bluuebunny/crossref_metadata_2025 huggingface dataset:
Video-microscopic measurement of cell-substratum traction forces generated by locomoting keratocytes
Authors: Oliver Tim , Leonard Michelle , Lee Juliet , Ishihara Akira , Jacobson Ken
Published: June 2020

We are using video-enhanced light microscopy to investigate the pattern and magnitude of forces that fish keratocytes exert on flexible silicone rubber substrata. Our goal is a clearer understanding of the way molecular motors acting through the cytoskeleton co-ordinate their efforts into locomotion at cell velocities up to 1 μm/sec. Cell traction forces were previously observed as wrinkles(Fig.l) in strong silicone rubber films by Harris.(l) These forces are now measureable by two independant means.

In the first of these assays, weakly crosslinked films are made, into which latex beads have been embedded.(Fig.2) These films report local cell-mediated traction forces as bead displacements in the plane of the film(Fig.3), which recover when the applied force is released. Calibrated flexible glass microneedles are then used to reproduce the translation of individual beads. We estimate the force required to distort these films to be 0.5 mdyne/μm of bead movement. Video-frame analysis of bead trajectories is providing data on the relative localisation, dissipation and kinetics of traction forces.
Brefeldin A affects the endomembrane system and vesicle trafficking in higher plants
Authors: Satiat-Jeunemaitre Béatrice , Henderson Jancy , Evans David , Crooks Kim , Fricker Mark , Napier Richard , Hawes Chris
Published: June 2020

In plant cells, as in animal cells, many macromolecules and membranes are transported by vesicle vectors through both the exocytotic and endocytotic pathways. In order to elucidate the mechanisms and molecular events of such trafficking we are using a set of drugs known to perturb membrane flow in plant cells in combination with immunocytochemical studies using a bank of monoclonal antibodies to various components of the endomembrane system and cell surface. In animal cells, one such drug, Brefeldin A, a fungal fatty acid derivative which causes disruption of the Golgi apparatus, has recently been used as a tool to dissect the mechanisms of vesicle flow from the endoplasmic reticulum to the Golgi apparatus and down the cisternae of the Golgi stack (1). It has been demonstrated that BFA also has a dramatic effect on the Golgi apparatus in higher plant cells (2,3,4).

In this paper we report on recent work on the disruption of the plant Golgi apparatus with BFA and the redistribution of endomembrane marker epitopes after drug treatment of roots and suspension culture cells.
Conformational characterization of nucleosome structure by Electron Microscopy
Authors: Czarnota Gregory J.
Published: June 2020

Chromatin structure at the fundamental level of the nucleosome is important in vital cellular processes. Recent biochemical and genetic analyses show that nucleosome structure and structural changes are very active participants in gene expression, facilitating or inhibiting transcription and reflecting the physiological state of the cell. Structural states and transitions for this macromolecular complex, composed of DNA wound about a heterotypic octamer of variously modified histone proteins, have been measured by physico-chemical techniques and by enzyme-accessibility and are recognized to occur with various post-translational modifications, gene activation, transformation and with ionic-environment. In spite of studies which indicate various forms of nucleosome structure, all current x-ray and neutron diffraction studies have consistently resulted in only one structure, suggestive of a static conformation. In contrast, two-dimensional electron microscopy studies and three-dimensional reconstruction techniques have yielded different structures. These fundamental differences between EM and other ultrastructural studies have created a long standing quandary, which I have addressed and resolved using spectroscopic electron microscopy and statistical analyses of nucleosome images in a study of nucleosome structure with ionic environment.
Gender determination of caucasoid hair using image analysis
Authors: Ball T.B. , Hess W.M.
Published: June 2020

It has been demonstrated that cross sections of bundles of hair can be effectively studied using image analysis. These studies can help to elucidate morphological differences of hair from one region of the body to another. The purpose of the present investigation was to use image analysis to determine whether morphological differences could be demonstrated between male and female human Caucasian terminal scalp hair.

Hair samples were taken from the back of the head from 18 caucasoid males and 13 caucasoid females (Figs. 1-2). Bundles of 50 hairs were processed for cross-sectional examination and then analyzed using Prism Image Analysis software on a Macintosh llci computer. Twenty morphological parameters of size and shape were evaluated for each hair cross-section. The size parameters evaluated were area, convex area, perimeter, convex perimeter, length, breadth, fiber length, width, equivalent diameter, and inscribed radius. The shape parameters considered were formfactor, roundness, convexity, solidity, compactness, aspect ratio, elongation, curl, and fractal dimension.
The versatility of the light microscope image analyzer
Authors: Cardell E.L. , Cardell R.R.
Published: June 2020

The centralization of resources means equipment and technical staff must be versatile since their services will be used by various investigators evaluating different applications and tissues. The Nikon Microphot light microscope (LM) equipped with MTI video camera, Gateway 2000 computer, Image-1 imaging system, and Sony Color Video Printer is helpful for the evaluation of sections of tissue from different projects, most of which are also electron microscope studies that benefit from LM analysis. The high resolution prints are immediately available to the researcher. The various computer manipulations facilitate the visualization of routinely stained sections and sections after autoradiography, β- galactosidase localization, and immunogold-silver cytochemistry, etc. Monochrome or color prints are possible with the display modes provided, or display modes may be edited as beneficial for a particular application. We have designed display modes that allow monochrome and color images in the same print. NIH Grant DK27097.
A modification of the Humphrey’s stain for epoxy sections: A suitable alternative to toluidine blue for routine section staining
Authors: Calomeni E.P. , Gunning W.T.
Published: June 2020

