bluuebunny/crossref_metadata_2025_split huggingface dataset:
Deep negative staining of cell whole mounts for high voltage electron microscopy
Authors: Malecki Marek , Ris Hans 
Published: June 2020

High voltage electron microscopy (HVEM) offers greatly enhanced beam penetration, reduced radiation damage, and increased resolution over conventional transmission electron microscopy (CTEM). Thus, HVEM is specially suitable for spatial organization studies on cell whole mounts rather than CTEM of cell freeze fracture replicas (Fig.2) or extracted cell thin negative stainings (Fig.3). These studies are particularly interesting, if the observations of a living cell can be correlated with the HVEM images of the same cell after either chemical fixation or rapid freezing. We present here a novel and simple procedure to study threedimensional organization of cell whole mounts with the aid of HVEM.Human neuroblastoma cells were grown on polystyrene films, These cells were gently extracted and fixed. Then, after brief washing, they were infiltrated with the staining and embedding mixture: 3% polyvinylpyrrolidone - as an embedment and 3% ammonium molybdate - as a negative stain (Fig.4). Finally, samples were air dried. Whole mounts were observed in our AEI Mark II 7 HVEM equipped with a rotating-tilting stage and operating at 1 MeV.
Analysis of electron-specimen interactions of thick biological specimens in Transmission Electron Microscopy at 200 KEV
Authors: Han Karen F. , Gubbens Alexander J. , Koster Abraham J. , Braunfeld Michael B. , Sedat John W. , Agard David A. 
Published: June 2020

The primary project of our laboratory is the investigation of chromatin structure by three dimensional electron microscope tomography. The goal is to understand how 30nm fibers fold into higher order chromatin structures. Three dimensional tomography involves the reconstruction of an object by combining multiple projection views of the object at different tilt angles. Due to the electronspecimen interaction and the characteristics of lens aberration in the electron microscope, however, the image is not always an accurate representation of the projected object mass density. In this abstract, we analyze the various types of electron-specimen interaction for thick biological specimens up to 0.7 microns thickness.Electron-specimen interactions include single elastic and inelastic, and multiple elastic and inelastic scattering. Of the imaging electrons, the single elastic and the plasmon electrons give rise to image intensities that can be linearly related to the projected object mass density. Multiply scattered elastic electrons contribute to an increase in background intensity. In addition, due to the chromatic aberration of the TEM’s objective lens, multiply scattered inelastic electrons cause a blurring of the image because of an effective broadening of the focus spread.
Image processing to extract line information from micrographs
Authors: Turner John H. , O’Keefe Michael A. , Paciornik Sidnei 
Published: June 2020

In processing microscope images, it is sometimes important to be able to extract the lengths and directions of linear features. For example, in characterizing transmission electron microscope images of faceted grain boundaries, the desired information is the length of grain boundary lying along each direction. In this case we require the sum of the lengths of the facets as a function of direction, but not their positions.The problem of having the computer recognize and extract lines from images is not trivial. In order to measure grain boundary lengths automatically, micrographs must be digitized and the linear features (e.g. the grain boundaries) binarized and reduced to a single pixel width. Then the problem requires finding two simple parameters for each line in the image, its direction and number of pixels. A new approach to this problem is to project the intensities of the source image in all possible directions, and to collect the projection results into a new two-dimensional “image” with one dimension representing the projection direction. This new image is the sinogram of the source image.
Effect of raised temperature on a stainless-steel surface by ambient atomic-force microscopy
Authors: Jung Pan S. 
Published: June 2020

We report images from atomic force microscopy (AFM) of a heated metallic surface performed under an ambient environment. The stainless steel surface was heated up to 121C, which is limited only by the capacitive heating element used (100W). We observed that the topography of the surface changes dramatically. It is found that at high temperatures, the surface appears flat compared with the presence of many round bumps observed on the same sample at room temperature when the ambient humidity is high. To the best of our knowledge, this is the first report on ambient AFM performed on a metallic surface above 100C.Most AFM systems adopt moving sample stages [1]. In this case, it is difficult to implement heavy heating heating elements and a heat reservoir block. For this experiment, the TAK3 from AT Corporation [2] is used. The sample stage is kept stationary and the heating element (the tip of a soldering iron) is fixed firmly on one side of a surface of the block using a screw.
Cross-sectional TEM specimen preparation from magneto-optical disk by ultramicrotomy
Authors: Shaapur F. , Kim M.J. , Lee Seh Kwang , Kim Soon Gwang 
Published: June 2020

