes P and S together explained 70-77% of the variation of cellular K+ content. Inclusion of the Na+ content data to the P and S data did not add further to the prediction of K+ content. These results are interpreted to indicate that cells maintain the majority of their K+ bound to protein and to nucleic acid and that “the pump” is not the primary determinant of the maintenance of cellular K+ content.
Novel epoxy/anhydride alternatives for biological electron microscopy: Physical and performance characteristis of embed 812 and LX 112 in combination with NSA/NMA/DMAE
Authors: Mascorro Joe A. , Kirby Gerald S.
Published: June 2020

Embedding media based upon an epoxy resin of choice and the acid anhydrides dodecenyl succinic anhydride (DDSA), nadic methyl anhydride (NMA), and catalyzed by the tertiary amine 2,4,6-Tri(dimethylaminomethyl) phenol (DMP-30) are widely used in biological electron microscopy. These media possess a viscosity character that can impair tissue infiltration, particularly if original Epon 812 is utilized as the base resin. Other resins that are considerably less viscous than Epon 812 now are available as replacements. Likewise, nonenyl succinic anhydride (NSA) and dimethylaminoethanol (DMAE) are more fluid than their counterparts DDSA and DMP- 30 commonly used in earlier formulations. This work utilizes novel epoxy and anhydride combinations in order to produce embedding media with desirable flow rate and viscosity parameters that, in turn, would allow the medium to optimally infiltrate tissues. Specifically, embeding media based on EmBed 812 or LX 112 with NSA (in place of DDSA) and DMAE (replacing DMP-30), with NMA remaining constant, are formulated and offered as alternatives for routine biological work.

Individual epoxy resins (Table I) or complete embedding media (Tables II-III) were tested for flow rate and viscosity. The novel media were further examined for their ability to infilftrate tissues, polymerize, sectioning and staining character, as well as strength and stability to the electron beam and column vacuum. For physical comparisons, a volume (9 ml) of either resin or media was aspirated into a capillary viscocimeter oriented vertically. The material was then allowed to flow out freely under the influence of gravity and the flow time necessary for the volume to exit was recored (Col B,C; Tables). In addition, the volume flow rate (ml flowing/second; Col D, Tables) was measured. Viscosity (n) could then be determined by using the Hagen-Poiseville relation for laminar flow, n = c.p/Q, where c = a geometric constant from an instrument calibration with water, p = mass density, and Q = volume flow rate. Mass weight and density of the materials were determined as well (Col F,G; Tables). Infiltration schedules utilized were short (1/2 hr 1:1, 3 hrs full resin), intermediate (1/2 hr 1:1, 6 hrs full resin) , or long (1/2 hr 1:1, 6 hrs full resin) in total time. Polymerization schedules ranging from 15 hrs (overnight) through 24, 36, or 48 hrs were tested. Sections demonstrating gold interference colors were collected on unsupported 200- 300 mesh grids and stained sequentially with uranyl acetate and lead citrate.
Sampling and analysis of the air-water interface with SEM and EDS of microlayers
Authors: Smith Randall W. , Dash John
Published: June 2020

The structure of the air-water interface forms a boundary layer that involves biological ,chemical geological and physical processes in its formation. Freshwater and sea surface microlayers form at the air-water interface and include a diverse assemblage of organic matter, detritus, microorganisms, plankton and heavy metals. The sampling of microlayers and the examination of components is presently a significant area of study because of the input of anthropogenic materials and their accumulation at the air-water interface. The neustonic organisms present in this environment may be sensitive to the toxic components of these inputs. Hardy reports that over 20 different methods have been developed for sampling of microlayers, primarily for bulk chemical analysis. We report here the examination of microlayer films for the documentation of structure and composition.

