bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of Scanning Electron Microscopy for observing glomerulus maturation in human kidney
Authors: Hyodo Toru , Naguro Tomonori , Kameie Toshio , Iino Akihiro , Miyagawa Ikuo
Published: June 2020

The ultrastructurg of the mammalian kidney has been explored by many authors, both in adult and fetal animals. As one of the most feasible means to elucidate the complicated three dimensional structure, scanning electron microscopy (SEM) has played an important role in the study of the kidney especially on the morphology of renal glomeruli. So far none of the authors has applied SEM to the human fetus for investigating the development of the glomerulus. In the present report SEM has been used to demonstrate the structure of human fetal metanephric glomeruli with special reference to the morphological differentiation of the podocyte.

Materials and Methods: The metanephric kidneys were collected from 4 human fetuses ranging from 11 to 14 weeks of gestation, 2-12 h after death following therapeutic or spontaneous abortions. All specimens were fixed with 2.5% glutaraldehyde in phosphate buffer and cut into small pieces with a razor blade.
Mechanisms of protein absorption in rat lung
Authors: Patton John S. , Gustafson Helen , Keller Gilbert-Andre
Published: June 2020

There is great interest in developing non-injectable methods for delivering recombinant proteins. The alveolar epithelium of rat lung is permeable to some proteins and it has been hypothesized that the extensive vesicle system of the Type 1 epithelial cells is the carrier mechanism for the transcytosis of proteins. To test this hypothesis, rats were exposed for 20 minutes to aerosols of recombinant human growth hormone-hGH (hGH), ferritin, and cationized ferritin. Their lungs were immediately fixed by vascular perfusion. Ferritin treated lungs were prepared by conventional techniques and examined directly. rhGH treated lungs were either incubated with anti-hGH immunoperoxidase conjugates, or thin cryo-sectioned and the cryosections were immunolabeled with anti-hGH immunogold conjugates. Both ferritin and cationized ferritin were excluded from the plasma membrane of Type 1 cells and from the plasmalemmal vesicles still connected with the surface of Type 1 cells. By contrast, macrophage plasma membrane and surfactant both bound cationized ferritin and both Type 2 cells and macrophages internalized ferritin and cationized ferritin. Immunoperoxidase labeling of lung tissue exposed to hGH revealed a uniform layer of reaction product tightly adhered to the alveolar luminal surface.
Structural analysis of the nuclear pore complex and its constituents unveils a pronounced asymmetry of its cytoplasmic and nucleoplasmic faces
Authors: Jarnik M. , Buhle E.L. , Reichelt R. , Engel A. , Aebi U.
Published: June 2020

The nuclear pore complex (NPC) is an elaborate supramolecular organelle implicated in nucleocytoplasmic transport, enabling small molecules (diameter ≤ 9 nm) to pass freely while mediating active transport of larger molecules. By conventional transmission electron microscopy (CTEM) the NPC consists of 8 radial spokes surrounding a central channel, often appearing plugged, sandwiched between two concentric, 8-fold symmetric rings. Based on quantitative scanning trasmission EM (STEM), the intact NPC has a mass of 125 MD, the cytoplasmic ring (RC) of 32 MD, the nucleoplasmic ring (RN) of 21 MD, the plug-spoke complex (PS) of 65 MD, and the central plug (P) of 13 MD, indicating that molecularly, the NPC may easily contain 100-200 different polypeptides. Here we document the structural asymmetry of the RC and the RN by CTEM.

Nuclear envelopes (NE) were manually isolated from Xenopus laevis oocytes and spread onto a carbon/colloidon-coated EM grid with the cytoplasmic side in contact. To obtain specimens comparable to those used for STEM, chemical fixation was omitted. Standard specimens were contrasted with 0.75% uranyl formate (UF) and air-dried.
Localization of pyroantimonate-precipitable calcium in biomineralization with special reference to the calcium carbonate crystals (otoconia)
Authors: Kawamata Seiichi
Published: June 2020

Biomineralized tissues (otoconia, bones and teeth) incorporate and/or liberate a lot of Ca ion. However, the direct visualization of ionic and loosely-bound Ca has not been successful because of the hardness of these tissues. This problem was partly overcome in the present study. The pyroantimonate technique and a new demineralization method were successfully combined to visualize pyroantimonateprecipi table Ca in the organs where CaCO3 crystals are formed.

Tree frogs, with or without 0.8% CaCl2 loading, were decapitated. Their spinal columns containing the endolymphatic sac were sliced and immersed in a fixative that contained 2% glutaraldehyde and 2% K-pyroantimonate in 0.05M K-phosphate buffer (pH 7.4). Rats (2 and 8 weeks of age) anesthesized with ether were perfused via left ventricle first with saline, followed by a fixative containing 3% glutaraldehyde and 2% K-pyroantimonate in 0.1M K-phosphate buffer (pH 7.4). The otolithic organs were removed and immersed in the same fixative. Specimens of the tree frogs and rats were postfixed in 1% OsO4 and 2% K-pyroantimonate.
Electron Microscopic studies on the antennal sensilla and the pheromone-producing glands of two important oil seed pests
Authors: Jamil Kaiser , Jyothi K.N. , Prasuna A.L.
Published: June 2020

Sensory mechanisms underlying insect chemical communication have been given considerable attention in recent years. A study on the ultrastructural details of the antennal appendages and the pheromone producing structures of Achoea janata L. and Ophiusa algira were studied with scanning electron microscope in order to understand the mechanisms of olfactory perception and pheromone release.

