bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ultrastructural preservation of the cortical region of sea urchin embryonic ectoderm
Authors: Nislow C. , Summers R. G. , Morrill J. B. 
Published: June 2020

Recent studies using immunocytochemical techniques have renewed interest in the sea urchin embryo as a model system for development, particularly with respect to cellular interactions during morphogenesis. The bulk of these studies have been performed with the light microscope (LM) using fluorescently labeled antibodies, (i.e. Wessel and McClay, 1986)Transmission electron microscopy (TEM) reveals that the cortex of the embryonic ectoderm includes membranous vesicles, yolk platelets, mitochondria, Golgi complexes, and the hyaline layer. The proximity of these compartments to the cell surface warrants a detailed study of their ultrastructure.We have examined the quality of preservation with simultaneous and sequential fixation using glutaraldehyde and osmium tetroxide. Also examined are the effects of several additives and the quality of preservation with Bouin's fixative, which has been used in immunocytochemical preparations.Embryos of Lytechinus variegatus were fertilized and reared according to the methods of Morrill and Santos (1986).
Development of propane jet freezing methods for cultured cardiac cells
Authors: Dalen H. , Lefurgey A. , Hawkey L. , Lieberman M. 
Published: June 2020

Physiologically viable and synchronously beating heart cells can be grown in varying geometric configurations for parallel biochemical, electrophysiological, ultrastructural and x-ray microanalytical studies. Our objective in these experiments was to develop a method by which heart cells grown to confluency either as a sheet, spherical aggregate or polystrand could be rapidly frozen prior to cryosectioning and electron probe x-ray microanalysis.A single-sided propane jet freezer, which has been shown to achieve excellent freezing of confluent cell sheets prior to freeze fracture, was constructed according to Pscheid et al.; a specimen holder was also designed and constructed which facilitated rapid transfer (<10 sec) of the various cultured cells from media to the jet freezer (FIG. 1a,b).
Preparation of cultured cell cytoskeletons for transmission and scanning electron microscopy
Authors: Goff Julie P. 
Published: June 2020

The cytoskeleton is comprised of microtubules, microfilaments and intermediate filaments that work collectively to regulate cell function and structure. Since there is agreement that the cytoskeleton plays a primary role in so many cell activities it is surprising to find conflicting opinions over its appearance, which seems to vary with the technique used to visualize the network. The microtrabecu1ar lattice described by Wolosewick and Porter contrasts sharply with the cytoskeletons produced by Heuser and Krishner, Ris, and others. These techniques require the use of either HVEM or special freeze drying and shadowing equipment. The procedure described here is an easy and reliable technique for visualizing the cytoskeleton using conventional TEM and SEM.For TEM, cells were grown on 200 mesh formvar coated gold grids adhered to glass coverslips. For SEM, cells were seeded onto Thermanox coverslips.
Quick-frozen chick embryos visualized by SEM following freeze-substitution
Authors: Allenspach A. L. , Clark P. J. 
Published: June 2020

The events of primitive streak regression and early axial formation have been described by SEM on embryos prepared using conventional chemical fixation. Chemically preserved embryos, however, are not suitable for purposes of performing analytical electron microscopic analysis (AEM) to survey for diffusible elements. Cryopreservation techniques for AEM studies require that specimens be very thin and that the freeze rates be very rapid. Convention allows that, under the best of conditions, ultrastructural preservation occurs to depths of 20-30 μm. Early chick embryos, properly supported, lend themselves to freeze techniques because of their thinness. We report on the depth of freeze and preservation of morphogenetic detail.Isolated chick embryos (28-33 hours) were rinsed in Hank's Balanced Salt Solution, mounted on donut-shaped filter paper supports and plunged quickly into a mixture of 30% isopentane/70% propane; the cryogen slush was held at -185C with liquid nitrogen. Specimens were then freeze-substituted using the procedure of Bridgman and Reese.
Ultrastructure of cultured endometrial epithelial cells grown on two extracellular matrices
Authors: Weidmann J. , Freund M. , McGeever B. 
Published: June 2020

