bluuebunny/crossref_metadata_2025 huggingface dataset:
Achieving atomic resolution in the Transmission Electron Microscope
Authors: Ponce F. A. , O'Keefe M. A.
Published: June 2020

In the past few years, the objective of achieving point resolution in the electron microscope comparable to the atomic separation in solids has been reached. The new high and mid-voltage instruments claim resolving powers of better than 0.20 nm, which is less than the nearest-neighbor distance in most solids. It is therefore relevant to consider the meaning of point resolution and the possibilities of achieving it. There are two important areas which are need to be considered: (a) electron optics, and (b) the specimen.
Kinematical electron diffraction simulations of quasicrystalline structures
Authors: Knowles K.M. , Stobbs W.M.
Published: June 2020

Since the initial report by Shechtman et al of a phase in a rapidly cooled Al-Mn alloy whose electron diffraction patterns exhibit icosahedral symmetry, a number of related alloys have now been discovered which exhibit this symmetry. There is now no doubt that such phases can be readily distinguished from multiply twinned particles. One interpretation of the structure of these phases is that they arise by a suitable atomic decoration of a three dimensional Penrose tiling (3DPT), in which space is filled aperiodically by prolate and oblate rhombohedra with equal sides and angles of ± acos. The diffraction patterns from the tilings themselves are known to exhibit the required symmetries. However, the details of the ‘correct’ atomic decoration remains an open question, although a variety of approaches have been tried. Solving this occupancy problem is of particular interest in HREM image interpretation, since if the appropriate decoration turns out to depend on the local environment, it will be desirable to assess the relative importance of dynamical and projection effects.
Real-time computer simulation of electron microscope images via a digital television frame store system
Authors: Krakow W.
Published: June 2020

Digital television frame store devices and software packages have made it possible to obtain images directly from electron microscopes, photographic prints and transparencies in real time and obtain the power spectrum (optical diffraction pattern) and filtered image of various electron micrographs. Enhancements have been added to the Fourier analysis program package which include the use of offset filter functions and the computation of high resolution electron microscope images by including the microscope lens aberration phase shifts and illumination conditions. Because of the use of the frame store and a large mainframe computer, it is possible to have several orders of magnitude gain in image computational speed which makes real-time interactive computations possible.
Simulations of high-resolution images of amorphous silicon films
Authors: Fan G. Y. , Cowley J. M.
Published: June 2020

It is well known that the structure information on the specimen is not always faithfully transferred through the electron microscope. Firstly, the spatial frequency spectrum is modulated by the transfer function (TF) at the focal plane. Secondly, the spectrum suffers high frequency cut-off by the aperture (or effectively damping terms such as chromatic aberration). While these do not have essential effect on imaging crystal periodicity as long as the low order Bragg spots are inside the aperture, although the contrast may be reversed, they may change the appearance of images of amorphous materials completely. Because the spectrum of amorphous materials is continuous, modulation of it emphasizes some components while weakening others. Especially the cut-off of high frequency components, which contribute to amorphous image just as strongly as low frequency components can have a fundamental effect. This can be illustrated through computer simulation. Imaging of a whitenoise object with an electron microscope without TF limitation gives Fig. 1a, which is obtained by Fourier transformation of a constant amplitude combined with random phases generated by computer.
Microstructure of an extruded rapidly solidified Al-8Fe-2Mo alloy
Authors: Donlon W. T. , Allison J. E. , Shinozaki S.
Published: June 2020

Light weight materials which possess high strength and durability are being utilized by the automotive industry to increase fuel economy. Rapidly solidified (RS) Al alloys are currently being extensively studied for this purpose. In this investigation the microstructure of an extruded Al-8Fe-2Mo alloy, produced by Pratt & Whitney Aircraft, Goverment Products Div. was examined in a JE0L 2000FX AEM. Both electropolished thin sections, and extraction replicas were examined to characterize this material. The consolidation procedure for producing this material included a 9:1 extrusion at 340°C followed by a 16:1 extrusion at 400°C, utilizing RS powders which have also been characterized utilizing electron microscopy.
Electron diffraction patterns from the T1-phase in Al-Li-Cu alloys
Authors: Eikum A. K. , Narayanan G. H.
Published: June 2020

Aluminum-lithium-copper alloys of commercial interest are strengthened by precipitation of several metastable phases that include δ (Al3Li), δ' (Al2CuxLi(1-x)) and T1' (Al2-xCuLi). The analysis of diffraction patterns from δ (L12 superlattice) and δB' (BCT lattice) phases is fairly straight forward. However, interpretation of diffraction patterns from the T1' phase is more complex. The observed patterns cannot be readily understood in terms of the hexagonal structure of the equilibrium T1 phase, which recently prompted a discussion of T1' based on an orthorhombic structure. This paper presents an alternate interpretation of the observed selected area diffraction patterns from T1'.
Domain coarsening by grain boundary migration in ordered Ni4Mo
Authors: Vasudevan K. , Kao H. P. , Brooks C. R. , Stansbury E. E.
Published: June 2020