TEM characterization and microanalysis of the recording media is crucial and complementary to new material system development as well as quality control applications. Due to the type of material generally used for supporting the medium, i.e., a polymer, conventional macro- and microthinning procedures for thin foil preparation are not applicable. Ultramicrotorny (UM) is a viable option and has been employed in previous similar studies. In this work UM has been used for preparation of XTEM samples from a magneto-optical (MO) recording medium in its original production format.The as-received material system consisted of a 4-layer, 2100 Å thick medium including a 300 Å TbFeCo layer enveloped by silicon nitride protective layers supported on a 1.2 mm thick × 135 mm (5.25 in.) diameter polycarbonate disk. Recording tracks had an approximate pitch of 1.6 μm separated by 800 Å deep peripheral grooves. Using a Buehler Isomet low-speed diamond saw, 1 mm wide and 20 mm long strips were cut out of the disk along the recording tracks.
Comparison of high-angle take-off and low-angle take-off EDX detector geometry of the HF-2000 FE-TEM
Authors: Sato Y. , Hashimoto T. , Ichihashi M. , Ueki Y. , Hirose K. , Kamino T. 
Published: June 2020

Analytical TEMs have two variations in x-ray detector geometry, high and low angle take off. The high take off angle is advantageous for accuracy of quantitative analysis, because the x rays are less absorbed when they go through the sample. The low take off angle geometry enables better sensitivity because of larger detector solid angle.Hitachi HF-2000 cold field emission TEM has two versions; high angle take off and low angle take off. The former allows an energy dispersive x-ray detector above the objective lens. The latter allows the detector beside the objective lens. The x-ray take off angle is 68° for the high take off angle with the specimen held at right angles to the beam, and 22° for the low angle take off. The solid angle is 0.037 sr for the high angle take off, and 0.12 sr for the low angle take off, using a 30 mm2detector.
Visualizing the intracellular membrane system of yeast cells using a laser scanning confocal microscope
Authors: McCoy Karen L. , Dillin Andrew G. , McCracken Ardythe A. 
Published: June 2020

Progress has been made in developing new preparative techniques for electron microscopic visualization of the intracellular structures of yeast. In addition, development of the laser scanning confocal microscope (LSCM) has provided improved resolution for fluorescent microscopy. We asked whether the LSCM in combination with new preparative techniques could be used for comparable investigative research of the intracellular organizaton of the yeast cell.To investigate this possibility, a BioRad MRC600 LSCM equipped with a krypton/argon laser and integrated computer imaging capabilities, was used to study various dipliod strains of the yeast Saccharomyces cerevisiae. Cells were treated with the lipophilic, cationic fluorescent dye DiOC6 (3,3’-dihexyloxacarbocyanine iodide), which has been used to visualize intracellular membrane structures, and in particular the endoplasmic reticulum of mammalian cells and living yeast cells. Since one of our interests is the intracellular localization of proteins in the yeast cell, we utilized transformed yeast cells expressing a human gene encoding a protein that inappropriately accumulates in the endoplasmic reticulum (ER).
Loss of membrane Structure and cytoplasmic volume in glutaraldehydefixed neutrophils compared to cryofixed-freeze-substituted neutrophils
Authors: Gilbert Charles S. , Parmley Richard T. 
Published: June 2020

