bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron microscope tracing of acid mucopolysaccharides after administration of dihydrotachysterol
Authors: Regal Dan-Sook
Published: June 2020

Nonspecific calcinosis engendered by dihydrotachysterol ( DHT ) is associated with massive extraosseous calcium deposits, in connection with which acid mucopolysaccharides ( AM ) can be observed. Cardiac muscle and the vascular system are particularly affected. The acri-flavine-phosphotungsten acid ( PTA ) contrast-method of tracing AM is based on the affinity of acriflavine for basophile material in the tissue. An electron dense contrast is achieved by means of the complex of acriflavine, which is selectively bound in the tissue, and PTA.

Six month old albino rats were sacrificed 1, 2, 3, 5, 8, 10, 13, 17 and 20 days after per os-administration of 3. 0 mg DHT. Tissue samples from cardiac muscle and aorta were first fixed in 2 % glutaraldehyde phosphate-buffer, pH 7. 0 for two hours and then pre-incubated in darkness with 2. 0 x 10-2 M acriflavine in 50 % ethanol phosphate-buffer overnight.
Sarcolemmal permeability changes during early myocardial anoxia
Authors: Ashraf M. , Landa L. , Nimmo L. , Bloor C. M.
Published: June 2020

Following coronary artery occlusion, the myocardial cells lose intracellular enzymes that appear in the serum 3 hrs later. By this time the cells in the ischemic zone have already undergone irreversible changes, and the cell membrane permeability is variably altered in the ischemic cells. At certain stages or intervals the cell membrane changes, allowing release of cytoplasmic enzymes. To correlate the changes in cell membrane permeability with the enzyme release, we used colloidal lanthanum (La+++) as a histological permeability marker in the isolated perfused hearts. The hearts removed from sprague-Dawley rats were perfused with standard Krebs-Henseleit medium gassed with 95% O2 + 5% CO2. The hypoxic medium contained mannitol instead of dextrose and was bubbled with 95% N2 + 5% CO2. The final osmolarity of the medium was 295 M osmol, pH 7. 4.
The diagnosis of metabolic storage disease in skin biopsies (a light-, electronmicroscopic, and analytical study)
Authors: Jurecka W. , Gebhart W. , Lassmann H.
Published: June 2020

Diagnosis of metabolic storage disease can be established by the determination of enzymes or storage material in blood, urine, or several tissues or by clinical parameters. Identification of the accumulated storage products is possible by biochemical analysis of isolated material, by histochemical demonstration in sections, or by ultrastructural demonstration of typical inclusion bodies. In order to determine the significance of such inclusions in human skin biopsies several types of metabolic storage disease were investigated. The following results were obtained.

In MPS type I (Pfaundler-Hurler-Syndrome), type II (Hunter-Syndrome), and type V (Ullrich-Scheie-Syndrome) mainly “empty” vacuoles were found in skin fibroblasts, in Schwann cells, keratinocytes and macrophages (Dorfmann and Matalon 1972). In addition, prominent vacuolisation was found in eccrine sweat glands. The storage material could be preserved in part by fixation with cetylpyridiniumchloride and was also present within fibroblasts grown in tissue culture.
Freeze-etch studies of epiphyseal growth plate cartilage reveal matrix vesicles associated with calcification
Authors: Cecil Russell N. A. , Clarke Anderson H.
Published: June 2020

Unfixed proximal tibial epiphyseal growth plates were studied by freeze-etch to confirm the presence of extracellular calcifying matrix vesicles and to determine the substructure of matrix vesicle membranes as compared to plasma and other membranes of intact chondrocytes. Growth plates from 6-10 week old Sprague-Dawley rats were cut into 1x3 mm blocks whose long dimension was oriented either perpendicular or parallel to the long axis of the tibia. Some blocks were fixed at pH 7. 0 in 0. 2M cacodylate - buffered 2. 5% glutaraldehyde for 1 hour at 4ÅC. The blocks were immersed in 30% glycerol solution at 4ÅC for 1 hour, frozen in liquid nitrogen, and then fractured, etched for 2 minutes, and coated with platinum, carbon and 0. 2% Formvar solution. The replicas were cleaned with chromic acid, floated onto Formvar coated grids, and examined with a Phillips EM 300 electron microscope.

Fixed and unfixed specimens appeared similar in ultrastructure. Chondrocytes, matrix, and matrix vesicles were identified. In specimens fractured parallel to the long axis of the tibia, the reserve, proliferative, hypertrophic, and calcifying zones could be discerned as described by light and electron microscopy.
An immunoelectron microscopic examination of the intermediate filament protein, desmin, in exercise-damaged skeletal muscle
Authors: Waterman-Storer C.M.
Published: June 2020

Intense exercise has been shown to produce pathological changes in normal skeletal muscle ultrastructure. Eccentric exercise (muscle lengthening during active tension development) in particular has been shown to cause the most severe muscle damage, and studies of both human and animal tissue following eccentric exercise have documented disruption to the contractile apparatus. The disruption originates at the Z-disc, which appears broadened, smeared, or totally disrupted, with Z-discs of adjacent myofibrils out of register and running a “zig-zag” course transversely across the fiber. This condition is known as Z-line streaming. Several researchers have implicated the disruption of the intermediate filament system in the etiology of exercise-induced Z-line streaming, as these filaments are believed to link adjacent myofibrils at the level of the Z-disc. The intermediate filaments are composed predominantly of the proteins desmin and vimentin. This study utilized immunoelectron microscopic localization of desmin in order to elucidate the role of the intermediate filament system in Zline streaming of eccentrically-exercised skeletal muscle.
HVEM studies of the calcified bone interface with endosteal dental implants
Authors: Steflik D. E. , Song M. J. , Sjsk A. L. , Parr G. R. , Hanes P. J. , McKinney R. V. , Singh B. B. , Brewer P.
Published: June 2020

