bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
Philips electron microscopes: Applications trends—design advances
Authors: Thompson M.N.
Published: June 2020

Application trends in the 1990's will continue to be driven by the pursuit of materials characterisation to higher levels of structural and chemical resolution. Due to the information-limit and current-density limitations of LaB6 sources, further advancements will require better electron guns. The choice of guns includes Cold, Thermally-assisted Cold and Schottky Field Emitters. For SEM and STEM the Cold Field Emitter is a reasonable choice, because the primary criterion for small-probe techniques is current density. This logic doesn't apply to TEMs, which require both high current density for small probes and high total current for large-area illumination at various TEM magnification levels. Table 1 compares Schottky and Cold Field emitters in different applications and microscope construction. In view of its performance (Figs. 1 and 2), the Field Emission Gun is expected to have a major impact on TEM in the 1990's.
Configured detectors in STEM holography
Authors: Wang Shi-Yao , Mankos Marian , Cowley J. M.
Published: June 2020

In Gabor's in-line holography, the transmitted plane wave and the beam diffracted by the specimen form an interference pattern (hologram). In a STEM instrument, when the incident probe is scanning over the specimen, the hologram translates across the illuminated portion of the detector plane. The signal produced by the detector can be represented by integrating the product of the hologram with some detector sensitivity function. It had be pointed out long ago by Veneklasen that scanning the hologram over a fixed detector has in effect performed a reconstruction of the hologram simultaneously, and with some specially designed detector the aberration introduced by the objective lens is correctable. This scheme has been reraised recently and some preliminary work will be reported here.
HRTEM studies of ordering in lead magnesium niobate ceramics
Authors: Park K. , Salamanca-Riba L. , Wuttig M. , Viehland D.
Published: June 2020

Lead-based ferroelectric compounds with a cubic perovskite structure exhibit a high dielectric constant, a relatively low firing temperature, and a diffuse ferroelectric-paraelectric phase transition. Smolenskii et al. proposed that the distribution of two kinds of cations on equivalent crystallographic positions causes a statistical compositional fluctuation forming microdomains with different Curie temperatures which contribute to a broad phase transition through a wide temperature range. Therefore, the diffuse phase transition is largely influenced by the degree in which the cations are ordered on the B-site sublattice. The purpose of the present work is to study the ordered structure of Pb(Mg⅓Nb⅔)O3 ceramics by means of selected area diffraction (SAD) and high-resolution transmission electron microscopy (HRTEM). To get detailed information on the ordered structure, computer image simulations were also performed using a multislice method.
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).
Image restoration for biological specimens using external TEM control and electronic image recording
Authors: Typke D. , Hegerl R. , Kleinz J.
Published: June 2020

Images of non-crystalline biological specimens embedded in vitreous ice or carbohydrates have to be recorded at rather high defocus to obtain sufficient contrast. Therefore resolution is normally rather limited. Though it is well-known that image restoration from focus series can provide more complete information, this or related techniques have only rarely been used. However, the recent facilities of external TEM control and electronic image recording suggest the restoration technique should be revived and adapted to the needs of biological structure investigation. As autofocus methods have been shown to work rather accurately and focus steps can be calibrated in advance, it can be expected that the restoration can, in principle, be carried out on line and quasi blind. An additional advantage of image restoration from focus series is that it can be used, particularly in case of rather thick ice layers, to separate the phase part of the image function from most of the background due to multiple scattering by combining under- and overfocus images.
Molecular-weight factors affecting formation of the OBBD morphology in block copolymer blends
Authors: Spontak Richard J. , Smith Steven D. , Ashraf Arman
Published: June 2020

Microphase-separated diblock copolymers have been known since 1970 to exhibit three principal morphologies. These morphologies depend on the composition of the copolymer and include dispersed spheres of the minor component on either a BCC or FCC lattice, dispersed cylinders of the minor component on a hexagonal lattice, or alternating lamellae. Recent microstructural studies of starblock and diblock copolymers have shown that an ordered bicontinuous morphology is observed between the lamellar and cylindrical regimes. This microstructure is currently referred to as the ordered bicontinuous double-diamond (OBDD) morphology and is an example of the Pn3m space group. In poly(styrene-b-isoprene) (SI) diblock copolymers, it exists at approximately 62-66 vol% polystyrene (PS). Efforts aimed at producing this morphology by blending a copolymer with various PS homopolymers have also been successful, when the blend composition is 65-67 vol% PS and the molecular weight of the hompolymer (Mhps) is less than that of the styrene block in the copolymer (Ms). In this work, we have used transmission electron microscopy to elucidate some additional factors responsible for development of the OBDD and other bicontinuous morphologies.
Cryo-electron microscopy of deoxy-sickle hemoglobin fibers
Authors: Lewis Michael R. , Josephs Robert
Published: June 2020