Extracellular matrices (EMSA) have been used in tissue culture to mimic the in vivo growth of cells. ECMS have been made by investigators using collagen extraction procedures and now are commercially available either in pre-coated labware or as a liquid which can be poured to variable depths over the culture surface. Common constituents in all are the basement membrane collagens, laminin, and other minor proteoglycans. Cells grown on these matrices are thought to attach at a greater rate, have increased longevity, and mimic the in vivo characteristics. Endometrial epithelial cells were cultured on two commercially available ECMS and examined by transmission electron microscopy to see if any differences in morphology existed.Endometrial epithelial cells derived from menstrual blood were cultured in 25 cm2 polystyrene flasks pre-coated with Matrix ECM (Invitro International) and also Basement Membrane Matrigel (Collaborative Research).
Cellular projections seen by Scanning Electron Microscopy
Authors: Burmeister Veronika , Millikin Paul D. 
Published: June 2020

Scanning electron microscopy is an ideal tool for the visualization of projections in biological cell surfaces because it combines high resolving power with extraordinary depth of focus. To appreciate the inside structures of cellular projections transmission electron microscopy is ideal, since it enables the identification of intricate ultrastructures In this presentation we compare the size and ultrastructure of microvilli in a normal as well as pathological state in mesothelium, cilia of the nasal mucosa and pseudoprojections of spirochetes.Specimens were routinely processed: fixed in 2.5% Glutaraldehyde, rinsed in Millonig's Phosphate Buffer and carried through Ethanol to 100%; SEM specimens were then critical point dried and gold-coated. TEM specimens were put into Propylene Oxide and subsequently polymerized in Epon 812. Silversections were cut and stained in Uranyl Acetate and Lead Citrate. JEOL, JEM 100 C transmission and JSM 35 scanning microscopes were used.
Applications of freeze-fracture cytochemistry as applied to the study of tissues and cells
Authors: Kan Frederick W.K. 
Published: June 2020

The applications of freeze-fracture cytochemistry to the study of tissues and cells are described. Two approaches will be presented: label-fracture and fracture-label. In both techniques, colloidal gold probe is used as the marker system. The use of cytochemistry in combination with freeze-fracture for the study of distribution of receptor sites, antigens and glyco- conjugates on freeze-fractured plasma membranes as well as on fractured intracellular organelles is illustrated respectively by the two models described below. A variant of the fracture-label technique, Backscattered Electron Imaging of labeled tissues following freeze-fracture cytochemistry, is also introduced as a novel method for the 3-dimensional visualization of the in situ distribution of receptor sites on freeze-fractured membranes.1.Molecular demarcation of surface domains in boar spermatozoa as visualized by label-fracture cytochemistry.The label-fracture technique has been previously described. In brief, boar sperm cells are fixed, labeled and impregnated with glycerol.
A 300-kV field-emission Transmission Electron Microscope
Authors: Murakoshi H. , Ichihashi M. , Kakibayashi H. 
Published: June 2020

For the ultimate resolution of 0.1 nm, we have developed a 300 kV field-emission TEM (FE-TEM). Atomic resolution requires conventional TEM to have an accelerating voltage to 1MV or higher, if a thermionic electron gun is used. On the other hand, an FE-TEM allows atomic resolution at a medium accelerating voltage. A 200 kV FE-TEM with a (310)-oriented cold W field emitter has already been developed and commercially released as the Hitachi HF-2000. Its information limit is 0.155 nm.To improve the resolution of an FE-TEM, we increased the accelerating energy E from 200 keV to 300 keV to reduce the electron wavelength and the energy fraction ΔE/E. A ten-stage accelerator tube was designed to maintain stable operation at 300 kV. The external view of the accelerator tube is shown in Figure 1. It is shielded by three layers of electrodes made of permalloy to lessen the influence of the stray magnetic field.
Digital imaging for high-resolution electron holography
Authors: Allard L. F. , Nolan T. A. , Joy D. C. , Hashimoto T. 
Published: June 2020