Previous studies have suggested the prerequisite of bone interfacing successful endosteal dental implants. This is the concept of osseointegration of dental implants within the jaw. However, the ultrastructure of the actual bone interface is largely unknown. This current study is planned to elucidate with correlative high-voltage electron microscopy (HVEM) and conventional electron microscopy (TEM), the morphology of the interface of the support tissues to endosteal dental implants. All ultrastructural protocols involve undecalcified tissue sectioning.

After 2 months healing following extraction of all premolars, 120 root form and blade-type endosteal dental implants were placed into the mandibles of 30 adult mongrel dogs. This report will be restricted to 12 implants retrieved from animals euthanized after 5 months of unloaded healing. Following perfusion with buffered 3% glutaraldehyde the implants were block resected with their encasement of mandibular tissues, fixed by immersion for 24 hr in glutaraldehyde and postfixed with 1% OsO4 for 2 hr.
Quantitative mapping of skeletal muscle fibers by x-ray microanalysis: Effect of long-time storage of freeze-dried cryosections
Authors: T. High , R. Nassar , I. Taylor , B. Scherer , P. Ingram , J.R. Sommer
Published: June 2020

Electron probe X-ray microanalysis (EPXMA) is uniquely capable of performing quantitative topochemical analyses with high spatial resolution of elemental concentrations in cryosections from quickfrozen biological material. EPXMA requires very expensive instrumentation and maintenance, and data acquisition is very time consuming. For economic reasons, it is most desirable that any EPXMA facility serve several users who, while waiting for their turn, may need to store cryosections for a time. Storage of cryosections is associated with loss of structure and, under certain circumstances, also with changes in elemental distribution after short periods of time. We have re-examined several cryosections by EPXMA mapping that had been stored under vacuum for very long periods of time.

Materials and Methods:

Single, intact semitendinosus muscle fibers from R. temporaria were quick-frozen as previously described. The cryosections were freeze-dried in a Balzers FDU 101 and carbon-coated before external transfer to plastic desiccators in which they were stored under vacuum. Quantitative EPXMA maps were obtained as previously described (Figs.1A-F).

Results and Conclusions:

In the maps [Ca] was very high in the JSRs and, occasionally, (Fig. 1D) seen in hot spots that may correspond to spotty accumulations of negatively charged electron-dense material often seen in the free SR away from JSR. Overall, only small variations in elemental distribution or concentration were observed after storage for over one year; the significance of these variations (especially with such elements as Na) is difficult to assess because of the small number (4) of muscle fibers examined. The lighter region in the center of Fig. 1B is a result of slight etching due to extended static probes that were taken from this area in addition to mapping. JSR [Ca] remained remarkably constant after one year of storage. Freeze-dried cryosections revealed some smearing of structural detail, ranging from minimal to moderate, especially in the myofibrillar compartment (cf. Figs 1A,B). Our findings show that, when handled properly, carbon-coated cryosections can be stored over extended periods of time with minimal loss of structure or changes in elemental distribution. Thus, quantitative EPXMA image maps after long storage present a rather accurate representation of the original. Proper storage of freeze-dried cryosections is a viable procedure for the effective participation of independent research groups in multiple-user EPXMA facilities, access to which may occasionally require prolonged storage periods.
Detection of human cytomegalovirus infection in paraffin sections by modified Steiner silver stain for LM and TEM
Authors: Chien K. , Kim Y.K. , Shintaku I.P. , Van de Velde R.L. , Heusser R.C. , Geller S.A.
Published: June 2020

Human cytomegalovirus (HCMV) infection is encountered more frequently in surgical specimens due to the increased number of immunosuppressed patients (organ transplants or HIV positive). Previous studies showed that HCMV infected cells in paraffin sections can be precisely labelled by in situ hybridization or by Grocott's methenamine silver stain for LM and subsequently confirmed by TEM. However, further studies indicate HCMV infection can also be identified by modified Steiner silver stain3 but in a different manner.