Baier and Gucinski reported the use of Langmuir-Blogett films obtained on germanium prisms for infrared spectroscopic analysis (IR-ATR) of components. The sampling of microlayers has been done by collecting fi1ms on glass plates and teflon drums, We found that microlayers could be collected on 11 mm glass cover slips by pulling a Langmuir-Blogett film from a surface microlayer. Comparative collections were made on methylcel1ulose filter pads. The films could be air-dried or preserved in Lugol's Iodine Several slicks or surface films were sampled in September, 1987 in Chesapeake Bay, Maryland and in August, 1988 in Sequim Bay, Washington, For glass coverslips the films were air-dried, mounted on SEM pegs, ringed with colloidal silver, and sputter coated with Au-Pd, The Langmuir-Blogett film technique maintained the structure of the microlayer intact for examination, SEM observation and EDS analysis were then used to determine organisms and relative concentrations of heavy metals, using a Link AN 10000 EDS system with an ISI SS40 SEM unit. Typical heavy microlayer films are shown in Figure 3.
Transperineurial vessels in the peripheral nerves of humans
Authors: Beggs J.L. , Johnson P.C. , Cleary C. , Watkins C.J.
Published: June 2020

Nerve fibers in peripheral nerve (PNS) are grouped into fascicles. The perineurial (PN) sheath forms the outer cover of the fascicle and separates the epineurium from the endoneurial compartment which contains the conducting units (nerve fibers) of the PNS. The PN sheath not only serves as a physical protective layer for the vulnerable nerve fibers but also plays a role in maintaining a unique endoneurial environment with respect to endoneurial pressure and chemical composition. All blood vessels that serve the capillary plexuses of the endoneurium pass through the PN sheath. In this study on human nerves, we describe some of the anatomical relationships between PN cells and vascular elements as they pass through the PN sheath. A better understanding of the anatomical arrangement of transperineurial vessels might help explain the pathogenesis of some of the ischemic neuropathies.

Segments of nerves (lumbosacral plexus, sciatic, posterior tibial, peroneal and sural) from autopsy and sural nerves from biopsy were prepared for electron microscopy. One μm serial cross-sections of nerve fascicles were stained with paragon. Thin sections for EM were taken at desired levels in the thick-section serial sequence. 3-D reconstruction of 1 μm sections was accomplished using a program provided by Dr. Kinnamon of The High Voltage Electron Microscopy Lab in Boulder, Colorado.
High-resolution scanning electron microscopy in biology
Authors: Tanaka Keiichi
Published: June 2020

With the development of scanning electron microscope (SEM) with ultrahigh resolution, SEM became to play an important role in not only cytology but also molecular biology. However, the preparation methods observing tiny specimens with such high resolution SEM are not yet established.

Although SEM specimens are usually coated with metals for getting electrical conductivity, it is desirable to avoid the metal coating for high resolution SEM, because the coating seriously affects resolution at this level, unless special coating techniques are used. For avoiding charging effect without metal coating, we previously reported a method in which polished carbon plates were used as substrate. In the case almost all incident electrons penetrate through the specimens and do not accumulate in them, when the specimens are smaller than 10nm. By this technique some biological macromolecules including ribosomes, ferritin, immunoglobulin G were clearly observed.

Unfortunately some other molecules such as apoferritin, thyroglobulin and immunoglobulin M were difficult to be observed only by the method, because they had very low contrast and were easily damaged by electron beam.
Application of low-voltage, high-resolution SEM in the study of complex intracellular structures
Authors: Ris Hans
Published: June 2020

Cellular architecture is a dynamic web of complex macromolecular assemblies accomplishing the diverse functions of a cell. Conventional electron microscopy on ultrathin sections or negatively stained preparations can provide little information on more extended three dimensional assemblies. High voltage and intermediate voltage TEM provide high resolution in much thicker specimens but are limited by problems of contrast and overlap of structures. In recent years new SEMs have become available that provide the high topographic contrast and three dimensionality of SEM at a resolution comparable to conventional TEM. I have used the low voltage high resolution SEM Hitachi S-900 at the Madison IMR and shall show some examples that illustrate the usefulness of LVSEM in structural cell biology. Most striking is the new information obtained about the nuclear pore complex (NPC). This structure is extremely important in controlling the selective and unidirectional transport of large molecules into and out of the nucleus.
Fibrinogen receptor movement on the ventral surface of platelets
Authors: Goodman S.L. , Lai Q.J. , Park K. , Albrecht R.M.
Published: June 2020