It is a goal of electron microscopy to eliminate film as the recording medium for electron microscope images in favor of direct digital recording. At present, there are commercially available digital TV systems (e.g. ref. [2]) based on CCD slow scan technology that provide 1M pixel images (i.e. 1k × 1k arrays). Such systems have proven useful for recording standard high resolution images and are sufficient to replace film for most standard electron microscopy. However, the newly developing technique of electron holography requires an advanced digital imaging capability, because the process of reconstruction of amplitude and phase images from a hologram necessarily gives final images that are only one-quarter the size of the original image. For a minimum desired 512 × 512 pixel reconstructed image, the original image should be 2k × 2k, requiring a CCD array with 4M pixels.Electron holograms which are recorded for reconstruction of aberration-corrected images with improved resolution (approaching 0.1 nm) require hologram fringes spaced on the order of 0.3 nm.
Electron trajectories in electrostatic fields
Authors: Czyzewski Zbigniew , Joy David C. 
Published: June 2020

Electron microscope use an electron beam to obtain various kind of information about specimen. The electron beam is focussed by electrostatic and magnetic fields and electron detectors employ electrostatic fields to attract or deflect electrons. In many cases the demand to calculate the electron trajectories in a fast and visual way is very strong. One of the most important questions is the problem of the secondary electron (SE) trajectories inside the SEM chamber and the effect of sample charging on detector yield. This is especially important in the low voltage SEM when investigating an uncoated, non-conductive specimen. A relatively large number of calculated trajectories gives a possibility to optimize SE detector placement as well as detector bias.The main problem is solving the Laplace equation in a 3-D space. In the 3-D space composed of cubic cells of dimension Δ3, the Laplace equation takes the following form:
Reflected polarized darkfield imaging of bone surfaces
Authors: Sato Masahiko , Herring Janice , Kim John , Lilly Eli ,   Company
Published: June 2020

Reflected polarized light microscopy (Fig. 1A) was used previously to generate high contrast images of birefringent and light scattering samples, including bone surfaces and autoradiographic specimens. We now present a modification (Fig. 1B) of the Gullberg system with improved sensitivity for the characterization of bone specimens and quantitation of silver granules on autoradiographic specimens. Reflected imaging techniques were useful to generate high contrast images superior to transmitted light strategies, and both of the strategies presented can be adapted easily to any fluorescence microscope.Reflected light produced images free of refractile noise from materials through the thickness of the specimen which detracts from transmission darkfield imaging of silver grains and brightfield imaging of bone surfaces. The use of crossed polars also eliminated noise from stray light reflected off of internal microscope elements. The rotatable lambda/4 plate mounted on the objective front element (Fig. 1A) allowed considerable manipulation of image contrast, permitting dual imaging of silver granules, birefringent tissues in autoradiographic specimens and the surface topography of bone specimens by rotating the lambda/4 plate to 45°.
Retarding-field electron energy analyzer with a two-tube adapting lens for voltage contrast
Authors: Słówko Witold 
Published: June 2020

In order to obtain a quantitative voltage contrast in SEMs various secondary electron energy analyzers are applied, often of a very sofisticated design. However, retarding field analyzers with planar or spherical fields are most popular because of their small dimensions which enable to place them below the objective lens. Spherical analyzers ensure better energy resolving power but contain too many grids (3-4) shadowing a sample when it is scanned.It is possible to design an analyzer equipped only with one grid which would save the advantages of the spherical one. Relatively good resolving power of the spherical analyzer results from the fact that electrons approach the retarding grid in perpendicular direction. The same effect can also be obtained for a planar grid when an electron lens is created below it and a sample is placed at the lens focus. The presented analyzer has been equipped with a two tube electron lens (Fig.1). The first tube plays simultaneously a role of the extraction electrode.
A control and imaging system for dedicated STEM
Authors: Colling J A , von Harrach H S 
Published: June 2020