Modified Steiner silver stained paraffin sections can be processed for TEM as follows: Coverslips are removed by immersion in xylene. A few drops of 1% OsO4, freshly dissolved in xylene, is then pipetted onto each slide for 5 minutes. Aqueous OsO4 can not be used as it will destain the sections instantly. Slides are rinsed in xylene, xylene/acetone and then 100% acetone. The sections are infiltrated with epoxy resin/acetone, embedded in epoxy resin, polymerized and subsequently separated from slides by heat as previously described.
Choroid plexus carcinoma in childhood: An Light and EM study
Authors: Sasi Maya , M Harb Joseph
Published: June 2020

The normal choroid plexus consists of plexiform pial vessels covered by an epithelial layer which is modified from ependymal cells. Neoplasms of the choroid epithelial cells constitute less than 1% of intracranial neoplasms in all age groups and 2-5% in the pediatric age group. The choroid plexus tumors are divided into two categories, with the benign tumors known as the choroid plexus papillomas and their malignant counterparts being the choroid plexus carcinomas. Only 35 cases of choroid plexus carcinomas have been reported in the literature and only 9 of those included ultrastructural descriptions. In this report, we describe the light and electron microscopic features of three additional cases of this rare neoplasm.
Ultrastructural diagnosis of plasmacytoma on a fine-needle aspirate
Authors: Siew S. , deMendonca-Calaca W.
Published: June 2020

A 36 year old man presented with a mass in the chest and multiple “hot” focal lesions were identified on bone scan. Fine needle aspiration was performed of the chest mass. Routine histology showed the presence of some bundles of dense fibrous tissue and a diffuse infiltration of mononuclear cells, which varied in size and nucleo-cytoplasmic ratio. The smaller cells had eccentric hyperchromatic nuclei. Nucleoli were noted in the larger cells. There was well marked cytoplasmic vacuolation of some of the cells. Mitosis was present. A small fragment of tissue was received for electron microscopy. Examination of 1 μm sections showed trabeculae of medium-large polygonal cells with eccentric nuclei and occasional nucleoli. Some irregularly shaped cells had well marked cytoplasmic vacuolation. Mitotic figures were present.
Human breast carcinoma (iron metabolism and the role of intracytoplasmic lumina)
Authors: Elliott Robert L. , Elliott Mary C. , Ledford Lindsay
Published: June 2020

Iron metabolism is very important in breast carcinoma. It is an essential micronutrient playing an important role in cell growth, energy metabolism and DNA synthesis. Iron bound transferrin interacts with a cell surface receptor, facilitating the transport of iron across the cell membrane. This is the transferrin receptor. Transferrin is a glycoprotein and is the chief iron transport protein in mammalian blood. These receptors are present in high numbers on proliferating and malignant cells and the higher the transferrin receptor level the more aggressive the tumor and greater the proliferative index. We have found that ferritin, an iron storage protein, is concentrated in breast cancer tissue and that anaplastic tumors have higher tissue ferritin levels (Fig. 1). Another study demonstrated that in anaplastic tumors ductal cells had abundant ferritin granules and prominent intracytoplasmic lumina, while mitochondria were sparse (Fig. 2).
Ultrastructural modifications of the pineal gland of aged rats: Role of calcium
Authors: Humbert W. , Pévet P.
Published: June 2020

The pineal gland, an important component of the neuroendocrine axis [review in Reiter] is known to play an important role in chronobiology and transduction of certain external stimuli. The purpose of the present investigation is to examine the pineal gland of young and aged rats and to determine structural and elemental alterations related to age.

28 months-old male Wistar rats and young adult males (2-3 months old) were used. Pineal glands were quickly removed from the brain after decapitation and fixed with 3% gl utaraldehyde in 0.1 M Na cacodylate buffer (1h) and postfixed in 1% OsO4 buffered with 0.1 M Na cacodylate. For ultrastructural localization of calcium the potassium pyroantimonate method was used following the method of Eisenmann et al. Calcium was analyzed using a CAMEBAX mi croanalyser with the TEM mode and fitted with WDS spectrometers.
Intracellular motility and actin polymerization induced by Listeria monocytogenes in infected cells
Authors: Sanger J.M. , Mittal B. , Southwick F.S. , Sanger J.W.
Published: June 2020

The bacterium, Listeria monocytogenes can be phagocytosed by macrophages and epithelial cells. Within an hour, the bacteria can assemble host cell actin into bundles or tails and move inside the living cells at rates up to 1.4μm/sec. In seeking to determine the mechanisms responsible for the intracellular movements of this bacterium, our working hypothesis is that actin polymerization is not only necessary for the movement but the polymerization reaction itself provides the force to move the bacteria in the host cytoplasm.

PtK2 cells, an epithelial cell line, were infected with Listeria and were then microinjected with fluorescently labeled cytoskeletal proteins (actin, alpha-actinin, filamin, tropomyosin, skeletal muscle light chains) and a control fluorescent protein (bovine serum albumin, BSA). Some infected cells were sequentially injected first with fluorescently labeled cytoskeletal proteins and then with proteins that bind monomelic actin (Vitamin-D binding protein; DNAase I; profilin).
Electron tomographic quantitation of mineral distribution in different collagen zones of calcifying tendon
Authors: McEwen B. F. , Landis W. J. , Song M. J.
Published: June 2020

The skeletal system of vertebrates is composed of hard tissue formed by the deposition of hydroxyapatite, a calcium phosphate salt, into an organic matrix that is principally collagen. Although bone and other vertebrate calcifying tissues have been well studied, the processes by which mineralization is initiated and regulated at the molecular level are incompletely understood. Recently these processes have been investigated through high resolution tomographic three-dimensional (3D) reconstruction of initial mineralization sites of calcifying tendon imaged by high-voltage electron microscopy (HVEM). Such tomographic reconstruction provided the first direct 3D images of crystallization sites and clearly established in vivo that, as indicated in earlier reports, initial crystals are thin, irregularly-shaped platelets rather than rod-shaped needles. The crystals develop in both length (along their crystallographic c-axis) and width but seem limited in thickness. Crystals in local tendon regions are approximately parallel to each other and fuse in coplanar alignments to form larger platelets within collagen. Crystal growth is thought to be initiated in hole zones of the protein and develop into adjacent overlap zones. In the present study, electron tomography has been used to quantitate the mineral distribution in collagen for the first time.
Immunocytochemical analysis of the secretory pathway of plant cells
Authors: Staehelin L.A. , Giddings T.H. , Kiss J.Z. , Lynch M.A. , Moore P.J. , Swords K.M.M. , Zhang G.F.
Published: June 2020