The fibrinogen receptor (glycoprotein Ilb/IIIa) on the blood platelet surface is important in platelet adhesion, cohesion, and thrombus formation. Previously, we showed that the fibrinogen receptors of surface adherent platelets become motile once ligand is bound. Subsequent to the binding of fibrinogen to GP Ilb/IIIa, either as soluble fibrinogen or conjugated to colloidal gold, the receptor-ligand complex moves from areas overlying the peripheral regions of the platelet to regions overlying more central areas. This movement occurs in relatively straight lines and requires the intact actin and myosin cytoskeleton. This receptor’s motility may be involved in clot retraction and thrombus formation. In vivo, blood platelets contact surfaces coated with fibrinogen and other proteins. Since GP Ilb/IIIa is an adhesion receptor we wished to determine if platelet contact with a fibrinogen coated surface would initiate movement of receptors on the surface-adherent or ventral face of the platelets, and if platelets move or redistribute surface adsorbed fibrinogen.
Immunocytochemical localization of lung surfactant proteins by cryoultramicrotomy and freeze-substitution embedding
Authors: Voorhout W.F. , Veenendaal T. , Haagsman H.P. , Slot J.W.
Published: June 2020

Lung surfactant is composed primarily of phospholipids but contains also about 10% proteins. Its main function is to decrease alveolar surface tension at low transpulmonary pressure to prevent alveolar collaps. Surfactant is stored in lamellar bodies in alveo1lar type II cells and is transformed after secretion in tubular myelin, a lattice-like structure.

We investigated the biogenesis of surfactant and the pathways that the large hydrophilic surfactant protein A (SP-A) and the small hydrophobic surfactant protein B (SP-B) follow in human lung by using two different immunocytochemical techniques, the cryo-ultramicrotomy method and a new post-embedding method.

In the non-embedded, ultrathin cryosections of the lung, prepared and immunolabeled for SP-A and SP-B as described before, it was impossible to achieve a satisfying preservation of lipid-rich structures like lamellar bodies (Fig. 1 and 2). Nevertheless SP-A and SP-B are detected in remnants of lamellar bodies (Fig. 1 and 2) and SP-A is further found to be present throughout the biosynthetic route, in some multivesicular bodies and over the apical plasma membrane of type II cells (Fig. 1).
Nucleolus-like bodies (Nuages) and annulate lamellae in spermatogonia of fish-tilapia, Oreochromis niloticus
Authors: Guha T. , Prentis P.F.
Published: June 2020

Type A spermatogonia in tilapia (Oreochromis ni1oticus) have been studied by electron microscopy. These are stem cells from which spermatogenesis beings in this species. In this paper we report presence of two cytoplasmic organelles, annulate lamellae and nucleolus-like bodies (nuages), in type A spermatogonia in O. niloticus.

Testes were fixed in 2% gluteraldehyde for 4 hrs. at 4°C and then in 1% osmium tetroxide for 1 hr. at 4°C, both in 0.1M cacodylate buffer (pH 7.4). Fixed tissues were processed in the conventional way for electron microscopy. Thin sections of tissues were stained by uranyl acetate and lead citrate. These were examined in a Carl Zeiss electron microscope operated at 40kV.

Nucleolus-like bodies (nuages) have been reported in rat spermatocytes, early postimplantation rat embryos, fish and amphibian oocytes and guppy (fish) spermatogonia. Annulate lamellae have been found in fish spermatogonia and oocytes. Type A spermatogonia (Figs. 1,2,3,4) in O. niloticus show presence of nucleolus-like bodies (nuages) and annulate lamellae in the cytoplasm.
Direct nucleotide mapping of human chromosomes by imaging Secondary Ion Mass Spectometry
Authors: Hallégot P. , Girod C. , Le Beau M.M. , Levi-Setti R.
Published: June 2020