The Ni4Mo alloy has a short-range ordered fee structure (α) above 868°C, but transforms below this temperature to an ordered bet structure (β) by rearrangement of atoms on the fee lattice. The disordered α, retained by rapid cooling, can be ordered by appropriate aging below 868°C. Initially, very fine β domains in six different but crystallographically related variants form and grow in size on further aging. However, in the temperature range 600-775°C, a coarsening reaction begins at the former α grain boundaries and the alloy also coarsens by this mechanism. The purpose of this paper is to report on TEM observations showing the characteristics of this grain boundary reaction.
Microstructdre of laser-hardened medium carbon/chromium steels
Authors: Kusinski J. , Thomas G.
Published: June 2020

Research on the effect of heat-treatment on microstructure and mechanical properties of Fe/Cr/C/X steels show that improvements in toughness, with little or no change in strength may be realized by proper design of a composite microstructure to achieve fine grained martensite surrounded by stable austenite films. Laser processing can be expected to further modify the mechanical properties of these steels because it involves exteme rates of heating and cooling that cause unusually high restraints in the heated layer of the material. The ultrafine. and homogeneous structure and residual stresses developed in the laser-heated (LH) layer may improve fatigue, hardness, abrasive and erosive wear and corrosive resistance of the material.
Characterizatiom of spinodally decomposed Fe-30.1% Cr-9.9% Co: Part 1
Authors: Horton L. L. , Miller M. K. , Spooner S.
Published: June 2020

Transmission Electron Microscopy (TEM), Atom Probe Field-Ion Microscopy (APFIM) and Small Angle Neutron Scattering (SANS) have been used to characterize the microstructure of a commercial Fe-30.1 at.% Cr-9.9 at.% Co alloy. One goal of this investigation was to demonstrate the complementary nature of these techniques in solving a difficult materials problem, the characterization of fine scale spinodally-decomposed structures. The alloy was solution treated at 925°C for 140 min followed by water quenching, isothermal aging at 525°C for times of 2, 8, 24, 72, 192, or 485 h and a final water quench. TEM characterizations were performed with Philips EM400T/FEG, Philips EM430T and JEM 120C instruments.
Transmission Electron Microscope characterization of a slip propagation event in alpha brass
Authors: Bamford T.A. , Clark W.A.T. , Wagoner R.H.
Published: June 2020

Slip propagation through grain boundaries is a process which is, in part, responsible for grain size strengthening. TEM characterization of a slip propagation event combined with quantitative stress analysis allows determination of the critical stress and micromechanism for the event. Such an analysis is presented here.

Commercially pure alpha brass(30 w/o Zn) was rolled to 125um and was then annealed in a dry hydrogen atmosphere at 800°C for one hour. The material was lightly deformed, and samples prepared using standard electopolishing techniques. This was then examined in a JEOL 200CX operating at 200keV.
ESEM - A multipurpose surface Electron Microscope
Authors: Danilatos G.D.
Published: June 2020

Over recent years a new type of electron microscope - the environmental scanning electron microscope (ESEM) - has been developed for the examination of specimen surfaces in the presence of gases. A detailed series of reports on the system has appeared elsewhere. A review summary of the current state and potential of the system is presented here.

The gas composition, temperature and pressure can be varied in the specimen chamber of the ESEM. With air, the pressure can be up to one atmosphere (about 1000 mbar). Environments with fully saturated water vapor only at room temperature (20-30 mbar) can be easily maintained whilst liquid water or other solutions, together with uncoated specimens, can be imaged routinely during various applications.
Intracellular colloids as charge conductors for uncoated SEM samples
Authors: Handley D. A. , Jeevanandam V. , Chien S.
Published: June 2020

Evaporated gold or gold-palladium are used as surface coatings to prevent electrical charging or thermal distortion to specimens during SEM viewing. However, during evaporation, metal coatings may induce specimen heating, damage delicate surface structures, and obscure fine detail. To avoid these inherent limitations, we developed a method for forming gold colloids within cells of chemically fixed specimens. Correlative studies of thin section TEM and surface SEM of carotid artery samples are provided to demonstrate the efficacy of this method.
Chromium deposition by sputtering
Authors: Specht R. C. , Lutz G. J.
Published: June 2020

Previous works have shown the benefits of depositing ultrathin films (0.5-3.0 nm) of chromium onto SEM specimens to permit improved resolution at higher magnification. The finer grain structure of the chromium permits closer replication of the specimen surface topography than is possible with the conventional 10 nm of gold. This paper presents the results of an ongoing program at Denton Vacuum to develop guidelines for successful, repeatable deposition of ultrathin chromium films by sputtering.