The Golgi apparatus of plant cells is the site of synthesis of complex polysaccharides and glycoproteins. Following their synthesis, the glycoproteins are sorted and then delivered either to vacuoles or to the plasma membrane or the cell wall, whereas all of the complex polysaccharides are secreted into the cell wall. Little is known about how these different synthetic pathways are organized within plant Golgi stacks, and where the secreted molecules are assembled into the cell walls. To address these problems, we have 1) reexamined the structure of plant Golgi stacks in high pressure frozen and freeze-substituted root tips of Arabidopsis and Nicotiana, as well as in sycamore maple suspension culture cells, and 2) used immunocytochemical methods to localize specific types of complex polysaccharides and glycoproteins in root tip cell walls and in Golgi stacks of high pressure frozen sycamore cells.
Localization of acid phosphatases in root cap of rice plant
Authors: Chen Y. R. , Huang Y. F. , Chen W. S.
Published: June 2020

Acid phosphatases are widely distributed in different tisssues of various plants. Studies on subcellular localization of acid phosphatases show they might be present in cell wall, plasma lemma, mitochondria, plastid, vacuole and nucleus. However, their localization in rice cell varies with developmental stages of cells and plant tissues. In present study, acid phosphatases occurring in root cap are examined.

Sliced root tips of ten-day-old rice(Oryza sativa)seedlings were fixed in 0.1M cacodylate buffer containing 2.5% glutaraldehyde for 2h, washed overnight in same buffer solution, incubated in Gomori's solution at 37° C for 90min, post-fixed in OsO4, dehydrated in ethanol series and finally embeded in Spurr's resin. Sections were doubly stained with uranyl acetate and lead citrate, and observed under Hitachi H-600 at 75 KV.
New views inside cells with a digital imaging microscope
Authors: Fay F. S. , Fogarty Kevin , Tuft Richard , Carrington Walter
Published: June 2020

Many current questions in cell biology revolve around questions regarding how changes in cell function are caused by changes in their molecular composition. Given that cells are highly organized structures often carrying out diverse functions in different compartments, it follows that changes in specific cell functions must involve highly localized changes in molecular composition.

We have been involved in the development of the digital imaging microscope as a tool to investigate the distribution of molecules inside single living cells. The system measures fluorescence of probes that are highly fluorescent and specific for a molecule or ion of interest and utilizes a wide-field rather than a confocal microscope to produce an image of fluorescence in a single cell. The image is captured by a high quantum efficiency, low noise cooled CCD, thereby providing ultrahigh efficiency in the acquisition of fluorescent images. By utilizing very powerful light sources, the system is capable of generating an image with good signal-to-noise ratio in a millisecond or less, thereby allowing one to follow extremely rapid changes in molecular or ion distribution in single cells.
Induction of chromatoid bodies in trophozoites of Entamoeba invadens growing in log phase at high CO2 tension
Authors: Ramirez-Bon E. , Villarreal-Treviño L. , Morales-Vallarta M.
Published: June 2020

Entamoeba invadens is an ameba that parasites reptiles; it is very similar in morphology and physiology to Entamoeba histolytica. Due to this, it is used as an - experimental model for amebiasis research, specially in the differentiation process, since E. invadens can be induced to “in vitro” encystment, which is not possible — with E. histolytica.

Chromatoid bodies, are arrays of helical structures formed by the aggregation of ribosomes, that are found only in cystic forms of E. invadens, in trophozoites cultured in slow growing conditions, or in the presence of protein synthesis inhibitors. This paper reports the formation of chromatoid bodies in trophozoites of E. invadens in active growing phase and high CO2 tension.
Morphometric granule analysis of the human neutrophil in a patient with Chediak-Higashi syndrome
Authors: Gilbert C. S. , Parmley R. T.
Published: June 2020

The neutrophils of Chediak-Higashi Syndrome (CHS) have a characteristic giant abnormal peroxidase-positive granule (PPG). This giant PPG has been thought to be the product of the fusion of all or part of the primary (azurophil) or PPG population with or without involvement of the secondary (specific) granules which are peroxidase-negative (PNG). Immunocytochemical studies have shown that the giant PPG contains lactoferrin a marker for the PNG, whereas biochemical studies of isolated CHS granules failed to localize PNG markers to the giant PPG band or show PNG depletion. Ultrastructural studies failed to show PNG depletion and considered PNG fusion to be rare. This study was undertaken to evaluate CHS neutrophil granules niorphometrically. Human peripheral blood neutrophils were collected from a male patient with CHS in heparin and fixed in 3% glutaraldehyde as a cell suspension and minced buffy coat. The cell suspension was stained for myeloperoxidase using 3,3'- diaminobenzidine (DAB) as substrate.
Structural analysis of biological macromolecular assemblies by scanning transmission electron microscopy
Authors: Boublik M. , Tumminia S. J. , Hellmann W. , Zhang Q. , Hainfeld J.F. , Wall J.S.
Published: June 2020