The relationship between chromosome banding patterns obtained by a variety of staining methods (G,Q,R, and C) and the actual nucleotide content of the different bands, has been the subject of extensive investigations over the past 20 years and has been critically reviewed. Although a number ofin vitroexperiments have shown preference of certain stains for specific bases, the presence of proteins in chromosomes and their interaction with the DNA, has to some extent obscured a direct correlation between banding patterns and base content. Even the widely useful differential staining following substitution of the thymidine in DNA by the analog bromodeoxyuridine (BrdU), is not independent of the presence of proteins. Clearly, it would be desirable to examine physical methods capable of assessing DNA base composition in chromosomes, free of chemical interactions. One of these methods, already successfully applied involves the3H-labelling of nucleotides and subsequent detection by autoradiography. An alternative approach, the subject of this report, is capable of attaining much improved spatial resolution and consists of the detection of isotope-labelled nucleotides by secondary ion mass spectrometry (SIMS).
Structure and Function of Nonorthodox Chromatins: II. Preliminary Results on the Localization of Helix-Stabilizing Histone-like Proteins of Thermophilic Archaebacteriae
Authors: Bohrmann B. , Arnold-Schulz-Gahmen B. , Villiger W. , Gyalog R. , Kellenberger E. , Searcy D. , Suryanarayana T.
Published: June 2020

Sulfolobus acidocaldarius and Thermoplasma acidophilum prefer extreme living conditions of low pH (<3) and/or high temperature (>60°C) for optimal growth. To resist thermal denaturation of DNA it appears to be associated with helix stabilizing proteins.

The chromosomal position of archaebacterial cells (the nucleoid) can be detected as ribosome-free space in which the DNA is located, as is shown by immunocytochemistry (fig. 1a,b). Similar to most eukaryotes the protein- DNA-ratio of their chromatin is relatively high (approx. 1:1 weight by weight), so that no aggregation occurs during dehydration for electron microscopy (fig. 2). Despite this aggregation-insensitivity, (2 and poster 1) the distribution of immunocytochemical label of the DNA bound proteins of Sulfolobus acidocaldarius (hsp's: A,B,C,C') is not exclusively connected with the bulk DNA of the nucleoid; only one of these proteins (C') seems to be associated with the bulk DNA.
Electronmicroscopic localization of ribonucleic acids in ciliate Bursaria truncatella during cell division
Authors: Popenko Vladimir , Cherny Natalya , Yakovleva Maria
Published: June 2020

Highly polyploid somatic nucleus (macronucleus) of ciliate Bursaria truncatella under goes severe changes in morphology during cell division. At first, macronucleus (Ma) condences, diminishes in size and turns perpendicular to longitudinal axis of the cell. After short time, Ma turns again, elongates and only afterwards the process of division itself occurs. The biological meaning of these phenomena is not clear.

Localization of RNA in the cells was performed on sections of ciliates B. truncatella, embedded in “Lowicryl K4M” at various stages: (1) before cell division (Figs. 2,3); (11) at the stage of macronucleus condensation; (111) during elongation of Ma (Fig.4); (1111) in young cells (0-5min. after division). For cytochemical labelling we used RNaseAcolloidal gold complexes (RNase-Au), which are known to bind to RNA containing cell ularstructures with high specificity. The influence of different parameters on the reliability and reproducibility of labelling was studied. In addition to the factors, discussed elsewhere, we found that the balance of mono- and bivalent cations is of great significance.
Low-Viscosity, Intermediate-Viscosity, and High-Viscosity Alternatives in the Family of Epoxy Resin Embedding Media...... Your Choice
Authors: Mascorro Joe A. , Kirby Gerald S.
Published: June 2020

The epoxy resins have been successfull as embedding media because of their demonstrated ability to preserve morphological detail, minimally extract cell components, infiltrate tissues, and polymerize uniformly without inducing shrinkage artifacts. These embedding media also produce blocks with favorable sectioning and staining qualities and sections capable of withstanding the heat and vacuum encountered in the electron microscope column. Many epoxy resins are available today and several have been evaluated in past works. The versatility of these resins allows them to be “matched” with various embedding media components (ie., DDSA, NMA, NSA, DMP-30, DMAE) in order to design a final embedding product with a predetermined viscosity. This work evaluates the resins Embed 812, LX112, Polybed 812, Eponate 12, and the traditional Epon 812 in “low”, “intermediate”, or “high” viscosity alternatives formulated with different combinations of anhydrides and catalysts.