Cryofixation instantly preserves cell processes without chemical alteration and freeze-substitution (FS) preserves cell constituents. Investigation has shown that cell membranes and microfilaments appear better preserved in cryofixed-freeze-substituted (CFFS) specimens, including basophils. However, the cells have not been looked at morphometrically nor have neutrophils been looked at specifically.Peripheral blood was collected in heparin from four male donors by venipuncture. Blood was centrifuged at 500 x g for 45 min to separate the granulocytes using a one-step density gradient approach, and then washed in RPMI 1640. Cells were split and half were resuspended in RPMI with 10% DMSO, cryofixed using the Eiko RF-2 and stored in liquid N2 until FS processing in 4% OsO4 in acetone with embedment in Spurr’s resin. The other half were resuspended and fixed in 3% glutaraldehyde in 0.1M Cacodylate, pH 7.35 for 1 hr at 4-6°C. Cells were dehydrated, embedded in Spurr’s resin, counterstained with UALC and examined using a Zeiss EM109.
Proliferation of microtubules from discrete electron-dense bodies in the ciliary dendritic portion of mechanosensory setae of copepod antennae
Authors: Weatherby Tina M. , Lenz Petra H. 
Published: June 2020

Sensory setae on the appendages of most arthropods contain microtubules in their innervating dendrites. Both mechano- and chemoreceptors are of ciliary origin. Mechanoreceptive dendrites typically contain more microtubules than do chemoreceptive dendrites, perhaps reflecting a role for microtubules in stiffening the setae. The presence of microtubules may also increase the sensitivity of mechanotransduction.The overall arrangement of calanoid copepod mechanoreceptors has been described elsewhere. In calanoid copepods the arrangement of microtubules in the distal ciliary segment of the dendrites is dramatic: as many as 3000 microtubules per dendrite are found in parallel rows (fig. 1,3a). These bundles of microtubules extend some distance into the setae themselves. The basal bodies within these dendrites are especially electron-dense and give rise to a circlet of 9 +	0 microtubules (fig. 2,3f)- Just distal to the circlet electron-dense material begins to form adjacent to the microtubules (fig. 3e), eventually forming elongate electron-dense bodies radiating outward (fig. 3d).
A freeze-fracture-etched study of the physarum polycephalum plasma membrane during early formation of the macroplasmodial stage
Authors: Taylor Randolph W. , Treadwell Henrie 
Published: June 2020

The plasma membrane of the Slime Mold,Physarum polycephalum, process unique morphological distinctions at different stages of the life cycle. Investigations of the plasma membrane ofP. polycephalum, particularly, the arrangements of the intramembranous particles has provided useful information concerning possible changes occurring in higher organisms. In this report Freeze-fracture-etched techniques were used to investigate 3 hours post-fusion of the macroplasmodia stage of theP. polycephalumplasma membrane.Microplasmodia ofPhysarum polycephalum(M3C), axenically maintained, were collected in mid-expotential growth phase by centrifugation. Aliquots of microplasmodia were spread in 3 cm circles with a wide mouth pipette onto sterile filter paper which was supported on a wire screen contained in a petri dish. The cells were starved for 2 hrs at 24°C. After starvation, the cells were feed semidefined medium supplemented with hemin and incubated at 24°C. Three hours after incubation, samples were collected randomly from the petri plates, placed in plancettes and frozen with a propane-nitrogen jet freezer.
Collagen ratios in exercise-trained and pressure-overloaded rat hearts
Authors: Burgess ML , Price RL , Abel FL , Jones GP , Buggy J , Terracio L , Borg TK 
Published: June 2020

The cardiac extracellular matrix (ECM) is composed of components known to affect cardiac function. Specifically, collagens (types III and I) provide a network integral to cardiac myocyte metabolism and function. Collagens promote the recoil action following contraction and maintain vascular patency during contraction. Chronic pressure-overload adversely affects cardiac compliance and function, and ultimately leads to heart failure. Fibrosis is common in pressure-overloaded hearts, partially accounting for the ventricular “stiffness” and reduced function. Chronic exercise training, however, is identified by enhanced cardiac function. Currently, little is known about ECM cellular biology and of exercised hearts and its relationship to cardiac function (1). This study examined the morphology, biochemistry and function of chronically exercised versus pressure-overloaded rat hearts to further delineate the role of the ECM in cardiac function.Male Sprague-Dawley rats were adapted to treadmill running and randomly divided into four groups: coarctation of abdominal aorta (Coarc) to induce pressure-overload, sham operated and sedentary controls (Cont) and exercise-trained (XT).
Lobular and cellular distribution and content of glycogen synthase in periportal and pericentral hepatocytes of the rat liver
Authors: Giffin Bruce F. , Morris Randal E. , Drake Richard L. , Cardell Robert R. 
Published: June 2020