We have abandoned the use of toluidine blue as a routine stain for light microscopy of epoxy embedded tissues. As an alternative, we utilize a three-component stain described by Humphrey and Pittman (1974) with minimal modifications to render the procedure substantially more timely. Numerous publications in the early 60’s through mid 70’s proposed a variety of polychromatic stains for light microscopic evaluation of epoxy sections. Most of these reports cited the difficulties involved in using a reliable method of staining epoxy sections in a timely and non-cumbersome fashion. One of the most successful stains to address these concerns is toluidine blue, first described by Trump el al. in 1961, which is undoubtedly the stain of choice in most laboratories. We have found that the differentiation of stained tissues when using the Humphrey’s stain is superior to that rendered by toluidine blue. This stain differentiates cellular detail from connective tissue in brilliant fashion as most cells will appear in various shades of blue (quite similar to shades produced by toluidine blue) and collagen appears a vivid deep pink to bright red.
Scanning electron microscopy of plant cells using a variable-pressure SEM and cryogenic techniques
Authors: Yamada M. , Ueda K. , Kuboki K. , Matsushima H. , Joens S.
Published: June 2020

Use of variable Pressure SEMs is spreading among electron microscopists The variable Pressure SEM does not necessarily require specimen Preparation such as fixation, dehydration, coating, etc which have been required for conventional scanning electron microscopy. The variable Pressure SEM allows operating Pressure of 1˜270 Pa in specimen chamber It does not allow microscopy of water-containing specimens under a saturated vapor Pressure of water. Therefore, it may cause shrink or deformation of water-containing soft specimens such as plant cells due to evaporation of water. A solution to this Problem is to lower the specimen temperature and maintain saturated vapor Pressures of water at low as shown in Fig. 1 On this technique, there is a Published report of experiment to have sufficient signal to noise ratio for scondary electron imaging at a relatively long working distance using an environmental SEM. We report here a new low temperature microscopy of soft Plant cells using a variable Pressure SEM (Hitachi S-225ON).
Confocal scanning laser microscopy applied to quantify gap junction expression and discriminate different connexin isoforms
Authors: Gourdie Robert G , Green Colin R , Thompson Robert P , Rothery Stephen , Peters Nicholas S , Severs Nicholas J
Published: June 2020

The principles of confocal scanning laser microscopy, and the ability of the technique to provide serial optical sections free from out-of-focus blur for three-dimensional image reconstruction, are now well known to most microscopists. Current studies have entered a new phase — how best to exploit the high definition digital images for maximizing information in specific research applications. Here we report methods for the quantitative analysis of gap junction content per unit volume of tissue and per cell, and for qualitative analysis of the expression of different gap junctional proteins (connexins) in the heart.

The first requirements for developing these methods are properly characterized antibodies that, under standardized conditions, give reproducible results in immunofluorescence staining protocols. The strategy as applied to cardiac gap junctions takes account of the unique organization of the junctions into intercalated disks at the end-on abutments between myocytes. Serial optical sectioning techniques are used to prepare projections revealing the entire gap junction population of en face-viewed disks.
Determining DNA strand breaks using the laser scanning microscope
Authors: Winters M.K. , Fairbairn D.W. , Standing M.D. , O’Neill K.L.
Published: June 2020

The single cell gel assay has been proven to be an effective, fast and accurate method of determining the amount of DNA single or double stranded breaks. The possible uses of this assay have already reached into some aspects of DNA damage caused by chemicals or other destructive agents. It has yet to reach even deeper into mutagenesis and carcinogenesis, both of which may show single or double stranded breaks to the DNA. Here we explore the effects that different concentrations of H2O2 have on the DNA of cells. The assay is performed by suspending cells in a low melt point agarose, spreading the agarose out on a frosted slide and letting it gel over ice. The cells are then treated with H2O2 to induce single strand DNA breakage. The cells are lysed in an alkaline environment, which separates the DNA into single strands and then electrophoresed. The gel is then stained with ethidium bromide.
Intracellular EDEMA does not adversely affect the ability to cryopreserve intact hearts
Authors: Hastings CL , Carlton RD , Lightfoot FG , Tryka AF
Published: June 2020

The earliest ultrastructural manifestation of hypoxic cell injury is the presence of intracellular edema. Does this intracellular edema affect the ability to cryopreserve intact myocardium? To answer this guestion, a model for anoxia induced intracellular edema (IE) was designed based on clinical intraoperative myocardial preservation protocol. The aortas of 250 gm male Sprague-Dawley rats were cannulated and a retrograde flush of Plegisol at 8°C was infused over 90 sec. The hearts were excised and placed in a 28°C bath of Lactated Ringers for 1 h. The left ventricular free wall was then sliced and the myocardium was slam frozen. Control rats (C) were anesthetized, the hearts approached by median sternotomy, and the left ventricular free wall frozen in situ immediately after slicing. The slam frozen samples were obtained utilizing the DDK PS1000, which was precooled to -185°C in liguid nitrogen. The tissue was in contact with the metal mirror for a dwell time of 20 sec, and stored in liguid nitrogen until freeze dry processing (Lightfoot, 1990).
Pyospermia and spermophagy in semen of the red wolf
Authors: Koehler James K. , Platz Carrol C. , Waddell Will , Jones Michael H.
Published: June 2020