Sickle cell disease is caused by the self assembly of deoxy-sickle hemoglobin (HbS) into fibers which rigidify and distort red cells. Fiber assembly is auto-catalytic in that the surface of existing fibers acts as a nucleating template for the assembly of new fibers (heterogeneous nucleation). Thus fibers are found typically as bundles of aligned particles. Understanding fiber-fiber interaction is crucial to understanding the pathology of fiber formation. Moreover, disrupting fiber-fiber interactions would likely ameliorate the severity of the disease. We have obtained images of both laterally associated and isolated fibers by cryo-electron microscopy (Figure 1). Optical diffraction indicates that laterally associated fibers tend to have much better long range order than isolated fibers. This observation suggests that deoxy-HbS fibers are more disordered when they are not mechanically coupled by inter-fiber contacts. In this study we assess the effects of fiber-fiber interactions on torsional disorder of deoxy-HbS fibers.
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.
Electron tomography’s remaining problems: How to fill the wedge in Fourier space and how to see what you’ve got in real space
Authors: Frank Joachim , McEwen Bruce F.
Published: June 2020

Thanks to new accelerated graphics hardware, reconstructions of subcellular structures obtained by high or intermediate voltage electron tomography are beginning to spin around on screens in laboratories and conference lecture halls. The clarity of these representations suggests that intricate questions such as the detailed mechanism of chromosome transport during mitosis and the higher-level organization of DNA in the chromosome can now be addressed by electron tomography. However, what is being shown is in most cases the result of a tedious process of interaction with the original reconstruction volume, with tools that are still extremely clumsy and inefficient. In the case of a complicated object embedded in a complex, structured environment, such as presented by the kinetochore reconstruction, we estimate that more than 80% of the entire effort is being spent on interpretation, with the remaining time going into the preparation, electron microscopy, scanning, and 3-D reconstruction. Interpretation includes attempts to isolate the relevant structure from its surroundings, to recognize its most significant features, and to produce a satisfactory representation.
Alleviation of pain and depression by specific neural transplants: Fine structural correlates
Authors: Pappas George D. , Sagen Jacqueline
Published: June 2020

We have been interested in the use of neural transplants mainly as a local source of neuroactive substances, rather than as a replacement for damaged neural circuities. In particular, we have been exploring the possibilities of reducing pain by transplants of opioid peptide producing cells, and reducing depression by transplants of monoamine-producing cells. For the past several years, work in our laboratory has demonstrated in both acute and chronic pain models that transplantation of adrenal medullary tissue or isolated chromaffin cells into CNS pain modulatory regions can reduce pain sensitivity in rodents. Chromaffin cells were chosen as donor source since they produce high levels of both opioid peptides and catecholamines, substances which independently, and probably synergistically, reduce pain sensitivity when injected locally into the spinal cord. The analgesia produced by these transplants most likely results from the release of both opioid peptides and catecholamines, since it can be blocked or attenuated by opiate or adrenergic antagonists, respectively. Furthermore, CSF levels of met-enkephalin and catecholamines are increased by the transplants.
Ultrathin frozen sections for Electron Microscopy: Some personal recollections
Authors: Christensen A. Kent
Published: June 2020

In the mid-1960s, while on the faculty of the Anatomy Department at Stanford, I was particularly interested in the cell biology of steroid-secreting cells. I had studied the ultrastructure of these cells, and was anxious to trace the pathways of steroid hormone synthesis and of the secretion from the cell. An invitation to speak at an international steroid congress in Milan, Italy, in May 1966, afforded me an opportunity to travel in Europe before the meeting started. During that trip I had a very enjoyable visit with Dr. Wilhelm Bernhard, in the Paris suburb Villejuif. He had developed means of cutting ultrathin frozen sections (UFS) of fixed tissue on a Sorvall MT-1 ultramicrotome maintained in a freezer at about −35°C. As the sections were cut, they floated off on a solution of dimethyl sulfoxide and water, from which they were picked up on EM grids, treated for cytochemistry, stained with uranyl acetate, and then viewed by EM.
Biomaterial infections: staphylococcal adherence to antibiotic impregnated automatic implantable cardioverter defibrillator (aicd) devices
Authors: Edmiston Charles E. , Uyeno David , Goheen Michael P.
Published: June 2020