For fixation of pheromone producing glands of A.Janata and O.Algira the posterior region of the abdomen was pressed sufficiently and flooded with carnoys fluid for complete excedation of the 9th abdominal along with the ovipositor. The above specimens as well as the antennal appendages were then fixed in 4% glutaraldehyde in phosphate buffer of 7.2 pH dehydrated in increasing grades of ethylalcohol and critical point dried. The specimens were then coated with gold in vacuum evaporator and observed in a Hitachi S-520 evaporator and observed in a Hitachi S-520 SEM operated at 20KV.
New ultrastructural findings about Borrelia burgdorferi by cryofixation, negative staining, thin sectioning and scanning techniques
Authors: Hayes S. F. , Corwin M. D. , Schwan T. G. , Dorward D. W. , Burgdorfer W.
Published: June 2020

Characterization of Borrelia burgdorferi strains by means of negative staining EM has become an integral part of many studies related to the biology of the Lyme disease organism. However, relying solely upon negative staining to compare new isolates with prototype B31 or other borreliae is often unsatisfactory. To obtain more satisfactory results, we have relied upon a correlative approach encompassing a variety EM techniques, i.e., scanning for topographical features and cryotomy, negative staining and thin sectioning to provide a more complete structural characterization of B. burgdorferi.

For characterization, isolates of B. burgdorferi were cultured in BSK II media from which they were removed by low speed centrifugation. The sedimented borrelia were carefully resuspended in stabilizing buffer so as to preserve their features for scanning and negative staining. Alternatively, others were prepared for conventional thin sectioning and for cryotomy using modified procedures. For thin sectioning, the fixative described by Ito, et al.
Gene molecular and ultrastructural study on the transforming activity of human papillomavirus genes by engineered recombinant retrovirus infection
Authors: Si Jingyi , Lee Kun , Zhang Wei , Han Ricai , Chen Lianfong , ming Zhao Wei , Song Guoxing , Liu Shide , Jia Liping , Mai Yongyan , Zeng Yi
Published: June 2020

Human papillomavirus(HPV) type 16 is the most prevalent viral type and its E6 and E7 early genes might play a key role in the carcinogenesis of cervical cancer in Chinese women. In order to elucidate the role of HPV-16 in the development of genital cancer, the present study was designed to transfect NIH3T3 cells with HPV-16 whole early genes and its E6, E7 genes separately. Besides the ordinary calcium phosphate/DNA coprecipitation technique, a newly designed recombinant retrovirus containing HPV-16 genes were used to infect cells for transfer the aim genes as shown in Fig.1.1). The cells transformed by recombinant retrovirus containing whole early gene and its E6-E7 respectively appeared more refractive, round shaped with rapid growth and loss of contact inhibition. Hundreds of cell colonies presented in cells transformed by the recombinant retrovirus after selected culture for one week, compared with the routine method, only a few colonies till 3 weeks. By using such a genomic engineering technique,the transforming activities have been shown to be most efficient. 2). The tumorigenicity of transformed cells: Transformed cells HZIP16-3T3 and HZIP16K-3T3, as well as negative controlled cells, NIH3T3 and pZIP-3T3 which was infected by recombinant retrovirus containing no HPV-16 gene, were transplanted into nude mice respectively. Tumours were formed in all mice within 15 days after inoculation of transformed cells (Fig.2). Whereas, no tumour was found in all controlled animals as long as 50 days. 3).
High-resolution analysis and probable non-viral origin of lattice structures found in rotavirus preparations
Authors: Weibel Josef , Fred Clark H , Maul Gerd G.
Published: June 2020

Acute gastroenteritis causes death in millions of cases, and many are due to viruses, mostly rotavirus. Through appropriate hygiene and health care, deaths from gastroenteritis have dropped considerably in the United States. Price and coworkers tested by electronmicroscopy of stool samples 537 specimens in 1988 and found 19% of them to contain viruses most commonly rotaviruses. The morphological criteria for the actual rotaviruses cannot be mistaken. However, they also used the appearance of the lattice structures to diagnose rotavirus infection. The same description of these lattice structures to be diagnostic for rotavirus was shown by Miller. The only possible material to contain similar hexagonal patterns were bacteriophage tails. Here we report that bacterial walls of an unidentified bacterium contain these lattices also. Virally-enriched preparations of stool samples from children with acute gastroenteritis are routinely tested for rotaviruses in early Spring each year.
Ultrastructural studies on the interaction of haemophilus influenzae with human endothelial cells in vitro
Authors: Ferguson D.J.P. , Virji M. , Kayhty H. , Moxon E.R.
Published: June 2020