The anhydride NSA and the tertiary amine catalyst DMAE are more fluid and less viscous than their counterparts DDSA and DMP-30.
Neuron model system: Correlative confocal laser scanned microscopy and membrane physiology
Authors: Szarowski Donald H. , Fejtl Michael , McCauley Paul , Carpenter David O. , Turner James N.
Published: June 2020

Confocal laser scanning microscopy (CLSM) has been used to correlate morphology and membrane physiology in cultured neurons, providing a model system for studying physiologic and pathologic conditions. Ion channels are studied by patch-clamp methods as a function of receptor stimulation and toxic excitatory amino acids, including those implicated in Alzheimer’s dementia. Glial cells are often closely associated with the neurons, and are difficult to detect in living cultures due to the relative sizes of glia and neurons (5-20 μm versus 125 μm), compounded with the fact that they are thick phase objects. Groups of glia can also be confused with neurons. Thus it is difficult to select appropriate cells and/or cell regions for patch-clamping. We are correlating physiology and conventional light microscopy with CLSM to determine the role of glia, and neuron surface geometry on the ability to establish Gigaohm membrane-micropipette seals. Morphology of the system as observed by CLSM is presented here.
Correlated Confocal and Intermediate Voltage Electron Microscopy of Myofibrillogenesis in Muscle Cells: Methods and Preliminary Results
Authors: Ishikawa Harunori , Peachey Lee D. , Schultheiss Thomas , Holtzer Howard
Published: June 2020

Immunofluorescence studies have helped to elucidate the process of molecular assembly into cross-striated myofibrils in cultured cardiac and skeletal muscle cells by localizing accurately identified proteins. However, our understanding of how such immunofluorescence images are related to fine structural organization is limited. High and intermediate voltage electron microscopes can image relatively thick specimens, including whole cells, and confocal scanning laser fluorescence microscopy provides thin optical section images of similarly thick specimens. It seemed natural for us to combine these two approaches in a correlated electron and light microscopic analysis of myofibrillogenesis in cultured cardiac muscle cells.

Cardiac myocytes from 7-9 day chick embryos, cultured for 3-5 days on glass coverslips carrying gold EM grids covered with formvar/carbon films, were fixed with 2% formaldehyde for 3 min., permeabilized with 5% Triton for 30 min., and stained with two antibodies conjugated with FITC and rhodamine. These specimens were first observed by epi-fluorescence and then by confocal laser scanning microscopy (Sarastro, Stockholm, SWEDEN).
Reorganization of the Actin Cytoskeleton in Human Fibroblasts Induced by PDGF and Neomycin
Authors: Bell Paul B. , Lindroth Margaretha , Hedberg Karin , Fredriksson Bengt-Arne , Wasteson Åke
Published: June 2020

Among the earliest effects on cells of platelet derived growth factor (PDGF), a growth-stimulating factor normally occurring in blood, are stimulation of lamellipodia formation and induction of changes in the actin cytoskeleton. We have investigated these phenomena by time lapse video (TLV) and fluorescence light microscopy (FLM), and high resolution scanning electron microscopy (SEM) using two strains of human diploid fibroblasts: ICIG-7 from embryonic lung and AG1523 from foreskin, grown either on coverglasses or on carbon-stabilized Formvar-coated gold electron microscope grids. Cells were maintained in serum-free culture medium for 48 hours prior to addition of PDGF (5-20 ng/ml), neomycin sulfate (0.7-7 mM), or neomycin followed by PDGF. For TLV cells were filmed continuously before, during, and after addition of experimental reagents. For FLM and SEM cells were either fixed directly in 4% paraformaldehyde or 2.5% glutaraldehyde (GA) respectively, or extracted in 0.5% Triton X-100 in microtubule stabilizing buffer (Tsb) and then fixed.
Use of Thick Histochemical Preparations, Whole Mount Cultured Cells or Thick Sections from Embedded Tissues, for Three-Dimensional Observation by Ultrahigh Voltage Electron Microscopy
Authors: Nagata Tetsuji , Usuda Nobuteru , Ma Hongjun
Published: June 2020

We have observed very thick biological specimens, both whole mount cultured cells and thick sections from embedded tissues, which were stained with histochemical reactions for specific cell organelles, by means of high or ultrahigh voltage electron microscopy (UHVEM).