The red wolf (Canis rufus) inhabited the Southeastern United States until the early 1900's when aggressive hunting and a shrinking primitive habitat virtually eradicated the species. C. rufus was certified as an endangered species in 1967 and was essentially extinct in the wild by 1980. About 200 animals are preserved in zoos and captive breeding facilities where efforts are underway to increase the stock. Since a shrinking gene pool and captive stress may reduce reproductive vigor, we undertook an electron microscopic examination of red wolf semen used for artificial insemination at the Graham, WA breeding facility of the Point Defiance Zoo.

Animals were anesthetized with 175 mg. Telazol, IM before electroejaculation using a rectal probe Semen was washed in PBS prior to fixation in 1.25% glutaraldehyde in 0.1 M cacodylate, post fixed in OsO4, dehydrated in alcohol and propylene oxide and embedded in Epon 812. Some samples were incubated in capacitation or maintenance media for several hours before fixation as above.
Confocal and Electron Microscopy of cultured cardiac myocytes
Authors: Simpson D.G. , Price R.L. , Terracio M. , Terracio L. , Borg T.K.
Published: June 2020

Early in heart development cardiac myocytes are spherical in shape, intercellular junctions are distributed at irregular intervals around the periphery of the cell, and myofibrillar organization is essentially random. As myocytes mature, they undergo extensive morphogenesis during which the phenotype changes to a tubular rodlike shape, cell junctions congregate at the distal ends of cells to form intercalated disks, and myofibrils become organized in parallel arrays typical of striated muscle. Although not fully understood, it is known that these changes are a result of interactive processes between intracellular components of the cytoskeleton, integrin membrane receptors, and the extracellular matrix (ECM).

In vivo studies on the process of cardiac myocyte maturation and myofibrillogenesis are difficult because of the complex biochemical environment of the intact animal and the many extra- and intracellular interactions which are required for proper development and myofibrillogenesis. Unfortunately, in previously available in vitro modelling systems, isolated myocytes spread out over the culture substratum, assume a stellate nonpolar shape, and myofibril organization remains essentially random.
High-resolution SEM studies of the attachment of Giardia trophozoites to the microvillous border of cultured intestinal epithelial cells
Authors: Gavin Muriel , Wells Carol L. , Erlandsen Stanley L.
Published: June 2020

Trophozoites or the free-swimming form of the intestinal protozoan,Giardia, resides within the host small intestine where they attach to the microvillous border by means of a grasping organelle, the ventral adhesive disc. Several mechanisms have been proposed to explain trophozoite attachment including contractile activity, microtubular coiling-uncoiling, hydryodynamic activity of the ventral flagella, and activated lectin binding.

We have developed a model for investigating the mechanism of attachment ofGiardiatrophozoites to the microvillous border of cultured intestinal epithelial cells. Caco-2 cells, a human colonic intestinal cell line was grown for 14 days on glass cover slips in Delbeccos' modified Eagle's medium.Giardiatrophozoites (Be-1;IP-0482:1) were cultured in TYI medium for 3 days, then harvested and resuspended in modified Eagle's medium prior to incubation for 30 minutes on Caco-2 cells. Coverslips were rinsed with warm Hanks balanced salt solution, fixed overnight in 3% glutaraldehye - 0.1 M cacodylate buffer, pH 7.2, dehydrated in ethanol, and critical point dried in CO2.
Appearance of myofibroblast during the healing of experimental corneal wound
Authors: Ishizaki Masamichi , Wakamatsu Kyoko , Matsunami Takakuni , Yamanaka Nobuaki , Saiga Toshikazu , Kao Winston W.-Y.
Published: June 2020

It is well known that corneal stroma cells (keratocytes) can transform to fibroblasts during the corneal wound healing. We have studied the expression of α-smooth muscle actin, vimentin and desmin in fibroblastic cells of the alkali-burneo and lacerated corneas in the rabbits by means of immunohistochemistry and electron microscopy.

Methods. Rabbits were anesthetized, and central corneal alkali-burn and laceration were produced. The injured corneas healed for 1 day to 45 days, and 18 days embryonic rabbit corneas were immunostained with monoclonal antibodies against α-smooth muscle actin, vimentin and desmin. For transmission electron microscopy, two techniques were used for the staining of ultra-thin sections: 1) uranyl acetate and Reynold's lead citrate and 2) the tannic acid method of Kajikawa.