Haemophilus influenzae is a human pathogen which causes meningitis in children. Systemic H. influenzae infection is largely confined to encapsulated serotype b organisms and is a major cause of meningitis in the U.K. and elsewhere. However, the pathogenesis of the disease is still poorly understood. Studies in the infant rat model, in which intranasal challenge results in bacteraemia, have shown that H. influenzae enters submucosal tissues and disseminates to the blood stream within minutes. The rapidity of these events suggests that H. influenzae penetrates both respiratory epithelial and endothelial barriers with great efficiency. It is not known whether the bacteria penetrate via the intercellular junctions, are translocated within the cells or carried across the cellular barrier in 'trojan horse' fashion within phagocytes. In the present studies, we have challenged cultured human umbilical cord_vein endothelial cells (HUVECs) with both capsulated (b+) and capsule-deficient (b-) isogenic variants of one strain of H. influenzae in order to investigate the interaction between the bacteria and HUVEC and the effect of the capsule.
Adherence and development of a smooth-walled filamentous bacterium associated with equine cyathostomes
Authors: Els H.J. , Krecek R.C. , Henton M.M. , de Wet S.C.
Published: June 2020

Zebras host large numbers and an extensive diversity of nematode species including cyathostomes. Communities of microorganisms colonize the anal and vulvar pores on the posterior extremities of female cyathostomes recovered from Burchell's zebras, Equus burchelli (Fig. 1). Cyathostomes with attached filamentous microorganisms were processed for scanning (SEM) and transmission (TEM) electron microscopy using standard methods. The adherence and in situ development of a filamentous bacterium is described (Fig. 2). This filament is designated as a smooth-walled multicellular organism which adheres to the cyathostome cuticle by means of a cauliflower vegetative cell complex and gives rise to branched mycelia (Fig. 3). The adherence appears to be superficial and no adverse reaction was noted at the sites of attachment to the cyathostome.
Comparison of air driven and gun powder driven microprojectiles in gene transfer system
Authors: Jones Johnnye M. , Chee Paula P.
Published: June 2020

The successful delivery of foreign material into plant tissues using gun powder driven microprojectile-mediated gene transfer system has been reported. In the gun powder driven microprojectile-mediated gene transfer system, the tungsten microprojectiles coated with the foreign genetic material is accelerated by an explosion of gun powder (Fig.1). The main disadvantages of the gun powder driven microprojectile are: variability of the velocity of the spent gas which can cause cell damage and random frequency of transformation, and the build up of unburned gun powder in the apparatus which can causeor create safety problems. An improved microprojectile for use in the genetic engineering ofplants which involves the use of air or gas pressure from a conventional pressurized air or gas supply as the accelerator has been developed. (Fig. 2). A comparison of the air driven and gun powder driven microprojectile is illustrated in electon micrographs of plant tissues bombarded by both methods (Figs. 5-8).
Resin mould technique for Scanning Electron Microscopy of floral initiation and development in Proteaceae
Authors: Dupee Steven
Published: June 2020

This paper illustrates the use of a replicating technique with SEM for distinct floral morphogenesis in two flower species. The initiation of floral apices and flower development have been studied intwo important cut flower crops, Telopea speciosissima and Protea cynaroides. The replicating technique using a dental impression material and Spurr's resin gives good results in monitoringthe development of Protea flower heads. When coitpared with other techniques, this one was preferred due to simpleprocedures, lack of shrinkage and collapse of cells, very little distortion and significant detail of individual flowers.
Development of spermatia in the red alga Caloglossa leprieurii preserved for TEM by freeze substitution
Authors: Lingle Wilma L. , Porter David , Darley Marshall
Published: June 2020

The red algae have been difficult to preserve for TEM observation. In an effort to overcome the limitations of conventional, aqueous fixations, we used rapid freezing techniques that are superior in preserving delicate and transient membrane features. Laboratory cultures of the red alga Caloglossa leprieurii were prepared for TEM by rapid freezing in liquid propane followed by substitution at -80° in 2% osmium tetroxide and 0.05% uranyl acetate in dry acetone for 65 hours. While in substitution fluid, the tissue was allowed to come to room temperature over a 6 hour period. The fluidwas replaced with dry acetone, followed by two 30 minute washes. Infiltration with Embed 812 without accelerator was performed at 4° on a tumbler in increments of 12.5% changed every 12 hours. Five percent increments were utilized from 75% to 100%. Three 24 hour exchanges with 100% resin including accelerator took place alternately under vacuum at room temperature for 8 hours, then at 4°on a tumbler for 16 hours. The tissue was cured at 60°.
High-pressure freezing and freeze substitution of teliospores of the rust fungus Gymnosporangium clavipes
Authors: Roberson Robert W.
Published: June 2020

The use of cryo-techniques for the preparation of biological specimens in electron microscopy has led to superior preservation of ultrastructural detail. Although these techniques have obvious advantages, a critical limitation is that only 10-40 μm thick cells and tissue layers can be frozen without the formation of distorting ice crystals. However, thicker samples (600 μm) may be frozen well by rapid freezing under high-pressure (2,100 bar). To date, most work using cryo-techniques on fungi have been confined to examining small, thin-walled structures. High-pressure freezing and freeze substitution are used here to analysis pre-germination stages of specialized, sexual spores (teliospores) of the plant pathogenic fungus Gymnosporangium clavipes C & P.