A new digital control system has been developed for the HB600 series of cold field-emission STEM’S manufactured by VG Microscopes. The philosophy behind this control scheme has been to develop a system which is both user-friendly for non-experts in STEM/TEM operation as well as offering access to all functions for unusual modes of operation.The control scheme, as shown in Fig.1, is based on two computers; one for instrument control, using a multi-tasking, real-time operating system (OS/2) with a graphical user interface (Presentation Manager), the other for control of image acquisition and display.The instrument control computer controls the high voltage power supply, column and stepper motor electronics via opto-isolated serial lines (RS232). The system is interrupt driven and therefore minimises the computer traffic which has the potential for causing interference in highly stable systems. An Ethernet link connects the two computers and a link to an external computer can also be implemented.
Scanning Electron Microscopy image analysis of fiber glass insulation
Authors: Talbot H. , Jeulin D. , Hobbs L.W. 
Published: June 2020

Glass wool, made of chemically bound glass fibers which trap stagnant air, has been used in a variety of applications as phonic and thermic insulation material. Mechanical and insulating qualities of the material depend heavily on the characteristics of the fibers themselves. Aside from the composition of the glass, the length and diameter distributions of the fibers within the material can be related to these characteristics. A fast and reliable means for obtaining these size distributions is therefore a crucial issue for quality control.Scanning electron microscopy (SEM) imaging was used to access sub-micron dimensions of the fibers; and image analysis, as opposed to manual procedures, was used for obtaining measurements.Samples of clean raw fibers (free of binder) were dispersed with ultrasound into a pH 9 aqueous solution and later deposited on a track-etched polycarbonate filter with 0.2 μm pore size. The filter was then dried, coated, and observed in BSE mode in a standard SEM, or left uncoated and observed in an environmental SEM (ESEM).
Automated data collection for electron tomography
Authors: Agard D.A. , Koster A.J. , Braunfeld M.B. , Sedat J.W. 
Published: June 2020

Three-dimensional imaging has become an important addition to the variety of methods available for research on biological structures. Non-crystalline samples can be examined by high resolution electron tomography which requires that projection data be collected over a large range of specimen tilts. Practical limitations of tomography are set by the large number of micrographs to be processed, and by the required (and tedious) recentering and refocusing of the object during data collection; especially for dose sensitive specimens. With automated electron tomography a number of these problems can be overcome. First, the images are recorded directly in digital format, using a cooled slow scan CCD camera, and, with automatic tracking and correction for image shift and focus variation, a pre-aligned dataset is obtained, with every image recorded under well defined imaging conditions.At UCSF, we use intermediate voltage electron tomography to study higher-order chromatin structure. Of central interest is elucidating the higher-order arrangement of the 30nm chromatin fiber within condensed chromosomes through several phases of the cell cycle and, in collaboration with Chris Woodcock, the structure of the 30 nm fiber.
Automatic vs computer-assisted 3D reconstruction
Authors: Russ John C. 
Published: June 2020

Images from light or electron microscopy of serial sections must be aligned and corrected for distortion before use in three-dimensional reconstruction. As computer rendering supplants physical modelling, algorithms are desired for automatic alignment and rectification, requiring rotation and rubber-sheeting or warping. A new image can be constructed from each digitized original with polynomial equations relating the pixel coordinates in the new image to those in the original. Interpolation between the pixel values is used to prevent aliasing of edges. Calculating the equation coefficients requires matching points in the serial sections, plus some external or a priori information about the cutting distortion. The difficulties in finding corresponding points suggest that human interaction may be needed to locate the proper matching points, while the computer carries out the more mundane but important tasks of pixel interpolation and rendering.Two approaches to automatic alignment have direct counterparts in the related problem of matching points in stereo pair images.
Computer-aided methods for 3-D visualization of serial sections and thick biological specimens
Authors: Ellisman Mark , Martone Maryann , Soto Gabriel , Masliah Eleizer , Hessler David , Lamont Steve , Young Steve 
Published: June 2020