Results. Antibody against α-smooth muscle actin reacted with fibroblastic cells in injured corneas (Fig 1) but not with stromal cells in normal and embryonic corneas. Antivimentin antibody strongly reacted with the fibroblastic cells in injured corneas (Fig 2) and stromal cells in normal and embryonic corneas.
Effects of Higher-Order Laue Zone reflections on structure images
Authors: Kim H. S. , Sheinin S. S.
Published: June 2020

The importance of image simulation in interpreting experimental lattice images is well established. Normally, in carrying out the required theoretical calculations, only zero order Laue zone reflections are taken into account. In this paper we assess the conditions for which this procedure is valid and indicate circumstances in which higher order Laue zone reflections may be important. Our work is based on an analysis of the requirements for obtaining structure images i.e. images directly related to the projected potential. In the considerations to follow, the Bloch wave formulation of the dynamical theory has been used.

The intensity in a lattice image can be obtained from the total wave function at the image plane is given by:

where ϕg(z) is the diffracted beam amplitide given by

In these equations,the z direction is perpendicular to the entrance surface, g is a reciprocal lattice vector, the Cg(i) are Fourier coefficients in the expression for a Bloch wave, b(i), X(i) is the Bloch wave excitation coefficient, ϒ(i)=k(i)-K, k(i) is a Bloch wave vector, K is the electron wave vector after correction for the mean inner potential of the crystal, T(q) and D(q) are the transfer function and damping function respectively, q is a scattering vector and the summation is over i=l,N where N is the number of beams taken into account.
Analysis of photographic emulsions for High-Voltage Electron Microscopy
Authors: Kremer James R. , Furcinitti Paul S. , O’Toole Eileen , Richard McIntosh J.
Published: June 2020

Characteristics of electron microscope film emulsions, such as the speed, the modulation transfer function, and the exposure dependence of the noise power spectrum, have been studied for electron energies (80-100keV) used in conventional transmission microscopy. However, limited information is available for electron energies in the intermediate to high voltage range, 300-1000keV. Furthermore, emulsion characteristics, such as optical density versus exposure, for new or improved emulsions are usually only quoted by film manufacturers for 80keV electrons. The need for further film emulsion studies at higher voltages becomes apparent when searching for a film to record low dose images of radiation sensitive biological specimens in the frozen hydrated state. Here, we report the optical density, speed and relative resolution of a few of the more popular electron microscope films after exposure to 1MeV electrons.

Three electron microscope films, Kodak S0-163, Kodak 4489, and Agfa Scientia 23D56 were tested with a JEOLJEM-1000 electron microscope operating at an accelerating voltage of 1000keV.
Modulation electron energy loss spectroscopy and its application of quantitative analysis
Authors: Luo Suichu , Dunlap John R , Joy David C
Published: June 2020

Electron energy loss spectroscopy (EELS) gives an inportant insight into the variety of excitations a sample may undergo when irradiated by an electron beam. The focus of this work was to simulate electronic excitations within the energy range from a few to several hundred eV. Our recently developed modulation scheme, combines both convolution and deconvolution techniques, to provide quantitative information about elementary inelastic scattering processes without knowledge of sample parameters such as thickness or optical constants.

In the low energy loss region of the spectrum the primary excitation mechanisms include interband transitions, and surface and bulk plasmons. In general these individual excitation events overlap in the spectrum. A FFT convolution procedure was developed where the basic inelastic processes may be represented by the dielectric theory . The dielectric function ε is used to describe both single excitations and collective excitations, whereHere ωp2=4πNe2/m is the bulk plasmon frequency, N is number of free electrons per unit volume, e and m are the charge and mass of the electron respectively and ω0is a constant which is finite for a bound state but zero for a free electron.
Determination of refractive index of solids by dispersion staining method: An analytical approach
Authors: Su Shu-Chun
Published: June 2020

When immersion liquids are used to determine the refractive index (RI) of non-opaque solids, the dispersion staining method is a simple, effective and precise way to determine the matching wavelength, λ0, at which the RI of an immersion liquid equals that of the solid. A series of equations has been derived to calculate the RI at any given visible wavelength such as nF, nD and vie, i.e. the RI’s at Fraunhöfer spectral lines F (486 nm), D (589 nm), and C (656 nm), from λ0 data obtained by the dispersion staining method. Two methods have been established: the Single Liquid Method and the Double Liquid Method.

The Single Liquid Method is applicable for solids with known dispersion coefficients, which is defined as (nF-nC). This method uses only one immersion liquid whose RI is close to the RI of the solid to be measured so that a match between the liquid and the solid occurs in the visible range.
High-resolution field emission SEM of bulk biological samples: Relationship between different metal coatings, their thicknesses, and quality of backscatter electron versus secondary-electron imaging
Authors: Frethem Chris , Wells Carol , Carlino Vince , Erlandsen Stanley L.
Published: June 2020

High resolution visualization of cell surfaces in bulk biological samples can be obtained by low voltage FESEM using either SE or BSE imaging methods and the preferred selection of low accelerating voltages (1-4 keV) has been based on theoretical considerations related to interaction volumes, electron charge, and potential radiation damage to the surface produced by direct exposure to the electron beam. Fine metal coatings (≤1 nm) produced by ion sputtering for high resolution FESEM imaging are used to minimize charging and to increase SE contrast as well as BSE topographical contrast.