Dormant teliospores were incubated in drops of water at room temperature (25°C) to break dormancy and stimulate germination. Spores were collected at approximately 30 min intervals after hydration so that early cytological changes associated with spore germination could be monitored. Prior to high-pressure freezing, the samples were incubated for 5-10 min in a 20% dextran solution for added cryoprotection during freezing. Forty to 50 spores were placed in specimen cups and holders and immediately frozen at high pressure using the Balzers HPM 010 apparatus.
Microscopy studies of Tilletia teliospore sheaths
Authors: Gardner John S. , Hess W. M.
Published: June 2020

Teliospores of bunts of wheat and rice have a complex multilayered wall. The outer wall layer or sheath may be absent from some Tilletia controversa teliospores and may be difficult to characterize unless it is hydrated. It may also contain surface rodlets. The sheath has been characterized with freeze fracture and thin sectioning studies. By altering the sample processing procedures and by using thin sectioning the sheath can be used to distinguish T. caries teliospores from T. controversa teliospores which is important for wheat marketing. Earlier attempts were made to distinguish the two species using SEM at low kV settings without the use of special procedures to hydrate the sheath. When many samples of each species were studied, variations in wall structure within species were evident, but at 1-15 kV the electrons penetrated the porous outer sheath and imaged the impermeable exospore layer. The purpose of these investigations was to use SEM to study hydrated sheaths of samples of T. caries and T. controversa teliospores at different kV settings.
Staining terminal cisternae proteins in rat skeletal muscle triads: Selectivity of a new silver technique
Authors: J Tandler Carlos , de Iraldi Amanda Pellegrino
Published: June 2020

A selective silver staining of proteins within the lateral components of triads and diads in fixed striated muscle cells and in isolated sarcoplasmic reticulum vesicles using a mercuric acetate postflxation has been demonstrated by us.

In the present work we modify the technique in order to study the mechanism of the reaction . First, the mercuric postfixation step was omitted and replaced by a treatment with diluted ammonia, a procedure thought to wash out the excess of the reducing aldehyde fixative. Second, a treatment with mercuric acetate was employed on the ammonia treated muscles. The rat gastrocnemius muscle was fixed by immersion (12-24 h) in 3% glutaraldehyde, washed briefly with water and placed (12-24 h) into an ammonia:distilled water (1:4 v/v) solution. After washing with water, 1% acetic acid and water, muscles were treated overnight with 50% (v/v) ammoniacal silver nitrate at 37°C in darkness, washed with three changes of 5% (w/v) sodium sulphite and water, embedded in Poly B, sectioned and examined unstained with a Siemens Elmiskop I electron microscope.
Studies of anionic sites of the kidney by re-injection of polyethyleneimine (PEI) after disappearance of PEI-particles by initial administration
Authors: Nakono Kiyoko , Yoshimura Ashio , Oniki Hiroaki , Ideura Terukuni , Koshikawa Shozo
Published: June 2020

As anionic sites of glomerular basement membrane (GBM) stained by intravenous injection of PEI became obscure 2 hours after administration, we studied reappearance of the anionic sites upon re-injection of PEI.

The following two experimental groups were prepared: In group A; 10 rats were injected with 0.3ml of 0.5% PEI (MW=1,800) solution, and left nephrectomy was performed after 3 hours (group A-1, N=5) and 5 hours (group A-2, N=5). Each tissue samples was subjected to contrasting/fixation immersion, washing, post-fixation and staining. Immediately after nephrectomy, re-administration of 0.3ml of the same PEI solution was performed on each animal. Right nephrectomy was done 15 minutes after re-injection of PEI, and renal tissue was treated the same way as that of the left kidney (group A-1', group A-2'). In group B; PEI with a molecular weight of 70,000 was used. Injection of PEI solution, nephrectomy and treatment of renal tissues were carried out in the same way as for group A (group B-l and B-l', N=6; group B-2 and B-2', N=8).
Morphometric comparison of uranyl acetate/lead citrate-stained and peroxidase-stained neutrophil granules
Authors: Gilbert Charles S. , Parmley Richard T. , Kinkade Joseph M.
Published: June 2020

Although neutrophil granules can be broadly divided into peroxidase-positive and peroxidasenegative granules, recent studies have demonstrated significant heterogeneity in neutrophil granule morphology and physical density. The present study was undertaken to evaluate this heterogeneity morphometrically. Human peripheral blood neutrophils were collected from normal volunteers in heparin or EDTA and fixed in 3% glutaraldehyde as a cell suspension or minced buffy coat. An aliquot was stained for myeloperoxidase using 3,3’-diaminobenzidine (DAB) as substrate. All samples were post fixed in 1% OSO4 and embedded in Spurr low viscosity medium. Additionally neutrophil granules isolated according to the method of Rice, et al. were similarly embedded. Thin sections of morphologic preparations were counterstained with methanolic uranyl acetate and aqueous lead citrate (UALC) (Fig. 1), whereas DAB stained specimens were counterstained with LC only (Fig. 2). Attempts to combine UALC and DAB staining in the same section obscured the density imparted by DAB. LC failed to impart significant density to DAB-negative granules in DAB-LC stained specimens resulting in an underestimation of this granule population.
A rapid fixation method for transmission and Scanning Electron Microscopy using microwave irradiation
Authors: Hotta Yasuaki , Nozaki Masumi , Honda Nobuhiko , Kato Hiroyuki , Wei Wang Ying , Watari Nakazo
Published: June 2020

Recently, the validity of microwave irradiation (MWI) for electron microscopy has attracted special interest especially in Japan. Recently, we developed a new maceration method for scanning electron microscopy (microwave maceration) and a rapid polymerization method for tissue embedding using epoxy resin (microwave polymerization). Concerning the tissue fixations using MWI (microwave fixation), there are many problems which are not yet clear. In this study, we intend to reveal the effectiveness and mechanism of microwave fixation for scanning and transmission electron mi c roscopy.