Structurally-oriented biologists examine cells, tissues, organelles and macromolecules in order to gain insight into cellular and molecular physiology by relating structure to function. The understanding of these structures can be greatly enhanced by the use of techniques for the visualization and quantitative analysis of three-dimensional structure. Three projects from current research activities will be presented in order to illustrate both the present capabilities of computer aided techniques as well as their limitations and future possibilities.The first project concerns the three-dimensional reconstruction of the neuritic plaques found in the brains of patients with Alzheimer's disease. We have developed a software package “Synu” for investigation of 3D data sets which has been used in conjunction with laser confocal light microscopy to study the structure of the neuritic plaque. Tissue sections of autopsy samples from patients with Alzheimer's disease were double-labeled for tau, a cytoskeletal marker for abnormal neurites, and synaptophysin, a marker of presynaptic terminals.
Scanning probe metrology
Authors: Griffith Joseph E. 
Published: June 2020

Scanning probe microscopes have unusual advantages as measurement tools. They achieve high resolution simultaneously in all three dimensions, over almost any solid, in ambients ranging from high vacuum to fluid electrolytes. They offer the prospect of performing dimensional metrology at the atomic level with the calibration linked directly to crystal lattice constants. Application of these microscopes to measurement is not completely straightforward, however. As with optical and electron microscopes, accurate measurement is not possible without a thorough understanding of the instrument's properties. We discuss here two aspects of probe microscope behavior that affect position measurement because they exhibit strong nonlinearities.The piezo ceramic actuators commonly used to generate the probe motion are ferroelectric so they suffer from hysteresis and creep. Consequently, the probe motion must be independently monitored. We have adopted a capacitive scheme for monitoring the probe position in all three dimensions. This scheme allows the tube position to be measured to within 10 nm, though there are distortions caused by the tube bending that must be corrected.
Structural characterization of conducting polymer thin films using scanned probe microscopies
Authors: Musselman I. H. , Gray K. H. , Leasure R. M. , Meyer T. J. , Linton R. W. 
Published: June 2020

As alternatives to silicon-based devices, organometallic conducting polymer thin-films are being used to develop new applications involving electrocatalysis, sensors, optical storage devices, and electrochromic displays. The chemical and topographic homogeneity of these films may play an important role in their electrochemical properties, as well as in their ability to form array structures with high spatial control. Film microstructures can be revealed using a range of surface specific characterization techniques. In our laboratory, scanning tunneling microscopy (STM) and atomic force microscopy (AFM) have been used to assess the surface structure (roughness and thickness) of these films. Results of the scanned probe microscopies also provide insight into the effects of initial film surface roughness and chemical microhomogeneity on the sputter depth profiles obtained using secondary ion mass spectrometry (SIMS). Reductive electropolymerization techniques were used to prepare separate thin polymeric films on B-doped highly oriented pyrolytic graphite (HOPG) substrates from monomer solutions of [Ru(Me4bpy)2(vpy)2]2+ (Me4bpy is 4, 4', 5, 5' - tetramethyl - 2, 2' bipyridine, vpy is 4 - vinylpyridine) and [Fe((CH2Br)2bpy)3]2+ ((CH2Br)2bpy is 4, 4' - dibromomethyl - 2, 2' - bipyridine).
Direct phase determination in electron crystallography: small organic molecules
Authors: Dorset Douglas L. 
Published: June 2020

The quantitative use of electron diffraction intensity data for the determination of crystal structures represents the pioneering achievement in the electron crystallography of organic molecules, an effort largely begun by B. K. Vainshtein and his co-workers. However, despite numerous representative structure analyses yielding results consistent with X-ray determination, this entire effort was viewed with considerable mistrust by many crystallographers. This was no doubt due to the rather high crystallographic R-factors reported for some structures and, more importantly, the failure to convince many skeptics that the measured intensity data were adequate for ab initio structure determinations.We have recently demonstrated the utility of these data sets for structure analyses by direct phase determination based on the probabilistic estimate of three- and four-phase structure invariant sums. Examples include the structure of diketopiperazine using Vainshtein's 3D data, a similar 3D analysis of the room temperature structure of thiourea, and a zonal determination of the urea structure, the latter also based on data collected by the Moscow group.