The purpose of this work was to investigate the relationship between the thickness of metal coatings produced by ion beam sputtering and the quality of imaging obtained by SE and BSE of biological samples. Cells of Proteus mirabilus were immunocytochemically labeled with 12 nm colloidal gold as previously described, fixed in glutaraldehyde followed by post-fixation in osmium, and critical point dried in CO2.
A comparative SEM study of the release of three herpes virus progeny
Authors: Moninger Thomas , Rodriguez Jose
Published: June 2020

Human foreskin (HF) cells are permissive for three members of the herpesvirus family: herpes simplex virus (HSV), varicella-zoster virus (VZV) and human cytomegalovirus (HCMV). HSV and VZV belong to the alphaherpesvirinae subfamily, HCMV to the betaherpesvirinae. Cultures of the human melanoma line known as MEWO were also infected with VZV and HSV. The viruses cause different diseases in human beings.

In their interaction with cultured HF cells, HSV, VZV and HCMV differ in the effect they have on cell morphology (normally elongated), on the length of their replication cycle, and in the period of time during which progeny infectious virions are released, if they are released, since, unlike HSV and HCMV, infectious VZV virions are found only in small numbers in extracellular fluid from infected cultures. These observations led us to use scanning electron microscopy (SEM) to do a comparative study of progeny virion release with HF cells infected with one of the three herpesviruses under study.
Particle-number estimation with special emphasis on the disector and fractionator
Authors: Nyengaard Jens Randel
Published: June 2020

This abstract deals with how to stereologically estimate the number of particles in sections without making any assumptions about the size or shape of the particles. The usual term for this kind of particle estimation is design-based because by designing the study adequately any size or shape-assumptions are avoided in contrast to the obsolete model-based stereological methods. Design-based sampling of particles is performed when all particles have the same chance of being sampled, i.e., sampling particles with uniform probability. This points to the importance of proper particle sampling in stereology. Further, it is important to know what the dimension is of the sampled object, e.g., particle volume has a dimension of 3, particle length has a dimension of 1 whereas particle number has a dimension of 0. Thus, in order to make designbased particle number estimation 3-dimensional information is needed because the geometric characteristic of the 0-dimensional number is cardinality.
Measurement of the geometric properties of lineal features in 3-D microstructures
Authors: DeHoff R.T.
Published: June 2020

In most applications of stereology the focus of the measurements is upon the volume fraction Vv, surface area density Sv and number density Nv of three dimensional features like particles, grains, cells, porosity, organelles, etc. Most microstructures contain features that are essentially one dimensional; their characterization is largely ignored.

This presentation reviews the geometric properties that may be defined for space curves and edges in microstructures and the stereological relationships that, in an appropriately designed sampling scheme, provide unbiased estimates of these geometric properties.

Space curves have a length, usually reported as a line length density, Lv in stereological analyses. The local geometry near a point on a space curve is characterized by two properties, the curvature k, which is the rate of rotation of the tangent vector along the are length, and the torsionτ, which is the rate of rotation of the binormal vector.

Edges are space curves formed by the intersection of two surfaces.
Quantitative AFM of langmuir-blodgett films
Authors: Viswanathan R. , Garnaes J. , Schwartz D. K. , Zasadzinski J. A. N.
Published: June 2020

Because of their applications in the areas of non-linear optics, molecular electronics, and biosensors, Langmuir-Blodgett (LB) films have been extensively studied by a wide variety of scattering and spectroscopic techniques. The atomic force microscope has emerged as an important tool for studying LB films because of its unprecedented ability to quantitatively measure films with high resolution.

The unique abilities of quantitative AFM imaging motivated us to investigate the effect of incorporating different divalent metal cations and fatty acids of different chain length into films deposited on both amorphous and ordered substrates. Once substrate effects have been accounted for, multilayer Langmuir-Blodgett films of palmitic, stearic and arachidic acid salts show that the area per molecule is primarily controlled by the detailed interactions of the counterion with the carboxylic acid group. However, the lattice dimensions and symmetry are dictated by the close packing of the alkane chains, given the constraint of area per molecule set by the counterion.
EELS study of bulk Co and CoSi2
Authors: Wong Kaikee
Published: June 2020

CoSi2 can grow epitaxially on Si and the Si/CoSi2/Si heterostructure has potential uses in metal-based transistors. To fully utilize the device potential of CoSi2, it is important to develop a thorough understanding of its electronic properties. By comparing the Co L2,3 spectra of pure Co and CoSi2, the bonding between Co and Si atoms can be studied.