Liver and trachea taken from Wistar strain male rats were used in this experiment. The fresh livers and tracheae were cut into small pieces and microwave irradiated using a microwave processor (H2500, Bio-Rad) for 1 or 3 min in 2.5% g1utara 1dehyde (GL) bufferized fixatives regulated at pH 7.3 with 0.1M phosphate buffer solution. To prevent the rising of temperature during MWI, specially designed metal case containing water and ice was used (Fig.1).
The contribution of osmium tetroxide to the image quality and detectability of elements in cells studies by electron spectroscopic imaging
Authors: Stearns Rebecca C. , Hastings Cindy L. , Katler Marshall , Godleski John J.
Published: June 2020

Osmium tetroxide has been recognized as an excellent fixative which also contributes to the quality of TEM micrographs (1). In the preparation of biological materials for electron spectroscopic imaging (ESI) of 30 run sections with the Zeiss CEM902 electron microscope, OsO4 is usually used in fixation, but poststaining with heavy metals is not needed. However, the extent of the contribution of OsO4, to the images obtained had not been defined clearly, nor was it clear the extent to which the presence of OsO, might obscure detection of elements by ESI or EELS. To establish optimal liquid fixation for the preservation of soluble ions we monitored the loss of Fe ions during preparation. To determine the role of Os4O, in ESI imaging, hamster alveolar macrophages (AMS) were incubated with 0.ImM FeCLo for 15 minutes, then fixed with 2.5% glutaraldehyde in 0.IM K phosphate buffer in 0.01% CaCLo, then grouped into 6 samples varying the times and percentages of OsO, . One sample was not exposed to any OsO4.
Hepatotoxicity of rubber vulcanization fumes in rats
Authors: Rydzynski Konrad , Opalska Barbara , Czerczak Sławomir
Published: June 2020

Rubber vulcanization fumes are one of the hazardous agents that occur in the workplace atmosphere in the rubber factories. Vulcanization processes generate a whole spectrum of vapours and gases, which contain, among others, benzene, benzo|a|pyrene and nitrozamines. The aim of the present study was to evaluate potential hepatoxic effects of acute and subchronic inhalatory exposures in rats.

For that purpose Wistar rats were exposed in dynamic inhalation chambers to rubber vulcanization fumes for 4 hours, at the concentration of 2-3mg/l, (acute exposure) and for 6 hours daily, five times a week, for 28 days, at the concentration of 0.1mg/l (subacute exposure). Controls inhaled air at the same conditions. Liver samples were taken from rats directly after the end of both exposure types, and at 2nd and 14th day after acute exposure to study recovery processes, fixed, dehydrated and embedded according to conventional procedures. The number of fat-storing cells and Kupffer cells was estimated directly on JEOL JEM100C fluorescence screen screen at x2600 (measured area 7500 μm2), and relative volume of lipid droplets in hepatocytes and in fat-storing cells cytoplasm determined using point-counting analysis.
Biological and biomedical applications of collagenous biomaterials
Authors: Kato Yasushi P. , Dunn Michael G. , Silver Frederick H. , Wasserman Arthur J.
Published: June 2020

Collagenous biomaterials have been used for growing cells in vitro as well as for augmentation and replacement of hard and soft tissues. The substratum used for culturing cells is implicated in the modulation of phenotypic cellular expression, cellular orientation and adhesion. Collagen may have a strong influence on these cellular parameters when used as a substrate in vitro. Clinically, collagen has many applications to wound healing including, skin and bone substitution, tendon, ligament, and nerve replacement. In this report we demonstrate two uses of collagen. First as a fiber to support fibroblast growth in vitro, and second as a demineralized bone/collagen sponge for radial bone defect repair in vivo.

For the in vitro study, collagen fibers were prepared as described previously. Primary rat tendon fibroblasts (1° RTF) were isolated and cultured for 5 days on 1 X 15 mm sterile cover slips. Six to seven collagen fibers, were glued parallel to each other onto a circular cover slip (D=18mm) and the 1 X 15mm cover slip populated with 1° RTF was placed at the center perpendicular to the collagen fibers. Fibroblast migration from the 1 x 15mm cover slip onto and along the collagen fibers was measured daily using a phase contrast microscope (Olympus CK-2) with a calibrated eyepiece. Migratory rates for fibroblasts were determined from 36 fibers over 4 days.
Enzyme histochemical application and backscattered electron imaging to visualize the distribution of lymphatic capillaries
Authors: Kato Seiji
Published: June 2020

Previously, the author repeatedly confirmed the higher 5’-nucleotidase (5’-Nase) and lower alkaline phoaphatase (ALPase) activities in the wall of lymphatic capillaries reacted with the lead-based method relative to those of blood capillaries. The ALPase, on the other hand, is markedly higher in blood capillaries than in lymphatics. On the basis of these enzyme characteristics, the author has developed a 5’-Nase— ALPase double staining method to differentiate small lymphatics from blood capillaries at the level of the light microcsopy. Furthermore, we applied it to histochemical observation of the lead-containing reaction products of 5’-Nase in lymphatics on the same or adjacent cryostat sections using backscattered electron imaging (BEI) in scanning electron microscope (SEM). This paper presents a new applicability of 5’-Nase histochemistry by BEI-SEM to demonstrate the distribution of lymphatic capillaries in tissue blocks.
SEM immunohistochemistry using alkaline phosphatase-labeled antibody
Authors: Hashimoto Hisashi , Hoshino Kazumasa
Published: June 2020