Chromium can be deposited using a high vacuum evaporator by either evaporation of chips from baskets or sputtering. Sputtering is the more desirable method since it has the potential for better rate control, coverage, repeatability and least specimen exposure to radiant heat.
The use of convergent-beam electron diffraction to determine the influence of anodization of a metal substrate
Authors: Bowman Keith J.
Published: June 2020

Research by Gibala and co-workers has revealed that the application of thin oxide films can substantially reduce the flow stresses of bcc metals at low temperatures. Because the extent of this change in flow stress has been related to the stresses generated at the oxide/metal interface, the sign and magnitude of these stresses are of interest. The experiment described in this paper was used to ascertain the potential of using center-disk HOLZ lines from CBED experiments to measure elastic stresses induced by anodic film formation. The power of CBED for measuring small changes in lattice parameter has been discussed by Steeds.

An electron beam zone refined and vacuum annealed [110] tungsten single crystal was diamond sectioned to 200 μm. The foil was hand polished to about 100 μm and electrothinned in a Tenupol twin jet polisher with a 2% NaOH solution at 25°C.
Electron microprobe analysis: The upper limit of submicron spectroscopy
Authors: Hlava Paul F. , Chambers William F.
Published: June 2020

In the electron microprobe, a beam of high energy electrons is focussed to a fine point on the surface of a fairly thick specimen and the x rays produced are analyzed to determine the chemistry of the "point". The spa- cial resolution of this instrument for chemical analysis, then, is defined by the volume of material from which the x ray signal originates. This, in turn, is related to factors such as the diameter of the electron beam, the spreading of the electron beam as it penetrates into the sample and interacts with the atoms of the sample to generate x rays, and the extent to which these primary x rays penetrate beyond the region of electron beam interaction and generate secondary x rays by the process of fluorescence. Beam voltage, current, and diameter can all be easily controlled by the analyst. Once the electrons enter the specimen, however, the analyst loses control of their density. Each individual electron follows a unique, erratic path as it passes near, passes through, or collides with parts of the atoms in the specimen. Monte Carlo calculations are a means by which many investigators have tried to model the paths of individual electrons and the interation volume that large numbers of such electrons define.3 “* It is well known that the size and shape of the region into which the electron beam penetrates and expends its energy is controlled primarily by the average atomic number, atomic weight, and density of the specimen in the region of interest and the beam voltage.
AEM-based XEDS/EELS microanalysis: A materials science perspective
Authors: Zaluzec Nestor J.
Published: June 2020

In 1966 Elad and Nakamura first described the use of a solid state device made out of silicon for use as a detector system in discriminating x-ray photons of differing energy. Since that time the growth and applications of the now universally accepted Si(Li) detector and its analysis technique X-ray Energy Dispersive Spectroscopy (XEDS) on electron column devices has been almost phenomenal. Nearly 50% of the electron microscopes sold today are routinely equipped with this peripheral for use as either a qualitative and/or quantitative analysis tool. An even older analytical technique Electron Energy Loss Spectroscopy (EELS) was first explored in the early 1940's by Hillier and Baker, and then rejuvenated in the late 1960's by a variety of laboratory's worldwide. For both of these analytical methodologies, the applications run the gamut from such routine aspects as determining the nominal composition of a specimen, through the more specific application such as measurement of elemental segregation at boundaries and interfaces, to the more esoteric applications which, for example, attempt to determine the relative atomic occupancy of sites within a crystalline lattice. Each of these modes of operations is well within the grasp of any microscopist once he or she fully appreciates the methodology and limitations of the instrumentation, technique and data analysis procedure.
EDA and ELS in the AEM - A biological perspective
Authors: Johnson D. E.
Published: June 2020

The purpose of this symposium/tutorial presentation will be to assess the current state and future prospects of both energy dispersive x-ray analysis and electron energy loss analysis in the submicron spectroscopy of biological systems.

In the successful use of any analytical technique in a biological study, the specimen preparative steps are crucial to the ultimate validity of the results. Nowhere is this more true, than in the analytical electron microscope. Fortunately, current techniques of cryofixation, cryosectioning and analysis, including freeze drying, have developed such that morphology and elemental distributions are typically preserved to a spatial resolution at least as good as the analytical and electron optical instrumentation allow.
HRAEM investigation of europium intercalated titanium disulphide
Authors: Long N.J. , Hsu S.P. , Glaunsinger W.S.
Published: June 2020

The intercalation of TiS2 with europium-ammonia has been the subject of an extensive investigation. It was shown that there can be up to four intercalates (Eu3+, Eu2+, NH3 and NH4+), depending on the synthesis conditions and composition. The europium and ammonia intercalates should expand the interlayer separation (equal to c/3) by approximately 0.2nm and 0.3nm, respectively. We report below the results from preliminary attempts to image the intercalated structure directly (TEM) and to determine the valence state of the europium (EELS), using a Philips FEG 400ST with a Gatan energy loss spectrometer.
Use of a protective film to assure the integrity of asbestos samples collected for TEM analysis
Authors: Lentzen D. E. , Dunmyre G. R.
Published: June 2020