Glycogen and the enzymes involved in hepatic carbohydrate metabolism have a heterogeneous distribution throughout the parenchyma of the liver. Although the precise lobular localization for many of these enzymes has been determined, information on the distribution and content of glycogen synthase (GS) has not been published. These studies were undertaken to determine: (1) the lobular and cellular distribution of GS in the fed rat; (2) any relationship between glycogen patterns and the distribution of GS.Livers of fed male Spraque-Dawley rats were perfused with cold 30% sucrose and 10 um frozen sections collected on Vectabond treated slides. After fixation for 5 min in 4% paraformaldehyde at room temperature, tissue sections were incubated in polyethylene slide boxes with blocking buffer (PBS, 0.5% Triton X-100, 5% fetal calf serum, 2% bovine serum albumin, 0.95% fish gelatin, pH 7.5) overnight at 4°C. Tissue sections were incubated for 6 hours at room temperature with goat anti-rat liver GS diluted in blocking buffer (1:1000) followed by 5 nm gold conjugated rabbit anti-goat IgG overnight at 4°C and 3 hours with 5 nm gold conjugated goat anti-rabbit IgG at room temperature.
Peroxisome proliferation: Organelles or activity as an endpoint?
Authors: Taylor Catherine A. , Jarnot Bruce M. 
Published: June 2020

Peroxisome induction can be expressed as an increase in peroxisome area (proliferation) or as an increase in peroxisomal fatty acid oxidation (activity). This study compares proliferation and activity as endpoints for hepatic peroxisome induction by perfluorodecanoic acid (PFDA). Fluorocarboxylic acids such as PFDA represent a class of compounds possessing commercially important surfactant properties. A single 50 mg/Kg ip. dose of PFDA produces a characteristic “wasting syndrome” in male F-344 rats. Symptoms include hypophagia, weight loss, hepatomegaly, and delayed lethality. Hepatic studies reveal changes similar to those seen with the hypolipidemic agent clofibrate. These include mitochondrial disruption, endoplasmic reticulum and peroxisome proliferation, and increased peroxisomal acyl-CoA oxidase activity.Male Fisher-344 rats received a single ip. dose of 2, 20, or 50mg/Kg PFDA dissolved in 1:1 propylene glycol/water and were sacrificed 8 days post-dose. All control rats received an equal volume of vehicle ip. Animals were provided food and water ad libitum, except pair-fed controls which received the same restrictive food intake consumed by their weight-paired dosed partners (50mg/Kg PFDA group) to simulate the hypophagia associated with PFDA.
Intracytoplasmic lumina in ependymomas: An ultrastructural study
Authors: Ho Khang L. , Caccamo Dario V. , Garcia Julio H. 
Published: June 2020

Intracytoplasmic lumina (ICL) are round to oval, single membrane bound spaces bearing microvilli that may or may not contain secretory material. The morphogenesis of ICL remains controversial and their biological significance and diagnostic implication of this structure have not been fully explored. ICL have been reported in a variety of epithelial tumors outside the central nervous system, but this structure is rarely described in tumors of the central nervous system.We analyzed ultrastructural features of ICL in six ependymomas (1 from lateral ventricle, 3 from fourth ventricle and 2 from the spinal cord) and three myxopapillary ependymomas of the filum terminale. Two types of ICL were identified: (1) ICL with both microvilli and cilia and (2) ICL with only microvilli (Fig.1). Ciliated ICL were common in ependymomas of the ventricles whereas non-ciliated ICL were frequently seen in myxopapillary variant. Various stages of formation and development were observed in ciliated ICL (Fig. 2,3).
Effect of cyclical stretching on cultured rat cerebral vascular smooth muscle cells: A flow cytometric, confocal laser scanning, and TEM Study
Authors: Ghoneim Shams , Waldschmidt Thomas , Yoder Elizabeth , Moore Steven , Baumbach Gary 
Published: June 2020