A Si (111) wafer was treated by Shiraki cleaning before 500 Å of Co was deposited in an E-beam evaporator with a base pressure of 10-7 Torr. The wafer was then annealed in an UHV oven with a base pressure of 10-10 Torr at 950°C for 2 hrs. A standard TEM plan view sample was then prepared from the wafer by dimpling and ion-milling, both from the Si side to preserve the silicide. The stochiometry of CoSi2 was confirmed by SAD and EDS. EELS measurements were carried out in a VG-HB501A UHV STEM operating at 100 keV equipped with a serial EELS detector.
Elemental mapping with an energy-selecting imaging filter
Authors: Krivanek O.L. , Gubbens A.J. , Kundmann M.K. , Carpenter G.C.
Published: June 2020

Imaging filters produce energy-selected images in a few seconds, and chemical maps formed by processing of several images taken at different energy losses in typically less than one minute. On the other hand, imaging filters do not provide detailed spectra from each specimen point, and are vulnerable to artifacts due to variations in specimen thickness, and other effects influencing EELS background extrapolation and subtraction. These include diffraction contrast arising particularly in crystalline samples, edge overlap, and extended fine structures (EXELFS) in the pre-edge region caused by major edges at lower energies. We have therefore been exploring the practical usefulness of imaging filters on a range of specimens from materials science and biology. The results suggest that the imaging capability combined with full paralleldetection EELS performance delivers a very powerful experimental set-up.

Figure 1 shows an energy-filtered bright field image of a steel sample containing about 1 % Cu, obtained at 120 keV with the Gatan Imaging Filter (GIF) attached to a Philips CM12ST microscope.
Detection and resolution in infrared microprobe analysis
Authors: Reffner John A. , Martoglio Pamela A.
Published: June 2020

Detection and resolution are terms commonly used when describing infrared microprobe (IRM) analysis. However, the clear definitions of each are not always understood completely. It is the purpose of this work to define these terms as they apply to IRM analysis.

It is a well-known fact that, for optical imaging, the ability to resolve two separate points is dependent upon both the wavelength of radiation and the numerical aperture of the optical system. The implications of this law were first described in terms relevant to IRM analysis in 1987. In order to be visually resolved, the Rayleigh criterion states that two points must be separated by 0.61 l/NA (the radius of the central maximum of the Airy disk; see Figure 1). Since an IRM detects energy outside the central maximum, this criterion is not valid for spectral resolution. With a 15X objective (NA = 0.58) over the spectral range of 600 - 4000 cm-1 (16.7 - 2.5 μm), the diffraction-resolution limit (DRL) ranges from 2.5 - 15 μm.
Single-electron sensitivity with a lens-coupled CCD camera
Authors: Fan G.Y. , Mrosko Bruce , Ellisman Mark H.
Published: June 2020

A lens coupled CCD camera showing single electron sensitivity has been built for TEM applications. The design is illustrated in Fig. 1. The bottom flange of a JEM-4000EX microscope is replaced by a special flange which carries a large rectangular leaded glass window, 22 mm thick. A 20 μm thick layer of red phosphor is coated on the window, and the entire window is sputter-coated with a thin layer of Au/Pt. A two-lens relay system is used to provide efficient coupling between the image on the phosphor scintillator and the CCD imager. An f1.0 lens (Goerz optical) with front focal length 71.6 mm is used as the collector. A mirror prism, of the Amici type, is used to "bend" the optical path by 90° to prevent X-rays which may penetrate the leaded glass from hitting the CCD detector. Images may be relayed directly to the camera (1:1) or demagnified by a factor of up to 3:1 by moving the lens assembly.
Strategies for structure determination by electron crystallography: Application to tubulin
Authors: Downing Kenneth H.
Published: June 2020

Determination of protein structures at the atomic level by electron microscopy has long been a goal, and has finally become a reality. Progress on several protein structures has been steady, but the rapid flow of structures that was anticipated to follow the initial atomic model of bacteriorhodopsin (bR) has not yet appeared. We will discuss here some of the effects that have led to this slow rate of progress and things that still need to be done to speed up the process.

The first requirement for doing crystallographic work is the availability of crystals. To date only a small number of proteins have been crystallized to give high-resolution data. Many of the principles of 2-D crystal formation are clear, and the limitations are in the nature of the amounts of protein available for crystallization experiments. With more experience, crystal availability should no longer be a limitation This will be useful because we envision the day when structures can be solved, at least through the data collection and processing stages, in a matter of days rather than the present years.
Kinematical structure factors from dynamical diffraction?
Authors: Eades Alwyn
Published: June 2020

In convergent-beam diffraction it is a common observation that kinematically forbidden reflections show "dynamical extinctions", also known as Gjonnes-Moodie lines, G-M lines, black crosses or dark bars. These zeros of intensity can be understood as resulting from the pairing of multiple diffraction routes so that each pair cancels. If the multiple diffraction routes for a reflection that is not kinematically forbidden could be paired in the same way, we could locate a position in the convergent-beam disc where the intensity would depend only on the structure factor for that one reflection. This would be extremely valuable because it would provide electron diffraction with a greatly simplified method of solving crystal structures.

It turns out that no such condition can be found. Here, an outline of the argument is given. A full account will be given elsewhere.

The pairing of the multiple diffraction routes depends on the existence of symmetry relations between different reflections.
Measurement of lattice parameter at the nanometer scale using convergent-beam microdiffraction from a thermal emission source
Authors: Pike W. T.
Published: June 2020

With the advent of crystal growth techniques which enable device structure control at the atomic level has arrived a need to determine the crystal structure at a commensurate scale. In particular, in epitaxial lattice mismatched multilayers, it is of prime importance to know the lattice parameter, and hence strain, in individual layers in order to explain the novel electronic behavior of such structures. In this work higher order Laue zone (holz) lines in the convergent beam microdiffraction patterns from a thermal emission transmission electron microscope (TEM) have been used to measure lattice parameters to an accuracy of a few parts in a thousand from nanometer areas of material.