Air samples which have been collected on a Nuclepore™ filter for TEM asbestos and other particulate analysis may lose fibers or other particulate during normal transport and handling conditions. Various reports and observations confirm that the distribution of collected particulates may be altered due to static electricity charge effects, physical dislocation, and even some subsequent analytical procedures

A method to help assure particulate sample integrity during transport is presented here. It involves enveloping the freshly taken sample between two electron transparent films. The first film is evaporated onto a Nuclepore™ filter of the chosen diameter and pore size. Vacuum deposit about 40 nm of carbon onto the collection side of the filter without polymerizing the polycarbonate by overheating. Load this filter in the sampling cassette under clean room conditions and store for transport to the field. Next, cast an electron transparent film of collodion in amyl acetate on a clean water surface. Select a portion of the cured film that is even, colorless, and flawless to pick up on the base end of the middle piece of a second cassette.
Freeze-etch TEM of aqueous polymer gel network morphology
Authors: Zasadzinski J. A. N. , Prud'homme R. K.
Published: June 2020

The rheological and mechanical properties of crosslinked polymer gels arise from the structure of the gel network. In turn, the structure of the gel network results from: thermodynamically determined interactions between the polymer chain segments, the interactions of the crosslinking metal ion with the polymer, and the deformation history of the network. Interpretations of mechanical and rheological measurements on polymer gels invariably begin with a conceptual model of,the microstructure of the gel network derived from polymer kinetic theory. In the present work, we use freeze-etch replication TEM to image the polymer network morphology of titanium crosslinked hydroxypropyl guars in an attempt to directly relate macroscopic phenomena with network structure.
The formation of amorphous interlayers by solid-state interdiffusion between Pd-Er and Si
Authors: Boothroyd C.B. , Stobbs W.M.
Published: June 2020

Interest has been maintained in the way silicides form between a deposited metal and silicon because the relationship between the contact potential and boundary reaction remains obscure. This is partially because the cleanliness as well as the vicinality of the silicon surface can change the temperature dependence and homogeneity of the reaction products and thus the presence of localised strains in the silicon. Chemical surface analysis and electrical measurements thus need careful correlation with microstructural examination and it was with this in mind, at the suggestion of K.N. Tu, that we followed up the work of his group on the behaviour of a rare earth-noble metal alloy pair on silicon (Pd-Er/Si) with a microscopical study. The more interesting results are summarised below.
β-conductivity imaging of (Hg,Cd)Te LPE films
Authors: Moore T.M. , Beck J. , Liu H-Y. , Schaake H.F. , Weirauch D.F.
Published: June 2020

Conductive mode imaging of semiconductors in the SEM can include contributions to the image signal from the absorbed primary current, from the electron voltaic effect (e.v.e.), and from electron-beam-induced conductivity (β-conductivity). Under the conditions of low primary beam current, no internal source of e.m.f., and an externally applied bias, local variations in the β-conductivity signal can be detected. An earlier study has concluded that localized contrast in β-conductivity micrographs of Ge is primarily due to variations in the rate of carrier recombination.
The crystalline-amorphous-crystalline transformations in the formation of FeSi2
Authors: Vanderwalker D. M.
Published: June 2020

The crystalline to amorphous transformation which occurs as a result of interdiffusion of Au and La was first observed by Schwarz and Johnson. This study will examine the crystalline-amorphous-crystalline transformations in the formation of FeSi2.

A layer of Fe was evaporated on Si wafers when the chamber pressure was below 10-6 torr. The samples were prepared for TEM by mechanical and chemical thinning with 6/7 nitric acid 1/7 hydrofluoric acid solution. The TEM was performed on the JEM 200 CX at Stony Brook.
Defect structure in Co-W thin films
Authors: Admon U. , Dariel M.P , Grünbaum E. , Kimmel G.
Published: June 2020

Thin films of Co-W, 300-500Å thick, electrodeposited unto polycrystalline copper substrates (the details of the deposition process are given in ref. 1), consist of a background of small grains of size of the order of the film thickness, in which 10-20 times larger grains are dispersed. These large grains display a predominant [0001]hcp or [001] or [lll]fcc texture. The large grains are mostly faulted, the faults appearing as parallel stripes running across their whole width (Fig. 1). These stripes have been identified by SAD and DF as being alternating hep and fee platelets. The diffraction pattern (Fig. 2), taken from grain A in Fig.1, indicates a (0001)h||(001)c||substrate surface, with [1010]h||[110]c orientation relationship between the hep and fee platelets (relationship I). The streaking and the appearance of the {200} cubic spots as cross shaped satellites is attributed to the presence of stacking faults on the {111} planes.
Microanalytical investigation of sintered FE-Nd-B magnets
Authors: Ramesh R. , Bielicki T. A. , Thomas G.
Published: June 2020

Fe-Nd-B is emerging as an important hard magnetic alloy system. The magnetic properties, in particular, the coercivity, are strongly influenced by the microstruetura1 condition of the magnet and are subject to degradation by various factors which can be understood only with the use of analytical electron microscopy. This investigation focuses on the characterization of inclusions discovered within a magnet which exhibits a kinked B-H loop.(Fig.1). Based on the microstructural investigation, an appraisal is made of the effect of these particles on the magnetic properties.
Morphology of as-precipitated and dried powders in the PLZT system
Authors: Schwartz R. W. , Payne D. A.
Published: June 2020

High quality ceramic products, such as transparent electro-optic materials, require better control of chemical composition, powder particle size and morphology, than is offered by conventional mixed-oxide ceramic processing methods. Chemical preparation procedures can be used to prepare powders with the required characteristics of controlled size and morphology.