Acquisition of an infection following insertion of a biomaterial device is of great concern to surgeons. The costs associated with these infections are great, both to patients and the institution in which they occur. The concept of implanting a biomaterial resistant to bacterial adherence and colonization has been shown to be valuable in preventing infection and treatment of tissue contamination. The following investigation was conducted to study the efficacy of three bonding methods on the release of antibiotic and its impact on microbial colonization of AICD (Figure 1) patch material. Cefazolin, ciprofloxacin, and teicoplanin were bonded to silicone/titanium AICD patches using either Tridodecylmethylammonium chloride (TDMAC) antibiotic bonding, Silver-antibiotic bonding or TDMAC silver-antibiotic bonding. In vitro drug levels were determined by biological assay. Antibiotic bonded and drug-free (control) AICD material was exposed to standardized inoculum of staphylococcal test isolates. At 4, 8, 24 and 48 hours microbial colonization was determined following ultrasonic oscillation.
An examination of a strand of 30,000 and 70,000 year old mammoth hair
Authors: Hammond Ernest C. , Jones Curṫis
Published: June 2020

The Scanning Electron Microscopy Laboratory has received two strands of hair from a Mammoth. These strands were obtained from the Smithsonian Institution in Washington D.C. One 70,000 year old strand from North Yakutia which was a part of the former USSR, is a clear smooth yellowish strand. While, the other strand which is approximately 30,000 years old, under the Electron microscope seems to have a rough surface. This strand is from Muck, Alaska. The 70,000 year old strand from USSR has unusual scratches, but in comparison with human hair of today it seems to display some of the tree bark growth phenomenon observed in human hair. The 30,000 year old strand of hair seem to have unusual rocky structures attached to shaft with large pits and channels. X ray analysis of the 70,000 year old Russian strand seem to indicate large deposits of calcium, iron, copper, and sulfur. Further, the 30,000 year old strand shows copper, iron, calcium and potassium with large quantities of silicon and fluorine.
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.
Observation of ripple conformations of pβ’ phase of dimyristoyl phosphatidylcholine with the scanning tunneling microscope
Authors: Woodward John T. , Zasadzinski Joseph A. N.
Published: June 2020

The Pβ’ or ripple phase of lecithin-water systems was first observed with x-ray diffraction by Tardieu, Luzzati and Reman in the early 1970's. The ripple phase has been observed in numerous studies using x-ray diffraction, freeze-fracture electron microscopy, and other spectroscopies. Electron microscopy on lecidiins far above full hydration showed the existence of a second ripple conformation nearly twice the wavelength of the ripples observed below full hydration leading to the designation of Λ and Λ/2 ripples. Yet none of these measurements have been able to provide a direct unambiguous measurement of the ripple amplitude and conformation. By modifying the technique of freeze-fracture replication to make it compatible with the scanning tunneling microscope (STM) we have observed both the Λ/2 ripple conformation and, for the first time with an STM, the Λ ripple conformation of dimyristoyl phosphatidylcholine (DMPC) in water.
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.
Towards quantitative simulations of inelastic electron diffraction patterns and images
Authors: Wang Z. L.
Published: June 2020

A new dynamical theory has been developed based on Yoshioka's coupled equations for describing inelastic electron scattering in thin crystals. Compared to existing theories, the primary advantage of this theory is that the incoherent summation of the diffracted intensities contributed by electrons after exciting vast numbers of different excited states has been evaluated before any numerical calculation. An additional advantage is that the phase correlations of atomic vibrations are considered, so that full lattice dynamics can be combined in the phonon scattering calculation. The new theory has been proven to be equivalent to the inelastic multislice theory, and has been applied to calculate energy-filtered diffraction patterns and images formed by phonon, single electron and valence scattered electrons.

A calculated diffraction pattern of elastic and phonon scattered electrons for a parallel incident beam case is in agreement with the one observed (Fig. 1), showing thermal diffuse scattering (TDS) streaks and Kikuchi pattern.
Quantitative strain and structure factor phase measurement by cbed.
Authors: Spence J.C.H. , Zuo J.M.
Published: June 2020

CBED is highly efficient - a large amount of quantitative information is obtained from an extremely small volume. Different regions of the pattern contribute different kinds of information. Outer reflections and HOLZ line intensities may be refined for atomic positions and temperature factors, inner reflections for bonding effects, and HOLZ line positions for lattice parameters and strain. Recently we have automated the process of comparing elastically filtered intensities with Bloch-wave and dynamically corrected kinematic computations by combining them with the “Simplex” leastsquares refinement algorithm, as used in the Reitvelt method. Adjustable parameters may be structure factors, cell constants, atomic position parameters etc. Perturbation methods are used for weak beams and error matrix elements.