Separation of the resolution and contrast affecting components and the optimized placement of detectors for the collection of elastic (contrast-forming) electrons on Brookhaven dedicated STEM make it possible to quantitatively detect greater than 90% of the available elastically scattered electrons. The high contrast and superior signal-to-noise ratio associated with the STEM annular detector allow for the imaging of unstained freeze-dried biological macromolecular complexes (chromatin, viruses, nucleic acids) at radiation doses as low as 1 e/Å. Specimens prepared in this way are free of the main resolution-limiting conditions of conventional TEM i.e. staining , air drying and radiation damage. The image intensity of unstained specimens can be related to their local projected mass and used for calculation of the total mass and mass distribution within any selected particle. Elimination of staining makes it possible to use heavy metals as high-resolution markers for topographical mapping of components and/or functional sites on a particular macromolecular complex.
Brochosome production in the Malpighian tubules of the leafhopper Euscelidius variegatus (Homoptera: Cicadellidae): An EM study
Authors: Cheung Wilkin W.K. , Purcell A.H.
Published: June 2020

Brochosomes are dodecahedral structures found characteristically in the malpighian tubules of leafhoppers. They measure 0.4-0.5¼m in diameter and are made up of lipids and proteins. Practically no information is available concerning the function of these structures. The method of discharge and significance of these peculiar structures are little reported except the key contributions of Day and Briggs (1958) for Austroaqullia torrida and Smith and Littau (1969) for Macrosteles fascifrons. During the course of investigation about the transmission of a bacterium (BEV) inside the body tissues of the leafhopper Euscelidius varieqatus we find it worthwhile to study the malpighian tubules of the latter leafhopper and report on the method of discharge of brochosomes.
Immunogold labeling of Pneumocystis carinii for Electron microscopy
Authors: Goheen Michael P. , Bartlett Marilyn S. , Smith James W.
Published: June 2020

Studies of the biology of Pneumocystis carinii (PC) are of increasing importance because this extracellular pathogen is a frequent source of severe pneumonia in patients with acquired immunodeficiency syndrome (AIDS) and is a leading cause of mortality in these patients. Immunoelectron microscopic localization of antigenic sites on the surface of PC would improve the understanding of these sites and their role in pathenogenisis of the disease and response to chemotherapy. The purpose of this study was to develop a methodology for visualizing immunoreactive sites on PC with transmission electron microscopy (TEM) using immunogold labeled probes.

Trophozoites of PC were added to spinner flask cultures and allowed to grow for 7 days, then aliquots of tissue culture fluid were centrifuged at 12,000 RPM for 30 sec. Pellets of organisims were fixed in either 1% glutaraldehyde, 0.1% glutaraldehyde-4% paraformaldehyde, or 4% paraformaldehyde for 4h. All fixatives were buffered with 0.1M Na cacodylate and the pH adjusted to 7.1. After fixation the pellets were rinsed in 0.1M Na cacodylate (3X), dehydrated with ethanol, and immersed in a 1:1 mixture of 95% ethanol and LR White resin.
Artifact-free electron microscopic immunocytochemistry on rat brain tissue
Authors: Burmeister Veronika , Ludvig N. , Jobe P.C.
Published: June 2020

Electron microscopic immunocytochemistry provides an important tool to determine the ultrastructural distribution of various molecules in both normal and pathologic tissues. However, the specific immunostaining may be obscured by artifactual immunoreaction product, misleading the investigator. Previous observations show that shortening the incubation period with the primary antibody from the generally used 12-24 hours to 1 hour substantially reduces the artifactual immunostaining. We now extend this finding by the demonstration of artifact-free ultrastructural localization of the Ca2/calmodulindependent cyclic nucleotide phosphodiesterase (CaM-dependent PDE) immunoreactivity in brain.

Anesthetized rats were perfused transcardially with phosphate-buffered saline followed by a fixative containing paraformaldehyde (4%) and glutaraldehyde (0.25%) in PBS. The brains were removed, and 40μm sections were cut with a vibratome. The sections were processed for immunocytochemistry as described by Ludvig et al. Both non-immune rabbit serum and specific CaM-dependent PDE antibodies were used. In both experiments incubations were at one hour and overnight. The immunostained sections were processed for electron microscopic examination.
Search mode for rapid detection of virus particles
Authors: Bradfute O. E.
Published: June 2020

Electron microscopy is frequently used in preliminary diagnosis of plant virus diseases by surveying negatively stained preparations of crude extracts of leaf samples. A major limitation of this method is the time required to survey grids when the concentration of virus particles (VPs) is low. A rapid survey of grids for VPs is reported here; the method employs a low magnification, out-of-focus Search Mode similar to that used for low dose electron microscopy of radiation sensitive specimens. A higher magnification, in-focus Confirm Mode is used to photograph or confirm the detection of VPs. Setting up the Search Mode by obtaining an out-of-focus image of the specimen in diffraction (K. H. Downing and W. Chiu, private communications) and pre-aligning the image in Search Mode with the image in Confirm Mode facilitates rapid switching between Modes.
Electron spectroscopic imaging of chemical versus cryo-fixed myocardial cells
Authors: Rufner Robyn , Stearns Rebecca C. , Hastings Cindy L. , Marsh James D. , Godleski John J.
Published: June 2020