Precipitated hydroxides in the PLZT system (lead lanthanum zirconium titanate) were prepared in a constant volume continuous reactor, following a procedure suggested by Murata, which uses nitrate and chloride precursors, and ammonium hydroxide for pH control. After washing with deionized water, the powders were dried by either spray-drying or freeze-drying in liquid nitrogen.
High Resolution Electron Microscopy studies of the degradation process of the calcium A zeolite
Authors: Acosta N. D.R.
Published: June 2020

The change from the crystalline to the amorphous state of the sodium A zeolite, has been investigated by Bursill and by Thomas. From individual HREM pictures those authors suggest three different mechanisms responsible for the degradation process. In the present work the degradation of the calcium A zeolite, characterized also by Auger spectroscopy, was studied by HREM methods. The sample was under thermal treatment at 450°C during four hours in a vacuum line after which it was mounted in the microscope in a nitrogen atmosphere.
An analytical microscopic study of metals in fluidized catalytic cracking catalyst
Authors: Rainey Clifford S.
Published: June 2020

The spatial distribution of V and Ni deposited within fluidized catalytic cracking (FCC) catalyst is studied because these metals contribute to catalyst deactivation. Y zeolite in FCC microspheres are high SiO2 aluminosilicates with molecular-sized channels that contain a mixture of lanthanoids. They must withstand high regeneration temperatures and retain acid sites needed for cracking of hydrocarbons, a process essential for efficient gasoline production. Zeolite in combination with V to form vanadates, or less diffusion in the channels due to coke formation, may deactivate catalyst. Other factors such as metal "skins", microsphere sintering, and attrition may also be involved. SEM of FCC fracture surfaces, AEM of Y zeolite, and electron microscopy of this work are developed to better understand and minimize catalyst deactivation.
On the geometrical relationship between Kikuchi line position and excitation error in electron diffraction
Authors: Ling P. , Gronsky R.
Published: June 2020

All of the main features of the geometry of Kikuchi lines can be thoroughly explained using the treatment first proposed by Kikuchi in which the observed diffraction lines are considered to arise from the elastic iBraggi scattering of electrons that had been previously scattered inelastically by the specimen. The positions of the lines occur at the intersection of Kikuchi cones or Kossel cones with the Ewald sphere, giving an accurate indication of the orientation of the specimen relative to the incident beam direction, and providing a rapid means (inspection) of deducing the sign of the deviation parameter, s. Less obvious from these traditional presentations however is the actual magnitude of s. This paper presents an alternative geometrical construction employing multiple Ewald spheres to illustrate the phenomenon of multiple scattering which is responsible for the formation of Kikuchi lines, and to provide a straightforward derivation of the excitation error from the relative positions of the Kikuchi lines and their corresponding diffraction spots.
An integrated computer system for STEM data acquisition and processing
Authors: Hainfeld James F. , Monson Keith L.
Published: June 2020

Several laboratories have reported digital data acquisition and control systems for electron microscopes. The considerations involved in implementation of such a system have been reviewed. All of these systems have used a relatively small minicomputer, either as a stand-alone, or as a satellite processor linked to a larger computer, so as not to burden the large host with the mundane task of operating a microscope. In recent years however, hardware costs have decreased sufficiently that many laboratories have their own superminicomputer (e.g., one of the Digital VAX series), or share one within a small group of users.
An Experimental Study of the Hyperbolic Electron Mirror
Authors: Rempfer G. F. , Mauck M. S.
Published: June 2020

The spherical and chromatic aberrations of electron mirrors are of opposite sign from those of electron lenses. This important property makes it possible in principle to design mirrors for correcting aberrations in electron lens systems. The hyperbolic electron mirror is an ideal model for the study of electron mirrors since the trajectories can be determined analytically, and the results can be compared with experimental results for essentially the same mirror field. In an earlier paper we reported the results of a theoretical study of the hyperbolic mirror. In the present paper we report on the experimental aspects of this study.
Molecular cytochemistry of freeze-fractured cells
Authors: da Silva Pedro Pinto
Published: June 2020

I will describe four approaches that combine cytochemistry with freeze-fracture: 1) FREEZE-ETCHING; 2) FRACTURE-LABEL; 3) FRACTURE-PERMEATION; and 4) LABEL-FRACTURE. These techniques, in particular fracture-label, involve delicate points of interpretation and numerous validating controls. In the publications listed at the end, these issues have been addressed in detail.