Although the use of CBM to measure strain using a dedicated field emission scanning transmission electron microscope has already been demonstrated, the recording of the diffraction pattern at the required resolution involves specialized instrumentation. In this work, a Topcon 002B TEM with a thermal emission source with condenser-objective (CO) electron optics is used.
Theory of transmission low-energy electron diffraction
Authors: Qian W. , Spence J.C.H.
Published: June 2020

Interpretation of the images from a point source electron microscope requires a detailed analysis of transmission low energy electron diffraction. Here we present a general approach for solutions to the mixed Bragg-Laue case in transmission LEED (100-1000eV), based on the dynamical diffraction theory of Bethe. However, the validity of the dynamical diffraction theory to low energy electrons can be justified by its connection to the band theory for low energy crystal electrons.

Assume that the incident beam forms a plane wave and the crystal is a thin slab. According to Bethe, the total electron wavefield within crystal can be written as a linear combination of Bloch waves (equation 1). The Bloch wave excitation coefficients b(j) can be determined by matching the boundary conditions, the wave amplitudes Cg(j) and the wave vectors k(j) for each Bloch wave can be obtained by solving the time independent Schrodinger equations (equation 2).
Preparation of plan view and cross-sectional specimens for TEM
Authors: Goodhew Peter J
Published: June 2020

The preparation of thin specimens remains one of the most important aspects of electron microscopy. Over the forty years in which materials TEM has been practised the rate of introduction of radically new techniques has been very low. In the 1960s microscopists were using electropolishing, chemical polishing, mechanical polishing, ion beam thinning and ultramicrotomy, many of which are also covered in this symposium thirty years later. The last three decades have seen a process of refinement and automation so that success rates and areas of thin sample are both much higher in the 1990s than they were in the 1960s. However the preparation of good specimens still requires skill and an element of "art" remains. The increase in electron energy which helped microscopists to overcome limitations of specimen preparation in the 1970s has now (for very good reasons) stopped, so the basic specimen thickness requirements for standard microscopy are now stable.
Advances in ultramicrotomy for physical sciences applications
Authors: McMahon G. , Malis T.
Published: June 2020

As with all techniques which are relatively new and therefore underutilized, diamond knife sectioning in the physical sciences continues to see both developments of the technique and novel applications.

Technique Developments Development of specific orientation/embedding procedures for small pieces of awkward shape is exemplified by the work of Bradley et al on large, rather fragile particles of nuclear waste glass. At the same time, the frequent problem of pullout with large particles can be reduced by roughening of the particle surface, and a proven methodology using a commercial coupling agent developed for glasses has been utilized with good results on large zeolite catalysts. The same principle (using acid etches) should work for ceramic fibres or metal wires which may only partially pull out but result in unacceptably thick sections. Researchers from the life sciences continue to develop aspects of embedding media which may be applicable to certain cases in the physical sciences.
Long-term variations in individual particle types in the aerosol of Phoenix, Arizona, as determined using automated electron microprobe analysis and multivariate statistical techniques
Authors: Katrinak Karen A. , Anderson James R. , Buseck Peter R.
Published: June 2020

Aerosol samples were collected in Phoenix, Arizona on eleven dates between July 1989 and April 1990. Elemental compositions were determined for approximately 1000 particles per sample using an electron microprobe with an energy-dispersive x-ray spectrometer. Fine-fraction samples (particle cut size of 1 to 2 μm) were analyzed for each date; coarse-fraction samples were also analyzed for four of the dates.

The data were reduced using multivariate statistical methods. Cluster analysis was first used to define 35 particle types. 81% of all fine-fraction particles and 84% of the coarse-fraction particles were assigned to these types, which include mineral, metal-rich, sulfur-rich, and salt categories. "Zero-count" particles, consisting entirely of elements lighter than Na, constitute an additional category and dominate the fine fraction, reflecting the importance of anthropogenic air pollutants such as those emitted by motor vehicles. Si- and Ca-rich mineral particles dominate the coarse fraction and are also numerous in the fine fraction.
SEM study of structure and formation of the carbon cluster in the arc-discharge deposits
Authors: Zhou Dan , Seraphin Supapan , Jiao Jun
Published: June 2020

The richly structured surfaces of the graphite electrodes in carbon arc discharges have been the subject of numerous studies in the past, due to the strong technological importance of the carbon arc lamp. However, since they were not known until recently , no attention was paid to the existence of carbon nanocluster which the discharge produces. Our previous results show that the variations of the production processing conditions will affect the properties of the individual carbon nanotube. We present here a scanning electron microscopy (SEM) study of the topography of the deposits at the graphite cathode, and attempt to correlate the growth behavior of nanotubes to the structural elements of the cathode surface.