Since chemical fixation and dehydration of cells results in loss and/or redistribution of endogenous ions, cryofixation with a process that preserves ultrastructure and the localization of intracellular ions is critical. Myocytes and macrophages prepared by the LifeCell®process of slam-freezing and freeze drying with subsequent osmium tetroxide/paraformaldehyde fixation have exhibited excellent structural and compositional integrity when examined by Electron Spectroscopic Imaging (ESI) and by Electron Energy Loss Spectroscopy (EELS). However, it has not been established if the apparent enhanced structure, particularly of cristae and membranes within mitochrondria, is solely a result of lipid preservation or if fixation with osmium tetroxide vapors can explain the pronounced “thickness” of membranes. In this study the effects of freezeslammed, freeze-dried myocytes with two different processing regimes for postfixation were compared to myocytes prepared by conventional chemical fixation.
Histo-grade diamond knives may be an appropriate tool for high-voltage electron microscopy
Authors: Rock M.E. , Anderson J.A. , Binder P.S.
Published: June 2020

High voltage electron microscopy (HVEM) has been employed in various ways (whole mounts of cells stereo pair imaging, axial tomography, and serial sections for reconstruction) to elucidate three dimensional (3-D) ultrastructural data. The increased specimen thickness allows further data analysis unobtainable from ultra-thin sections. HVEM can reduce the number of sections needed in 3-D reconstructiortby approximately ten times over conventional transmission electron microscopy (CTEM). But increasing section thickness also increases wear on the diamond knife used to section. We have compared the serial sections obtained from a histo-grade diamond knife with those from an E.M. grade ultra-knife. Both sets of sections were cut 0.5 μm thick from the same block, and evaluated under the one million volt beam of the HVEM.
Role of PKC isozymes in water transport in toad urinary bladder
Authors: Mia A.J. , Oakford L.X. , Yorio T.
Published: June 2020

Protein kinase C (PKC) isozymes, when activated, are translocated to particulate membrane fractions for transport to the apical membrane surface in a variety of cell types. Evidence of PKC translocation was demonstrated in human megakaryoblastic leukemic cells, and in cardiac myocytes and fibroblasts, using FTTC immunofluorescent antibody labeling techniques. Recently, we reported immunogold localizations of PKC subtypes I and II in toad urinary bladder epithelia, following 60 min stimulation with Mezerein (MZ), a PKC activator, or antidiuretic hormone (ADH). Localization of isozyme subtypes I and n was carried out in separate grids using specific monoclonal antibodies with subsequent labeling with 20nm protein A-gold probes. Each PKC subtype was found to be distributed singularly and in discrete isolated patches in the cytosol as well as in the apical membrane domains. To determine if the PKC isozymes co-localized within the cell, a double immunogold labeling technique using single grids was utilized.
Immunoelectron microscopic demonstration of insulin in the iris and ciliary body of the neonatal rat eye
Authors: Morse D. E. , Pansky B. , Budd G. C. , Caswall L. A. , McCann P. S.
Published: June 2020

Evidence is sound that the beta cells of the pancreas are not the only source of insulin in vertebrates. Published data suggests that tissue or organ specific production of insulin or an insulin-like peptide occurs in adult brain, retina, pituitary, seminal vesicle and retinoblastoma. Recent observations from our laboratories demonstrate that insulin is present in the developing retina. The purpose of the present study was to examine the neonatal eye with immunocytochemical techniques at the electron microscopic level to better characterize the cells which contain insulin. Thin sections were treated by standard methods for indirect immunolabeling. The primary antibody was guinea pig anti-porcine insulin (1:800) and the secondary antibody was biotinylated guinea pig IgG (1:200). This was followed by extravidin conjugated to 10 nanometer gold particles (1:100).
Localization of actin in the salivary glands of the American dog tick,Dermacentor variabilis
Authors: Lamoreaux William , Sankhon Naby , Coons Lewis B.
Published: June 2020

The salivary glands of ixodid ticks are the primary organs of osmoregulation and the source of pathogen transfer from tick to host. Excess fluid is extracted from the blood meal, moved across the gut wall and into the salivary glands, where it is returned to the host. Previously it has been shownin vivoandin vitrothat the type III acinus alternately swells as it fills with fluid and then contracts as the acinus empties, and that cytochalasin D prevents contraction of the type III acini.

In this investigation, the rhodamine-phalloidin technique was used to localize actin filaments in the salivary glands of fed mated female ticks. Two microfilament inhibitors were used as controls. Samples were also taken for electron microscopy and dot blot analysis.
Image formation in the scanning electron microscope
Authors: Wells Oliver C.
Published: June 2020

Image formation in the scanning electron microscope (SEM) was discussed in theory by von Ardenne. It was reduced to practice by von Ardenne, Knoll, McMullan, Smith and Oatley and Smith. The basic ideas are as follows. An electron beam (EB) of diameter dEBis scanned over a solid specimen. This penetrates for a distance dPENbelow the surface. A signal is collected and modulates the brightness of a cathode ray tube scanned in sympathy with the EB. While the resolution is always limited by dEB, it is not always limited by dPEN.