1. FREEZE-ETCHING. I developed freeze-etching as a cytochemical approach to prove that membranes were split by freeze-fracture and to show that biological membranes were comprised of a bilayer membrane continuum interrupted by integral membrane proteins.1 - 4 In freeze-etching, the distribution of the marker over the membrane surface exposed by sublimation is compared to that of the intramembrane particles exposed by fracture. It is often required to aggregate the particles into domains larger than the labeling molecules (Fig. 1). This, and the need for freezing in distilled water, severely limits the application of freeze-etching.
Criteria for the evaluation of low-temperature Scanning Electron Microscopy
Authors: Beckett Alan
Published: June 2020

Low temperature scanning electron microscopy (LTSEM) has been evaluated with special reference to its application to the study of morphology and development in microorganisms. A number of criteria have been considered and have proved valuable in assessing the standard of results achieved. To further aid our understanding of these results, it has been necessary to compare those obtained by LTSEM with those from more conventional preparatory procedures such as 1) chemical fixation, dehydration and critical point-drying; 2) freeze-drying with or without chemical vapour fixation before hand.

The criteria used for assessing LTSEM for the above purposes are as follows:

1)

Specimen immobilization and stabilization

2)

General preservation of external morphology

3)

General preservation of internal morphology

4)

Exposure to solvents

5)

Overall dimensional changes

6)

Cell surface texture

7)

Differential conformational changes

8)

Etching frozen-hydrated material

9)

Beam damage

10)

Specimen resolution

11)

Specimen life
Ultrastructural diagnosis of a thyrotroph cell adenoma of the pituitary
Authors: Siew Shirley , Mueller Willys F. , Fratkin Jonathan D.
Published: June 2020

Thyrotroph cell adenoma is an extremely rare neoplasm, comprising less than 1% of pituitary tumors.

We report a case of a 42 year old woman, who had presented first at the age of 33 with the chief complaint of blurred vision in the left half of her visual field. Examination showed the presence of bitemporal homonymous hemianopsia and optic atrophy in the left eye. CT scan, skull x-ray and tomograms showed expansion of the sella turcica with an intrasellar and suprasellar mass. Pancerebral angiogram showed vascular displacement secondary to an avascular sellar and suprasellar mass. There was also a history of secondary amenorrhoea. The follicle stimulating and luteinizing hormone levels were low. Thyroid function tests were within normal limits. A transsphenoidal radical excision of a pituitary adenoma was performed and the pathological diagnosis was a chromophobe adenoma. The patient was followed with periodic CT scans. The present admission followed the finding of a large recurrent pituitary adenoma with recurrence of loss of visual fields. The serum prolactin was normal, T4 was low and TSH was normal.
Freeze etch replication of extracellular bacterial polymers adsorbed onto kaolinite
Authors: Barker William W. , Hurst Vernon J.
Published: June 2020

Freeze etch replication of experimental mixtures of euhedral kaolinite and pure cultures of polysaccharide and protein-producing bacteria reveals that surface textures formed by adsorption of extracellular bacterial polymers onto kaolinite surfaces resemble structures observed in natural estuarine clay rich sediments, where anhedral 0.1 micrometer clay surfaces are covered by anhedral masses and fibrous elements of organic material.

All samples, were cryofixed in a Balzers QFD101 propane jet and replicated in a Balzers 360M freeze etch device. Etching occurred at −100°C for 45 sec. and replicas were created with 15 nm Pt @ 45 °, 15 sec. C @ 90°. Following 24 h digestion in cone. HF, replicas were mounted on Formvar-coated 100 mesh Cu grids, carbon-coated for thermal stability, and examined in a Philips STEM 400.

Anionic mucopolysaccharide produced by pure cultures of encapsulated Klebsiella pneumoniae (ATCC 8044) readily adsorbed to both the basal (negative charge) and edge (positive charge) surfaces of kaolinite (Fig. 1).
Visualizing replicas of molecules made in a fully automated freeze-fracture machine
Authors: Branton Daniel
Published: June 2020

We have designed and constructed an automated, computer driven freeze-etch apparatus that optimizes all of the standard operating procedures through which biological samples are normally processed to create a replica suitable for electron microscopy. Although designed for automated freeze-etching, manual intervention is possible at any step and the apparatus may be used to facilitate any or several sequences of computer driven or manual steps (Figure 1) that render a high resolution replica of a frozen or dried biological specimen.