The starting material in this study is the carbonaceous deposit (i.e. slag) formed on the initial flat cathode as the anode is consumed during the arc discharge. The slag was carefully cleaved to make a SEM sample and the topography of the slag was characterized in both top view and cross-section.
Insertion of yttrium carbide into carbon nanoclusters by vapor transport during growth
Authors: Seraphin Supapan , Zhou Dan , Jiao Jun
Published: June 2020

Carbon nanoclusters have basic features in common, although they vary greatly in shapes. Concentric graphitic layers separated by 0.34 nm surround a hollow core a few nanometers in diameter. Encapsulating materials into this hollow core changes the physical properties drastically. The insertion offers the possibility to study materials of small dimensions in a closed-off environment. Technological promise is given for the production of nanowires, and other applications in which the nanoscale is of importance. Recent work takes a variety of approaches, inserting lead into nanotubes by capillary suction through open ends, or encapsulating microcrystals of LaC2 into the polyhedral cages. We report a method for filling carbon clusters of various shapes in situ through vapor transport from a composite anode of a graphite rod filled with yttrium oxide paste at a center hole.

Both materials pass through the arc discharge, and grow as composite carbon nanoclusters at the pure graphite cathode.
Electron sources: Past, present, and future
Authors: Joy David C
Published: June 2020

The electron source is the most important component of the Scanning electron microscope (SEM) since it is this which will determine the overall performance of the machine. The gun performance can be described in terms of quantities such as its brightness, its source size, its energy spread, and its stability and, depending on the chosen application, any of these factors may be the most significant one. The task of the electron gun in an SEM is, in fact, particularly difficult because of the very wide range of operational parameters that may be required e.g a variation in probe size of from a few angstroms to a few microns, and a probe current which may go from less than a pico-amp to more than a microamp. This wide range of operating parameters makes the choice of the optimum source for scanning microscopy a difficult decision.

Historically, the first step up from the sealed glass tube ‘cathode ray generator’ was the simple, diode, tungsten thermionic emitter.
The SEM in present-day semiconductor FABS
Authors: Bennett Marylyn Hoy
Published: June 2020

Computers, cars, HDTV, and microwave ovens: what great modern conveniences! Why do these marvels of technology work the way they do? The Scanning Electron Microscope has a lot to do with it. For the last decade, the shrinking geometries of integrated circuits have necessitated the use of the Scanning Electron Microscope (SEM) for inspection and metrology. The current device feature sizes in production are as small as 0.5μm, and current feature sizes under development are 0.25μm or smaller. The semiconductor industry depends on the SEM to tell us if our processes, whether production or developmental, are correct. Neither leading edge fabs nor developmental fabs could function very well without the SEM.

The uses of the SEM are many and varied, both inside and outside the fab. Inside the fab, there are many inspection steps to be performed. Although the optical microscope is still a viable option for, and is still routinely used for, in-line wafer inspection, the SEM is indispensable for inspection (Fig. 1) of small geometries such as 0.35μm lines and spaces and 0.5μm contact holes.
Effects of a thiol antioxidant on leucocyte adherence to aortic endothelium during atherogenesis: Quantitative SEM assessment
Authors: Robinson Keith A. , Medford Russell M. , Wayne Alexander R.
Published: June 2020

Dysfunction of arterial endothelial cells (EC) leading to adherence of circulating leucocytes (WBC) is a pivotal step in atherogenesis, preceding fatty streak formation. The attachment process is mediated at least in part by inducible endothelial-leucocyte adhesion molecules (ELAMs) on the EC surface which interact with WBC counterreceptors. Expression of VCAM-1, a monocyte selective adhesion molecule, by IL-1 and TNF stimulated vascular EC in vitro was shown to be inhibited by a thiol antioxidant (pyrrolidine dithiocarbamate, PDTC). In this study we analyzed the effect of PDTC administered to cholesterol-fed rabbits, on WBC attachment to lesion-prone sites of the aorta using SEM morphometry.

New Zealand White (NZW) rabbits (2-3 kg) were fed 1% cholesterol for 3 d. The day before feeding, venous cannulas were introduced, and PDTC (20 mg/kg) or vehicle (0.1 M PBS) was given twice daily throughout. The animals were euthanatized at 4 d and the aortas pressure perfusion fixed with buffered 2.5% glutaraldehyde after rinse with Ringer‘s solution containing heparin (10 u/ml).
STEM characterization of a 6Å diameter Mo3Se3 fiber
Authors: Hornbostel M. , DiSalvo F.J. , Hillyard S. , Silcox J.
Published: June 2020

LiMo3Se3 is a highly anisotropic conductor containing 6Å diameter one dimensional chains of Mo3Se3 triangles. This compound can be dissolved in polar solvents to produce solutions containing micron length Mo3Se3 fibers. Choice of solvent and solution concentrations allows some control of fiber diameters, from a few hundred angstroms down to the molecular limit of a single 6Å diameter chain. These fibers have been deposited from solution on holey carbon substrates by vacuum evaporation of the solvent to produce free-standing, one dimensional wires.

High resolution microscopy at 100kV was carried out in a VG HB501A STEM and confirms the presence of many different sized bundles extending all the way down to the single strands. Figure 1a shows an ADF image of a medium sized strand which commonly occured in a sample prepared with the solvent propylene carbonate. The flexibility of the fiber and its seeming attraction to the edges of carbon holes is apparent as it snakes its way along the surface.