Vascular hypertrophy during chronic hypertension has been linked to a variety of potential determinants including increased intravascular pressure, neurohumoral factors and genetic factors. Of the various determinants, one might assume that arterial pressure per se would play an especially important role. Nonetheless, the role of intravascular pressure has remained unclear. Based on findings in vivo, we recently proposed that increases in pulse pressure, as opposed to mean pressure, may be an important determinant of cerebral vascular hypertrophy (3).The goal of the present study was to further explore the possible link between pulse pressure and hypertrophy using a combination of tissue culture, fluorescent-activated cell sorting (FACS), transmission electron microscopy, and confocal laser fluorescent microscopy techniques. Cultured smooth muscle cells derived from rat cerebral microvessels were used for these experiments. Flex culture plates with rigid (control) or flexible (elastomer), collagen-coated bottoms were seeded with smooth muscle cells. After 48 hours to allow for attachment, the culture plates were placed in a Flexercell Strain Unit (model FX-2000) (1, 2), and exposed to continuous cyclical stretching at 20 k Pascals unit pressure and 60 cycles per minute for 5 days.
Properties and dose dependence of embedding media for cryo-automated electron tomography
Authors: Braunfeld M.B. , Koster A.J. , Sedat J.W. , Agard D.A. 
Published: June 2020

Electron tomography is well suited to the study of complicated, non symmetric biological structures. In our laboratory, we use intermediate voltage electron microscopic tomography to follow complex paths of chromatin fibers within intact sections of Hela telophase chromosomes. In order to accurately reconstruct these features at resolutions beyond 50Å, precise imaging conditions and data collection schemes have been developed and employed.To obtain useful high resolution information, the specimen needs to be well preserved. Data collection must also be accurate and self-consistent. However, a serious limitation has been radiation damage to the specimen during scanning, and data collection. Because of the high doses required for tomography, the standard approach has been to accept the inevitability of serious radiation damage, and to heavily pre-irradiate the sample in an attempt to provide stability and consistency during data collection.The use of fully-automated data collection methods allows a substantial decrease in beam dose, suggesting that the entire approach should be reevaluated.
Biomineralization in loricate green algae
Authors: Dunlap John R. , Walne Patricia L. 
Published: June 2020

Members of the green algal family Phacotaceae are characterized by the presence of an extracellular wall-like structure termed a lorica. A complex assemblage of mucopolysaccharides and metals, primarily calcium and iron, the lorica is considered to be a highly conserved structure. Hence, lorica morphology has served as a basic criterion for classification at both the generic and species levels. Although lorica development is thought to be genetically controlled in the phacotacean algae the extent of cellular control over development and mineralization is largely unknown. This study was initiated to gain a better understanding of lorica microarchitecture and mineralization patterns in this unusual group of organisms.Three members of the Phacotaceae are reported on here: Pteromonas, Phacotus and Dysmorphococcus. Samples for the present study were fixed in glutaraldehyde; some were post-fixed in osmium tetroxide and all were dehydrated in alcohol and embedded in Spurr's low-viscosity resin. Thin and thick sections were examined and analyzed on an Hitachi H-800 TEM/STEM operating at 200kV.
Design and performance of instrumentation for automated single-tilt-axis electron tomography
Authors: Koster A.J. , Chen H. , Clyborne W. , Sedat J.W. , Agard D.A. 
Published: June 2020

One of the driving questions in our group is into understanding how chromosomes are constructed from fibers of DNA wrapped around histones in their native form. To permit high resolution analysis of these highly complex fibers, we use intermediate voltage electron tomography. To obtain 50Å resolutions, we incorporate new approaches to overcome the resolution limiting factors determined by specimen fixation and staining techniques, data collection and 3D reconstruction algorithms.With our present instrumentation we can automatically collect a series of projection data of large, radiation sensitive objects with only a minimum of manual operation, with high accuracy and consistency. The images are recorded directly in digital format to overcome the time consuming task of digitizing negatives. Furthermore, the system offers automated eucentricity setting, automatic tracking of image shifts, and automatic focusing during data collection. Highly reliable data collection is ensured by closely monitoring the variation in image shift, defocus, average image intensity, and exposure time throughout the tilt series.