Scanning electron microscopic immunohistochemical observations have been performed mainly using antibodies labeled by either virus particle or colloidal gold. In order to observe immunoreactive sites using these markers, however, high magnification is needed, even when the backscattered electron image (BEI) mode is utilized. Using alkaline phosphatase (ALPase)-labeled antibodies, lead phosphate can be deposited on immunoreactive sites and is detectable as strong signals on the BEI. Recent progress in computer technology enabled us to superimpose two or more images using different colors and gray levels. In this study, our attempt was made to detect the localization of prolactin cells in the pituitary gland and of laminin in the kidney using ALPase-labeled antibodies, by superimposing immunoreactive sites indicated by the BEI over their surface structures shown by the secondary electron image (SEI).

Anterior pituitary glands obtained from female mice and kidneys from male mice were fixed by perfusion with 4% paraformaldehyde in 0.1M sodium phosphate buffer (SPB). After rinsing with 0.1M SPB, the fixed specimens were transferred into 50% dimethyl sulfoxide, frozen in liquid nitrogen and fractured, and rinsed with 0.1M SPB. The kidneys were pretreated with 0.1% pepsin in 0.01N HCl in order to have immunoreactivity against laminin restored.
Involvement of nucleus in the maturation of dengue-2 virus in C6/36 cells
Authors: Wang Jaang J. , Chen Cheng C. , Shaio Men F. , Liu Chia T. , Lee Chung S. , Yeh Ming Y.
Published: June 2020

The involvement of nucleus in the maturation processes of Dengue-2 virus in a mosquito cell line, C6/36 cells, has been identified by the electron microscopy and immunocytochemistry. The C6/36 cells were obtained from ATCC and maintained in MEM culture medium containing 10% fetal bovine serum at 28°C. The cell suspensions or cells grown on teflon-coated coverslips were infected with Dengue-2 virus (107/ml) for various time periods of 2 hours, 3, 6, 8, and 10 days. The cells were then fixed in buffered 1.5% glutaraldehyde, and washed in acetone before immunolabeled with monoclonal antibody. An indirect immunocytochemical labeling method of avidin-biotin complex (ABC) conjugated with peroxidase or gold particles (20 nm in diameter) and a flat embedding technique were used to localize the virus particles.

At early stages of infections (before 3 days), there were no virion particles detected. After 6 days and on of infections, cytopathic effect (CPE) was observed and showed positive immuno-peroxidase reactions under the light and electron microscopies.
Improved immunolabelling of ultrathin cryosections using antibody conjugated with 1nm gold particles
Authors: Nielsen Morten H. , Bastholm Lone
Published: June 2020

During the last 5 years the diameter of the gold probes used for immuno-cytochemical staining at the electron microscopical (EM) level has been decreased. The advantage of small diameter gold probes is an overall increased labelling density. The disadvantage is a lower detectability due to the low electron density of smaller gold particles consequently an inconvenient high primary magnification needed for EM examination. Since 1 nm gold particles are barely visible by conventional EM examination the need for enlargement by silverenhancement of the gold particles has increased.

In the present study of ultrathin cryosectioned material the results of immunostaining using 5 nm gold conjugated antibody and 1 nm gold conjugated antibodies are compared after silverenhancement of the 1 nm gold particles.

Slices of freshly isolated mouse pituitary gland were immersion fixed for 20 min in 2 % glutaraldehyde /2 % paraformaldehyde. Blocks cryoprotected with 2.3 M sucrose were frozen in liquid nitrogen and ultra-cryosectioned on a RMC cryoultra-microtome.
Use of antibody and lectin-colloidal gold conjugates to detect surface and extracellular glycoproteins produced in vivo by the Lyme disease spirochete Borrelia bergdorferi
Authors: Dorward David W. , Garon Claude F.
Published: June 2020

Despite the often serious pathological ramifications of Lyme disease, Borrelia burgdorferi, the causative agent, is rarely demonstrated in nor isolated from naturally-infected mammalian hosts. This dilemma has led to suggestions that a limited number of spirochetes expel extracellular bioproducts that may contribute to the clinical symptoms. Currently, no mammalian toxins have been attributed to B. burgdorferi, however, the production of extracellular vesicles by this bacterium was recently described. In order to assay the possible presence of such extracellular material in infected hosts, we developed a sensitive electron microscopic assay to detect and characterize B. burgdorferi products in biological samples.