Thick histochemical specimens, both whole mount cultured cells and semithin sections from embedded tissues were used. Two cell strains in culture, one established CHO-K1 cell line and the other primary culture cells of adult Wistar rat hepatocytes were used. Culture cells were seeded onto formval coated gold meshes and incubated in CO2 incubator. CHO-K1 cells were cultured in Ham’s F12 medium containing HRP (1 mg/ml), while rat hepatocytes were in L-15 medium containing Clofibrate (0.2 mM). The cultured cells were stained with DAB reaction, dried in a critical point dryer (Hitachi HCP-1). Thick sections (0.2-1.0 μm) from DEHP fed rat livers, fixed in 2.5% glutaraldehyde, stained with DAB reaction, postfixed in 1% osmium tetroxide and embedded in Epon, or semithin Epon sections (0.2μm) from the pancreases of 3H-thymidine or 3H-uridine injected mice, fixed doubly, radioautographed with Sakura NR-H2 emulsion.
Ultrastructural Study of a differentiating human colon carcinoma cell line
Authors: Polak-Charcon Sylvie , Hekmati Mehrdad , Ben Shaul Yehuda
Published: June 2020

The epithelium of normal human colon mucosa “in vivo” exhibits a gradual pattern of differentiation as undifferentiated stem cells from the base of the crypt of “lieberkuhn” rapidly divide, differentiate and migrate toward the free surface. The major differentiated cell type of the intestine observed are: absorptive cells displaying brush border, goblet cells containing mucous granules, Paneth and endocrine cells containing dense secretory granules. These different cell types are also found in the intestine of the 13-14 week old embryo.

We present here morphological evidence showing that HT29, an adenocarcinoma of the human colon cell line, can differentiate into various cell types by changing the growth and culture conditions and mimic morphological changes found during development of the intestine in the human embryo.

HT29 cells grown in tissue-culture dishes in DMEM and 10% FCS form at late confluence a multilayer of morphologically undifferentiated cell culture covered with irregular microvilli, and devoid of tight junctions (Figs 1-3).
Morphologic alteration in the muscles of patients with hypokalemic periodic paralysis or myopathy
Authors: Shimizu T. , Muranaka Y. , Ohta I. , Honda N.
Published: June 2020

There have been many reports on ultrastructural alterations in muscles of hypokalemic periodic paralysis (hpp) and hypokalemic myopathy(hm). It is stressed in those reports that tubular structures such as tubular aggregates are usually to be found in hpp as a characteristic feature, but not in hm. We analyzed the histological differences between hpp and hm, comparing their clinical manifestations and morphologic changes in muscles. Materials analyzed were biopsied muscles from 18 patients which showed muscular symptoms due to hypokalemia. The muscle specimens were obtained by means of biopsy from quadriceps muscle and fixed with 2% glutaraldehyde (pH 7.4) and analyzed by ordinary method and modified Golgimethod. The ultrathin section were examined in JEOL 200CX transmission electron microscopy.

Electron microscopic examinations disclosed dilated t-system and terminal cistern of sarcoplasmic reticulum (SR)(Fig 1), and an unique structure like “sixad” was occasionally observed in some specimens (Fig 2). Tubular aggregates (Fig 3) and honeycomb structure (Fig 4) were also common characteristic structures in all cases. These ultrastructural changes were common in both the hypokalemic periodic paralysis and the hypokalemic myopathy, regardless of the time of biopsy or the duration of hypokalemia suffered.
Ultrastructural Changes in Oral Mucosa After Topical Application of Nicotine
Authors: Laffoon J.E. , Kremer M.J. , Squier C.A. , Lesch C.A.
Published: June 2020

The potential health risks involved with the use of smokeless tobacco have been well documented. This habit involves the placement of tobacco containing 0.2-8.0% nicotine directly on the oral mucosa. It is possible that such levels of nicotine may have a directly injurious effect; we have examined this using pig oral mucosa, which has a similar oral mucosa to that of man.