The gradual evolution and improvement of scientific apparatus are commonplace, but there are some specific reasons why we sought to automate the freeze-etch process:

1) Many are dissuaded from using standard freeze-etch machines because operator training is time-consuming and using the apparatus is laborious;

2) Reproducible results are difficult to obtain and require conscientious attention to a mire of trivial details that are easily remembered by computers;

3) Numerous sub processes, e.g. the speed of fracturing, have not been systematically optimized because formidable record keeping is required to encompass the many subtle variations of conditions sustained by the specimens in a single freeze-etch run;
Comparison of IVEM and quick-freeze, deep-etch, rotary shadow images of the cell cytoplasm
Authors: Larabell Carolyn A.
Published: June 2020

The techniques of freeze-fracture and quick-freeze, deep-etch, rotary-shadow electron microscopy have provided information about cellular structures that cannot be obtained from thin section electron microscopy. Freeze-fracture replicas provide extensive views of membranes and the arrangement of integral membrane proteins, but filamentous structures such as the cytoskeleton and extracellular matrix are not visualized because they are embedded in ice. In order to visualize such fine structures, it is necessary to deeply etch (or freeze-dry) cells that have been rapidly frozen, thus revealing about 0.1 to 0.3 μm of three dimensional structure. Replicas of cells prepared in this fashion provide excellent high magnification views of the individual filaments and their interactions with the plasma membrane as well as with membrane-bounded organelles. Another excellent method for visualization of cytoskeletal structures is whole mount electron microscopy. In this paper, I describe views of the egg cytoskeleton obtained from visualization of the isolated egg cortex prepared as a whole mount at 400 kV and compare these results with those obtained from replicas of cells that have been rapidly frozen men fractured and deeply etched.
Molecular features of heterochromatin condensation
Authors: Hamkalo B.A. , Lundgren K. , Parseghian M.H. , Radic M.Z. , Saghbini M.
Published: June 2020

Eukaryotic chromosomes are nonuniformly condensed in both interphase and metaphase. This difference is most apparent in mitotic chromosomes in which centromeric heterochromatin is distinguishable from euchromatic arms because of a higher degree of condensation. This difference prevails in interphase and has led to the designation of this type of heterochromatin as “constitutive”. Differences in condensation presumably are a consequence of differential stability of higher order chromatin structure. Since higher order structure is likely to be due to distinctive DNA-protein and protein-protein interactions, our goal is to identify components of such interactions as a first step in understanding the molecular basis for differential chromatin condensation.

Analysis of the structure of the mouse major centromeric satellite DNA revealed the presence of stable DNA curvature which could be reversed if certain small ligands which recognize a sequence motif common to this highly repeated DNA were bound to the DNA. In addition, growth of cells in the presence of these ligands prevented complete condensation of centromere regions of metaphase chromosomes (Fig.1).
The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes
Authors: Swedlow Jason R. , Osheroff Neil , Karr Tim , Sedat John W. , Agard David A.
Published: June 2020

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.
Video-light microscopic analyses of centrosome separation in vivo during mitosis in vertebrate cells
Authors: Rieder Conly L. , Waters Jennifer , Cole Richard W.
Published: June 2020

The major microtubule (MT) organizing center of the vertebrate interphase cell is the centrosome. After replication near DNA synthesis it functions as a single unit until mitosis. At the onset of mitosis the cytoplasmic MT complex is resorbed and the replicated centrosomes separate to form the spindle poles. During this time each nucleates a radial array of dynamically unstable MTs known as an aster. After spindle formation the centrosomes separate again during anaphase B to elongate the spindle. The mechanism(s) by which the centrosomes separate during these two mitotic stages remain unknown. The most popular hypothesis is that interacting MTs of opposite polarity, derived from opposing asters, elongate and slide relative to one another through the action of MT plus-end directed motor proteins. Unfortunately, this hypothesis does not explain why centrosomes continue to separate in vertebrate cells when their MT arrays no longer overlap, as in anaphase-like prometaphase (ALP).

Both centrosomes are clearly visible in vivo during mitosis in newt lung cells. The goal of our study was to compare, with high spatial and temporal resolution, the behavior of centrosomes as they separate when their MT arrays overlap during prophase (Fig. 1) and anaphase B (Fig. 3), with when they no longer overlap during ALP (Fig. 2).
Cryo-enhancement of ultrastructural, immunological, cytochemical, and low-voltage SEM characterization of fungal growth and morphogenesis
Authors: Howard Richard J. , Bourett Timothy M.
Published: June 2020

Cells of filamentous fungi exhibit a characteristic somatic form, organized as a linear file of individual cells, known as the hypha. Growth of the fungus occurs primarily through expansion at the apex of the hyphal tip cell, where highly organized and localized exocytosis is thought to be responsible for the deposition of new cell wall. In pathogenic fungi, changes in the pattern of tip growth are the means by which specialized infection structures develop, enabling the fungus to adhere to and penetrate the host surface, establish infection and cause disease. The use of cryo techniques has provided a vastly expanded knowledge of the cellular bases underlying these important morphogenetic events.