Strains due to oxygen disorder near a grain boundary in YB2Cu3O7-x have been measured on a 2 nm scale with an accuracy of 0.015% using HOLZ line intersection measurements. From these, the oxygen deficit x may be mapped out. A CBED pattern near [0-12] was recorded using an EM400ST FEG microscope and digitized into SEMPER.
Calculated dynamical scattering in a fibrous protein structure
Authors: Dyson P.W. , Spargo A.E.C. , Tulloch P.A. , Johnson A.W.S.
Published: June 2020

Despite many technical, experimental and in principle difficulties, particularly in regard to radiation damage and stability of the structure at the atomic level, considerable progress has been made in the application of the combined electron microscopy/electron diffraction (EM/ED) technique to the solution of protein structures. The technique now represents a possible alternative to x-ray diffraction for those cases where the protein is difficult or impossible to grow as macrocrystals, provided that the molecule can be incorporated in microcrystals or two-dimensional ordered arrays. Encouraged by this we have recently recorded images and diffraction intensities from microcrystals of an alpha-helical coiledcoil protein extracted from the ootheca of the praying mantis. The ED data shows reflections out to beyond 2Å, though not uniformly in all directions. Fourier transforms of the corresponding digitised images show resolution out to nearly 3Å and this data is being used to phase the ED data. All data was recorded from frozen-hydrated specimens at 400kV in a super-fluid helium specimen stage of a JEM 4000EX electron microscope using spot-scan and low-dose imaging techniques. Further work aimed at collecting a 3-d data set to a resolution of 3Å using tilted crystals is continuing.
Thickness dependence of higher-order laue zone line positions at strongly dynamic zone axes
Authors: Mansfield John , Bird David , Saunders Martin
Published: June 2020

The positions of Higher Order Laue Zone (HOLZ) Lines, visible in the bright field discs of Convergent Beam Electron Diffraction (CBED) patterns, are frequently used to determine the lattice parameters or strain present in phases observed in the Analytical Electron Microscope (AEM). The technique usually relies upon the use of a “standard” sample of known lattice parameter that has the same crystal structure as the unknown phase and is of similar average atomic number. The relative movement of particular HOLZ lines in the unknown phase with respect to the standard are used to calculate the difference in lattice parameters between the standard and the unknown. The thickness of the samples is not taken into consideration. This paper describes the results of both computer simulations and cold stage experiments in the AEM which lead us to conclude that the HOLZ lines are not independent of thickness, particularly in the presence of highly dynamical zero layer diffraction effects.
Quantitative analysis of diffuse scattering with imaging plate
Authors: Shindo D. , Ohishi T. , Iijima S. , Hiraga K. , Oikawa T. , Kerskei M.
Published: June 2020

The excellent properties of the imaging plate (IP), i.e., a wide dynamic range and good linearity for the electron intensity, are promising for quantitative analysis of diffuse scattering since they allow a small dynamical diffraction effect to be evaluated with ‘dynamical factor’. In this paper, we first present accurate measurement of thermal diffuse scattering (TDS), which contributes dominantly to the background of electron diffraction patterns. Secondly, we present a method of extracting weak signal scattering intensities from the background, and apply it to the analysis of diffuse scattering caused from short-range ordered structures.

Electron microscope images were obtained with a JEM-2000EXII electron microscope. Image processing was carried out by using a computer system (ACOS 2020) at Tohoku University. Electron diffraction patterns were recorded by using the TEM-IP system (PIXsysTEM). The details of the IP data handling were presented in the previous paper.

Figure 1 shows an electron diffraction pattern obtained from an Au thin film ( t ∼ 40nm) at room temperature.
The nano-probe profile of the hf-2000 field-emission TEM
Authors: Hashimoto T. , Joy D.C.
Published: June 2020