Biopsies of porcine attached gingiva, buccal (B) and floor of mouth mucosa (FM) were incubated with either 2% or 6% nicotine in phosphate buffered saline (PBS) placed on the epithelial surface at 37°C for 1 or 2 hours. Controls were incubated with PBS at the same pH (7.5%) and osmolarity (450 milliosmoles) as the nicotine solution. The biopsies were prefixed in 2% paraformaldehyde, 2.5% glutaraldehyde, washed with cacodylate buffer, post fixed with 1% solution of osmium tetroxide in cacodylate buffer, dehydrated in graded alcohols, infiltrated, embedded and polymerized in Spurrs epoxy resin. Thin sections were prepared from all specimens and examined in the transmission electron microscope (TEM).
The Effect of Electron Microscopy on Pathological Diagnosis of Neoplasm
Authors: Zhou Xiaojun , Zhang Taihe
Published: June 2020

Although electron microscopy (EM) has contributed enormously to an understanding of the structural intricacies of tumor cells, the usefulness of EM in pathological diagnoses of neoplasms has not been readily appreciated by general pathologists. In the present study, 223 tumors submitted for EM diagnosis were analyzed in an attempt to gain further information concerning the contribution of EM to tumor diagnosis.

223 neoplasms were submitted to EM for their final diagnoses when histopathological diagnoses were obscure, which represented about of the total number of surgical tumor specimens. Most specimens were taken at the time of frozen section diagnosis and a small number of tissues were originally fixed informaldehyde. All of tissues were fixed with buffered glutaradehyde, postfixed with osmium tetroxide and embedded in Epon 812. Ultrasections were made after semith in sections were examined to verify that representative tumor tissues were present. Thin sections were stained with uranium acetate and lead citrate, and examined under JEM-1200 EX electron microscope. In selected cases, mainly with neuroendocrine tumors, nickel grid-mounted sections were subjected to post embedding immunoelectron microscopy (IEM) using protein A-gold for more detailed functional classification. Protein A-gold probes were prepared as Wang and co-workers described.
Studies on Ultrastructure and Cytochemistry of Right Ventricular Endomyocardial Biopsy
Authors: Mingyu Xia , Wengshu Ma , Xiangh Wu , Dong Chen
Published: June 2020

This paper describes morphological and cytochemistry changes of endomyocardial biopsy in 94 patients. The samples of myoicardium were taken from 32 patients with dilated cardiomyopathy, and sdudied with light and electron microscop. The cytochemical studies in some of these patients were performed at histological and ultrastructure level. This paper also reported the result of myocardial biopsy in 33 patients with serious dysrythmia.

The result of this controlled study indicates that morphological assessment in both cardiomyopathy and congenital or rheumatic heart diseases showed no special changes. In patients of dilated cardiomyopathy, the decreased activity of myosin ATPase was secondary to cardial failure. The change of succinate dehydrogenase (SDHase) was not significant with light microscopy. But ultrastructural localization of SDHase activity is valuable. Its activity was found to be localized in endomembrane and ridge of the mitochondria, the activity of this enzyme was decrease, normal, or increase. SDHase activity was more intense in cardial myocytes well-functioning, or ultrastructurally well preserved hearts.
Electron Microscopic alterations in the fourth ventricle choroid plexus of rabbits after cyclophosphamide and mitomycin-C treatment
Authors: Singh D.R. , Srivastava A.K. , Bajpai V.K.
Published: June 2020

Cyclophosphamide and mitomycin-C have withstood the test of time for the treatment of many human neoplasms. While desirable results are achieved with their use, severe untoward toxic effects do occur in many body organs. Choroid plexus damage in the brain, varying from severe odema and necrosis of individual villi to haemorrhagic infarcts, are reported by Levine and Sowinski (1973) and Wenk et al (1979) after cyclophosphamide administration in rats. These authors noticed the choroid plex us of the fourth ventricle most susceptible for the drug injury. Separate groups of adult male and female rabbits were given single intraperitoneal injections of cyclophosphamide (200mg/kg) and mitomycin-C(0.1 mg/kg). After 48hrs, the control and treated animals were anaesthetized by using 30mg/kg sodium pentobarbital. The fourth ventricle choroid plexus was fixed in situ with buffered Karnovsky (1965) fixative (pH 7.3). After post-fixation in 1 per cent osmium tetroxide the tissues were processed for convential transmission electron microscopy.