Secondary electron (SE) imaging. von Ardenne distinguished between the secondary electrons (SE) with an energy lower than 50 eV and the backscattered electrons (BSE) having energies between 50 eV and the primary energy E0. He proposed that SE that escape at the point of entry of the EB (now called SE-I) should provide a high resolution component to the SE image, while SE excited at the exit point (now called SE-II) escape from a larger area and so give rise to an image with a poorer resolution. (Also see Peters.6)
Development of a digital SEM
Authors: Yamada A. , Shibano A. , Harasawa K. , Kobayashi T. , Fukuda H. , Saito M. , Nielsen C. H. , Nakagawa S.
Published: June 2020

A newly developed digital scanning electron microscope, the JSM-6300, has the following features: Equipped with a narrower conical objective lens (OL), it allows high resolution images to be obtained easily at a short working distance (WD) and a large specimen tilt angle. In addition, it is provided with automatic functions and digital image processing functions for ease of operation.

Conical C-F lens: The newly developed conical C-F objective lens, having low aberration characteristics over a wide WD range, allows a large-diameter (3-inch) specimen to be tilted up to 60° at short WD, and provides images with low magnifications starting at 10*. On the bottom of the lens, a p n junction type detector is provided to detect backscattered electrons (BE) from the specimen. As the narrower conical 0L increases the secondary electron (SE) detector's field intensity on the specimen surface, high SE image quality is obtained.
Improving the signal-to-noise ratio of backscattered electron detectors at low beam accelerating voltages
Authors: Robinson V.N.E.
Published: June 2020

At high beam accelerating voltages, >5kV, backscattered electron (BSE) detectors rely upon the energy of the BSEs to generate the signal. At low beam accelerating voltages, BSEs do not have sufficient energy to generate a strong signal. This is further compounded because the electrons lose energy penetrating through a thin metal film, which is a dead layer on the surface of the detector. Noise bottleneck considerations1show that the bottleneck for the detection of low energy BSEs, is the signal generated when they impinge upon the detector. Consequently, the signal has to be enhanced before it is amplified. One method of achieving this is to use a multi-channel plate detector. Another is to increase the energy of the BSEs, prior to them impinging upon the detector. This can be acheived by accelerating the BSEs. after they emerge from the specimen, through the application of a positive voltage to the surface of the detector.
Vibrations of turbo-molecular vacuum pumps with high stable magnetic bearings operating within electronic microscope systems
Authors: Thomas C. A. , Reimer K. P.
Published: June 2020

In order to operate Electronic Microscopes vibration free, it is necessary to assemble the apparatus on a vibration free isolated desk. Similarly, strict requirements, regarding vibrations, are demanded from the attached vacuum pump which provides vacuum for the system. The rotor and rotating shaft of Turbo Molecular Pumps with mechanical bearings are connected to the pump housing by means of ball bearings. The inertia and vibration component resulting from the ball bearing dynamic effects produce the vibration results as indicated in Fig. 1.

In order to design a Turbo Molecular Pump with minimum vibration effects it was necessary to provide the pump with a magnetic bearing system containing 4 passive and 1 active controlled axis. This magnetic bearing system is less sensitive to vibration and simpler in assembly than other existing more complicated 5-axis active controlled versions. However it is more complication than the mechanical bearing type. The magnet bearing is provided with a touch-down bearing. Larger pump movements due to high or low frequency stimulation recieved via the microscope desk could result in forcing the shaft out of its magnet-bearing-centre position, the touch-down bearing now re-centres the shaft allowing immediate magnetic bearing centre recovery.
Imaging molecules and metals on metal surfaces by scanning tunneling microscopy
Authors: Chiang S. , Chambliss D. D. , Hallmark V. M. , Wilson R. J. , Brown J. K. , Wöll Ch.
Published: June 2020

Using an ultrahigh vacuum scanning tunneling microscope (STM), we have imaged naphthalene molecules adsorbed on Pt(111) and submonolayer metal coverages of Ni, Fe, Ag, and Au on Au(111). The STM is able to observe atomic scale features on both types of systems, giving information on the ordering and binding sites of atoms and molecules on the surface.

High resolution STM images of naphthalene on Pt(111) show the molecules as bi-lobed features with three discrete molecular orientations on the surface, 120° apart, as shown for the ordered layer in Fig. 1. The absolute orientation of the long axis of the molecules is observed to be parallel to the near-neighbor directions of the Pt(111) lattice. The sketch of the observed features, shown in Fig. 2, with the molecules overlayed arbitrarily onto on-top sites of a Pt(111) lattice, demonstrates that the molecules are located on 3x3 lattice sites, with separation of 4 lattice constants between domains. Although the (6x3) LEED pattern reported previously was reproduced, the proposed unit cell is seldom observed in the STM images.
Scanning tunneling microscopy ofE. coliRNA polymerase deposited onto an Au substrate by an electrochemical process
Authors: Keller Rebecca W. , Bustamante Carlos , Bear David
Published: June 2020

Under ideal conditions, the Scanning Tunneling Microscope (STM) can create atomic resolution images of different kinds of samples. The STM can also be operated in a variety of non-vacuum environments. Because of its potentially high resolution and flexibility of operation, it is now being applied to image biological systems. Several groups have communicated the imaging of double and single stranded DNA.