Hyphae are easily grown as a monolayer in culture, on pieces of cellophane overlying nutrient agar. These cell monolayers are easily manipulated for plunge freezing with minimal perturbation and no treatment necessary prior to quenching. In this sense, plunge freezing is superior to other cryo-fixation techniques that require pre-treatment or mounting of live specimens onto holders, hats or pistons.
Low-temperature evaluation of high-pressure frozen biological samples by TEM cryosections and high-resolution SEM
Authors: Müller Martin
Published: June 2020

The unique ability of electron microscopy in biology is its power to describe and integrate structural details down to molecular dimensions within the context of a complex living system.

Structural information closely related to the living state may be achieved by cryoimmobilisation techniques that vitrify the cellular water and, at the same time rapidly arrest all physiological processes.

High-pressure freezing (Müller and Moor 198, Moor 1987) is at present the only practical way of cryofixing larger non-pretreated samples up to a thickness of 500 μm. A very high yield of adequately frozen specimens was demonstrated in TEM using suspensions of microorganisms as well as plant and animal tissue (i. e. no detectable effects of ice crystal damage are visible after freeze substitution) (Studer et al. 1989). High-pressure freezing can vitrify biological samples sometimes up to a thickness of 200μm, as extrapolated from electron diffraction of cryosections (Michel et al 1991) The actual thickness that can be vitrified depends on the specimen, particularly on the presence of substances that exhibit cryoprotectant activity and also depends on the optimum transfer of pressure and cold to the sample and on the sample geometry (thickness and shape of the aquous layer, and the mass of the specimen planchettes necessery to protect the biological sample from direct pressure effects).
Microtubule organization in the siphonous green alga bryopsis: An ultrastructural observation with Electron Spectroscopic Imaging (ESI)
Authors: Wei Sheng Jun , Bishop Kyle
Published: June 2020

Transmission electron microscopy (TEM) has been routinely used to study the ultrastructure of microtubules (MT). However, we found that some MTs in the filamentous green alga we examined could not be easily recognized using conventional brightfield imaging, because of low contrast. Electronspectroscopic imaging (ESI) microscopy using the imaging electron energy filter solves many problems involving low contrast and feature resolution. By ESI, the contrast can be tuned and optimized so that images with higher resolution can be produced. In the present study we used ESI to improve the image quality in order to clearly resolve the MTs.

A specimen of the giant marine green alga Bryopsis sp. was prepared following a new modified double fixation procedure of our own to preserve cell structures (Fig. 1). In brief, the branched alga was cut into small segments of about 1 cm in length in 2% glutaraldehyde in 0.1 M phosphate at pH 7.2.
UV absorption microspectroscopy and alkali extraction to characterize aromatic constituents in plant cell walls
Authors: Akin D. E. , Morrison W. H. , Rigsby L. L.
Published: June 2020

Aromatic compounds bound to structural carbohydrates in plant cell walls limit the microbial utilization of otherwise biodegradable polysaccharides. Such limitations have important consequences in conversion of plant biomass to useful products and in carbon recycling. These aromatic constituents are chemically diverse, with further variations occurring in chemical linkages. Microspectroscopy and chemical analysis of alkali-treated plant walls was used to investigate aromatics.

Pearl millet (Pennisetum glaucum) stems (Fig. 1) were separated into pith parenchyma, pith vascular bundles, and rind (vascular and sclerenchyma cells combined). A normal (N) line and a counterpart brown midrib (bmr) mutant line, which has modified lignins, were both sampled. Ground (~ 1 mm) walls of these cell types were sequentially treated with 1M NaOH at 25°C for 20 h to remove ester-linked phenolic acids, and the residues were then treated with 4M NaOH in Teflon polyfluoroalkoxy copolymer vials which were placed in stainless steel vessels at 170°C for 2 h to remove more resistant ether-linked and polymerized aromatics.
Meiotic spindle assembly in cytoplasmic extracts of Spisula solidissima oocytes
Authors: Palazzo Robert E. , Vaisberg Eugeni , Cole Richard W. , Rieder Conly L.
Published: June 2020

Critical for karyokinesis in animal cells, is the assembly of a spindle-chromosome-aster complex which serves as the force-generation system for chromosome segregation. Spisula solidissima oocytes offer a unique system for the study of spindle assembly since they can be obtained in large quantities, are naturally synchronized in the cell cycle and can be induced to assemble meiotic spindles within 15 min after parthenogenetic activation. Oocytes contain a large nucleus [germinal vesicle (GV)] and are arrested at the G2/M border of meiosis I. No centrosomes or asters have been observed in oocytes at this stage by either electron microscopy or fluorescence microscopy using anti-tubulin antibodies. Observation of live oocytes with polarized light microscopy revealed that within 4-6 min after parthenogenetic activation, asters appeared and invaded the GV as it breaks down (GVBD). 15 min after activation a complete bipolar meiotic spindle was observed.

As a first step towards the development of an in vitro biochemical reconstitution system for the study of spindle assembly, concentrated lysates were prepared at various times following parthenogenetic activation and analyzed by phase-contrast microscopy.
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.