The probe profile is as important as the probe size for nano-meter area X-ray analysis. Assuming the electron distribution of the probe as the Gaussian, the probe size d is usually defined as the full width half maximum (FWHM) of the probe distribution, which is suitable for SEM/STEM resolution. However, for the X-ray analysis of thin specimens, the full width tenth maximum (FWTM) which contains 90 % of the incident electrons, and thus 90 % of the × rays generated, is more suitable. For a Gaussian probe FWTM = 1.82 × FWHM. Recent electron microscopes with higher than 200 kV acceleration voltage have enough image resolution to observe objects of a few nm such as precipitates at a grain boundary or small particles on a catalysis surface. They are so small that it have been difficult to aquire statistically sufficient amount of x-ray signals with thermionic TEMs. To obtain larger probe current, larger condenser apertures are used and the condenser lens excitation is stronger so that the demagnified electron source image is formed on the specimen.
Imaging dislocations with an annular dark-field detector
Authors: Liu J. , Cowley J. M.
Published: June 2020

High-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM) has been recently the subject of active research because of its successful applications to the characterization of supported catalysts, interface problems in MBE grown semiconductors, superconductors and X-ray multilayers. The characteristics of HAADF images are different from those of the TEM images. For perfect single crystals the HAADF signal is mainly generated from thermal diffuse scattering. HAADF technique has also been used to study dopant contrast effects in semiconductors and dislocations have also been observed with an ADF detector. In this paper we report a study of dislocation contrasts and their dependence on the inner collection angle of the ADF detector.

The STEM instrument used for the observations was the HB5 from VG Microscopes, Ld., modified by the addition of an ultra-high resolution pole piece (Cs = 0.8 mm) and a two-dimensional detector system. Post specimen lenses and various beam stops were used to change the inner (α) and outer (β) collection angles of the ADF detector.
Corrections for surface films in microanalysis by EDS and EELS
Authors: Bentley J. , Kenik E. A.
Published: June 2020

Common artifacts on analytical electron microscope (AEM) specimens prepared from bulk materials are surface films with altered structure and composition that result from electropolishing, oxidation, hydrocarbon contamination, or ion milling (preferential sputtering or deposition of sputtered specimen or support material). Of course, the best solution for surface films is to avoid them by improved specimen preparation and handling procedures or to remove them by low energy ion sputter cleaning, a capability that already exists on some specialized AEMs and one that is likely to become increasingly common. However, the problem remains and it is surprising that surface films have not received more attention with respect to composition determination by energy dispersive X-ray spectrometry (EDS) and electron energy loss spectrometry (EELS).

For EDS, an effective first-order correction to remove the contribution of surface films on wedge shaped specimens is to subtract from the spectrum of interest a spectrum obtained under identical conditions (probe current, diffracting conditions, acquisition live time) from a thinner region of the specimen.
Determination of the detection efficiency of an UTW detector and modelling of spectra used for the quantification of EDS analyses of light elements
Authors: Hovington Pierre , L'Espéiance Gilles
Published: June 2020

The increased use of Si(Li) energy dispersive x-ray spectrometers (EDS) with a windowless (WL) or ultra thinwindow (UTW) detectors has made the detection of light elements down to Be a routine laboratory procedure. The quantification of EDS spectra implies that the net intensity of the characteristic peak of interest must be determined accurately. For light-element x-rays for which the background is non linear and for which the Kα lines can often overlap with the L and M lines of transition metals, the simple determination of the characteristic intensity is not a straightforward operation and often requires sophisticated data processing. The use of a conventional least-squares fitting technique with prefiltering of standard spectra for deconvolution is made difficult by the non-linearity of the background, the presence of the triggered noise peak below 0.3 KeV, and the lack of appropiate standards. In addition to the difficulty associated with the determination of the net intensity, the uncertainties for the detection efficiency below 1 Kev has limited the use of standardless procedures for quantification.
A data base for energy loss cross sections and mean free paths
Authors: Egerton R.F.
Published: June 2020

For quantitative elemental analysis by standard EELS techniques, we require inner-shell cross sections integrated over an energy range Δ beyond the ionization edge. These cross sections are conveniently parameterized in terms of a dipole oscillator strength f(Δ) which is independent of incident energy Eo and collection semi-angle β.

Values of f(Δ) are obtainable from EELS measurements, from theoretical models and from photoabsorption data. Compilation of this data allows one to obtain a best estimate of a given cross section, together with an estimate of the probable error. Consistency is good for K-shells, allowing f(Δ) to be predicted to within 5% accuracy. At the opposite extreme, discrepancies of an order of magnitude exist for the N- and O-shells (Fig.1). In general, there is more consistency with regard to edge shape, as expressed by the ratio ϕ(Δ)=f(Δ)/f(100); see Fig.2.

If microanalysis is carried out by matching to stored edge profiles, the convenient quantity is the differential oscillator strength df/dE. Such data is available directly from atomic calculations and, for some edges, from experimental measurements; see Fig.3.