However, reproducibility is still the main problem with most STM results on biological samples. One source of irreproducibility is unreliable sample preparation techniques. Traditional deposition methods used in electron microscopy, such as glow discharge and spreading techniques, do not appear to work with STM. It seems that these techniques do not fix the biological sample strongly enough to the substrate surface. There is now evidence that there are strong forces between the STM tip and the sample and, unless the sample is strongly bound to the surface, it can be swept aside by the tip.
Atomic force microscopy of Langmuir-Blodgett monolayers and bilayers
Authors: Zasadzinski J. A. N.
Published: June 2020

Phospholipid monolayers and bilayers prepared by the Langmuir-Blodgett technique are simplified model systems of biological membranes. The molecular organization of these monolayers and bilayers has been intensely studied as they are excellent tools for understanding biological processes such as adhesion, fusion, protein structure and organization, etc. Recent structural studies have included electron diffraction of monolayers deposited on substrates and X-ray diffraction of monolayers at the air-water interface. Both diffraction techniques suggest that in the gel phase, the lipid hydrocarbon chains possess long range orientational order, but relatively short range positional order. As a result, L-B films in the gel state closely resemble the “hexatic” phases better known in thermotropic liquid crystals. However, both shortrange and long-range order have not yet been studied in a single Langmuir-Blodgett film.
An improved design for an Scanning Tunnelling Microscope (STM) for use in a Transmission Electron Microscope (TEM)
Authors: Lo W.K. , Spence J.C.H.
Published: June 2020

An improved design for a combination Scanning Tunnelling Microscope/TEM specimen holder is presented. It is based on earlier versions which have been used to test the usefulness of such a device. As with the earlier versions, this holder is meant to replace the standard double-tilt specimen holder of an unmodified Philips 400T TEM. It allows the sample to be imaged simultaneously by both the STM and the TEM when the TEM is operated in the reflection mode (see figure 1).

The resolution of a STM is determined by its tip radii as well as its stability. This places strict limitations on the mechanical stability of the tip with respect to the sample. In this STM the piezoelectric tube scanner is rigidly mounted inside the endcap of the STM holder. The tip coarse approach to the sample (z-direction) is provided by an Inchworm which is located outside the TEM vacuum.
Nanometer-sized tungsten tips for STM and AFM
Authors: Fotino Mircea
Published: June 2020

An essential requirement in the pursuit of atomic resolution by scanning tunneling microscopy (STM) or atomic force microscopy (AFM) is the use of a tip with an apex of dimensions comparable to or preferably smaller than those of the specimen to be identified. Although atomic resolution of mostly planar specimens has been obtained even with atomically blunt tips, the above requirement appears indispensable for determining 3D surface structures extending equally in all three dimensions.

Tips most commonly used so far in STM or AFM applications are made by grinding or by etching a thin and rigid Pt/Ir or W wire. Mechanical grinding occasionally leaves protruding spikes typically several tens of nm in radius that can play the role of scanning stilus even though neither adequately oriented nor particularly sharp and symmetric.
Confocal microscopy of the fertilization-induced calcium wave in sea urchins
Authors: Strieker Stephen A. , Paddock Stephen W. , Schatten Gerald
Published: June 2020

At fertilization, the egg undergoes a rapid rise in intracellular free calcium (=[Ca2+]i), which in turn triggers several important events, including cortical granule release and metabolic activation. In sea urchins, the fertilization induced increase in [Ca2+]i begins at the site of sperm fusion and sweeps through the egg in a wavelike fashion. Whether fertilization affects calcium levels only in the peripheral ooplasm or throughout the interior of the sea urchin egg is difficult to ascertain by conventional microscopic techniques, because out-offocus rays obscure subcellular organization in these relatively bulky cells. Recently, however, confocal microscopy has been introduced as a means of producing thin optical sections that reveal details not normally discernable by conventional light microscopy. In this paper, we use confocal laser scanning microscopy (CLSM) and time-lapse reconstructions to image calcium dynamics during fertilization in living sea urchin eggs labeled with the calcium-sensitive fluorescent dye fluo-3.
Three-dimensional confocal laser microscopy of fibronectin in normal and glaucomatous eyes
Authors: de Kater Annelies W. , Rand Allingham R. , Bowman Doug S. , Fay Fredric S.
Published: June 2020

Aqueous humor exits the anterior chamber of the eye through the trabecular meshwork into Schlemm's canal, a vessel which drains into the systemic circulatory system. The aqueous humor crosses the endothelial lining of Schlemm's canal by means of focal invaginations which form pores and allow digress of fluid into the canal. The trabecular meshwork provides a resistance to fluid flow resulting in a physiologic intraocular pressure in normal eyes. The region adjacent to Schlemm's canal appears to be the site of highest resistance to fluid flow in both normal and glaucomatous eyes. In eyes with primary open angle glaucoma (POAG) the resistance to fluid flow through the trabecular meshwork is abnormally high, causing an elevated intraocular pressure (IOP), which can lead to irreversible damage of the optic nerve and blindness. A definitive morphologic correlate of this disease has not been discovered. Immunohistochemical studies localizing fibronectin in unfixed frozen sections of human eyes have shown elevated levels of fibronectin in the eyes with glaucoma, however the specific distribution of this protein has not been determined due to poor tissue preservation.