Polyclonal rabbit serum was raised against purified B. burgdorferi vesicles and against an electrophoretically-purified 83 kilodalton (kDa) vesicle-associated protein. After purification of IgG from the sera, anti-vesicle F(ab’)2 fragments were produced and used to “activate” parlodion-coated grids. Such grids were incubated with cultured spirochetes, or with fluids and tissues from infected and non-infected mammals and ticks. Captured antigens were then labeled with anti-83 kDa IgG and Protein A-colloidal gold conjugates, and/or lectin-colloidal gold conjugates, then examined and characterized by electron microscopy.
Quantitative immunocytochemical localization of four immunodominant antigens of Toxoplasma gondii
Authors: Bonhomme A. , Boulanger F. , Lebonvallet S. , Bonnet N. , Bharadwaj L.M. , Pinon J.M.
Published: June 2020

Four immunodominant antigens frequently observed in congenital, acquired and reactivated toxoplasmosis have been localized on Toxoplasma gondii ultrathin sections using four monoclonal antibodies (Mab GII9, IV47, IE10 II38) which detect membrane and cytoplasmic antigens. Thin sections as well as the whole surface of RH tachyzoïtes of Toxoplasma gondii are immunolabeled with the different Mab and Biotin-streptavidin-colloïdal gold complex. The quantitative analysis of immunogold labeling was performed with the help of a semi-automatic procedure developped by us, using a digital image analysis system (Bio 500-BIOCOM, les Ullis, FRANCE). The number of gold particles per unit surface are quantified inside three cellular compartments defined on the video screen : surface membrane, submembrane area and rhoptries.

On the whole cell surface, proteins of 30 kDa (P30) detected with Mab GII9 were the major surface protein (Fig 1). By quantitative evaluation, they were 50 fold more abundant than P45-50kDa (recognized by Mab II38) proteins and 7 fold more abundant than P66-70kDa (recognized by Mab IE10) proteins. For the proteins of 28 kDa detected with Mab IV47 the labeling was very intensive in the submembrane area (Fig 3).
A description of the phospholipid arrangement in DOPE:DOPC mixture and its modification by trehalose as studied by freeze-fracture
Authors: Mariana Tihova
Published: June 2020

The effect of trehalose on the lamellar/HII phase transition in hydrated dioleoylphosphatidylethanolamine:dioleoylphosphatidylcholin mixtures as a function of temperature (t°) was investigated.

DOPE:DOPC/3:I or DOPE only (at concentrations 60wt%)(Avanti Pol.Lip.) were hydrated in 1.8M trehalose solution at t°>TH and shaken 5-10 times on a vortex mixer ai this t° by the technique of exhaustively cycling the t° through TH. Suspensions were equilibrated at the t° desired and quenching was achieved by liquid nitrogen jet freezing.

A gradual transition of HII phase into a new type of bilayer - sinusoidal ripple phase in the lipid mixture quenched from 22°C is shown in fig.1. The HII phase exhibits unusual appearance presented by bent cylinders situated at an angle to each other. The sinusoidal ripples described as a stable intermediate structure between Lα and HII phases is different from Pβ’ripple phase in saturated lipid-water systems. Upon cooling the ripple bilayers transform at 0°C into smooth lamellae (figs.2a,2b) with non typical fracture face.
A Morphologic Approach to Understanding Molecular Events During Development
Authors: Witte D. P.
Published: June 2020

Through the combined technologies ofin situhybridization and transgenic mouse analysis major advances have been made in the understanding of developmental biology. Recent advances in understanding embryogenesis have been accomplished by determining specific gene activation during development, determining the cell specificity of individual activated genes, characterizing the cis regulatory elements of the developmentally regulated genes, and disrupting the activity and functions of these genes during critical stages of development through targeted mutation.In situhybridization provides highly specific and sensitive detailed information of both the spatial and temporal pattern of endogenous gene expression during embryogenesis. The regulatory elements that determine the tissue specific and temporal related expression pattern of these embryonic genes are then identified and characterized byin situhybridization through the generation of transgenic mice which carry gene constructs with reporter genes, such as CAT, luciferase, or lac Z, linked to the flanking DNA sequences that exert either positive or negative influence over the expression of the gene in question. Finally once the expression patterns and regulatory elements have been characterized targeted ablation of the developmentally regulated gene can determine or provide important insight into function.
Molecular and Microscopic Analysis of Altered Gene Expression in Transgenic and Gene Targeted Mice
Authors: Jones W. K. , Robbins J.
Published: June 2020

Two myosin heavy chains (MyHC) are expressed in the mammalian heart and are differentially regulated during development. In the mouse, the α-MyHC is expressed constitutively in the atrium. At birth, the β-MyHC is downregulated and replaced by the α-MyHC, which is the sole cardiac MyHC isoform in the adult heart. We have employed transgenic and gene-targeting methodologies to study the regulation of cardiac MyHC gene expression and the functional and developmental consequences of alteredα-MyHCexpression in the mouse.

We previously characterized an α-MyHC promoter capable of driving tissue-specific and developmentally correct expression of a CAT (chloramphenicol acetyltransferase) marker in the mouse. Tissue surveys detected a small amount of CAT activity in the lung (Fig. 1a). The results ofin situhybridization analyses indicated that the pattern of CAT transcript in the adult heart (Fig. 1b, top panel) is the same as that ofα-MyHC(Fig. 1b, lower panel). The α-MyHC gene is expressed in a layer of cardiac muscle (pulmonary myocardium) associated with the pulmonary veins (Fig. 1c). These studies extend our understanding ofα-MyHCexpression and delimit a third cardiac compartment.
Phosphorus-mapping of isolated viruses by energy spectroscopic imaging (ESI): an experimental approach to discriminate mass-effects from the element signal
Authors: Richter K. , Tröster H. , Haking A. , Schultz P. , Oudet P. , Witz J. , Probst W. , Spiess E. , Spring H. , Trendelenburg M.
Published: June 2020

ESI uses the fact that the probability of an electron beam loosing a certain amount of energy by inelastic scattering is a function of the elemental composition of the specimen. However, to extract precise information about the elemental distribution from the specimen, the background intensity at the element sensitive energy-loss must be known. Since background increases with mass-thickness, adequate background-estimation is a crucial prerequisite for element mapping in isolated particles as otherwise the element signal conflicts with the contrast in mass-thickness.

To control the mass effect, a double layer of carbon film is used as an internal probe for phosphorus detection of the RNA in TYMV (turnip yellow mosaic virus). Phosphorus detection was performed at the P-L23-edge above 132 eV (energy-windows: 155/120/90 eV; window-size dE=15eV; irradiation dose per image approx. 105 e/nm2 EM Leo 912; detection device slow-scan ccd 14 bit). Virus particles (diameter 25 nm, ratio Protein/RNA 3/2; 6000 bases; ca. 20 P-atoms/pixel) and carbon film (thickness 10 nm) have about the same mass thickness. A second set of images taken before the P-edge (120/100/75 eV) was acquired as a negative-control for phosphorus. ESI-signals calculated from this set of images are interpreted as mass-thickness.
Cryo analytical microscopy: Multiple applications for plant structure and physiology
Authors: Huang C. X. , Ling L.E.C. , McCully M.E , Canny M.J
Published: June 2020

Carleton University’s cyo-analytical SEM facility deals with a very wide range of specimens from all the sciences. One of its major specializations is the study of plant structure and function, as illustrated by reference to particular research programs, for example: Stabilization of structures that cannot be preserved by conventional fixation and embedding methods. Many plant tissues are constructed of extremely fragile cell walls containing large vacuoles with high turgor pressures within, interspersed with large volumes of air or fluid. Plants which grow under water are a conspicuous example, requiring large internal open channels for the transport of gases to and from the roots. Other fragile tissues and those having cell walls that are impermeable to solvents and resins have been preserved in roots of desert monocotyledons, and in tree roots. Fluids in spaces between cells. We have pioneered the discovery that many intercellular spaces in plant tissues, always believed to contain air, are in fact filled with fluid. These spaces in sugarcane stems (Figs. 1 & 2) have been shown to contain both strong sugar solution, and an endophyte that lives on this sugar and fixes atmospheric nitrogen. The large air spaces (aerenchyma) in some roots, always considered an aeration system, have been shown to contain water some of the time, and to enhance diffusion of solutes in roots. We have also discovered that roots, always considered to be organs for collecting water from soil, also excrete water to the soil at night. Distribution of nutrient ions in plant tissues. Quantitative analysis of nutrient ions (especially potassium) in individual cells of roots, stems and leaves are opening up new perspectives on the acquisition, use and transport of ions in plants. Bubbles of air and water.
Pseudo aberration free focus for atomic resolution imaging of two adjoining crystals
Authors: Hashimoto H. , Endoh H. , Hashimoto M. , Song M.
Published: June 2020

The atomic structure images of two crystals on both sides of the interface made by conventional TEM are not so easy to be recorded at the same focus without the effect of spherical aberration of imaging lenses even though the spacings of the crystal lattices of both crystals are within Scherzer focus limit, because the specimen is generally larger than 1 Onm thickness and cannot be treated as weak phase objects and Bragg reflected waves from tow crystals generally cannot be in phase at the image plane. In TEM, the shift of the phases of several Bragg reflected waves from a crystal due to the spherical aberration (coefficient Cs) can be compensated by the defocus (Δf), which was named as aberration free focus (AFF). The current images of crystals on both sides of an interface are formed by the waves reflected from one or two kinds of lattice planes with low indexes.
Integration of Core-Edge Spectroscopy Methods for the Study of Polymers
Authors: Rightor E. G.
Published: June 2020

Core edge spectroscopy methods are versatile tools for investigating a wide variety of materials. They can be used to probe the electronic states of materials in bulk solids, on surfaces, or in the gas phase. This family of methods involves promoting an inner shell (core) electron to an excited state and recording either the primary excitation or secondary decay of the excited state. The techniques are complimentary and have different strengths and limitations for studying challenging aspects of materials. The need to identify components in polymers or polymer blends at high spatial resolution has driven development, application, and integration of results from several of these methods.
Characterization of Aluminum Bonding Pad Corrosion by Electron Microscopy
Authors: Klein J.E.
Published: June 2020

One leading factor of integrated circuit (IC) reliability today deals with the identification and prevention of aluminum bonding pad corrosion. Using three common IC industry conductors, namely aluminum (99.999%), aluminum-copper(0.5%) and aluminum-silicon(l .0%), samples were fabricated and analyzed by FE-SEM/EDS after various IC processing steps.

Samples consisted of boron doped (P type) <100> silicon wafers which were processed according to a standard fabrication procedure. Metal deposition was performed using a metal sputter system with an RF field and heated stage. To examine deposition temperature effects, stage temperature was varied from 200 to 300°C for each conductor type. Wafers were processed through normal fabrication with one wafer from each deposition temperature and metal type being examined after various processing steps. In addition, samples were also subjected to accelerated testing by exposure to a Pressure Cooker Test (PCT) at 120°C, 1.5 atm for 24 hours. In order to characterize the effects of oxide growth of the different metals, sputter deposited samples of each conductor as processed earlier but at one stage temperature, were microsectioned and examined by TEM.