bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron Microscopy in Tumor Diagnosis in 2001
Authors: Shelburne J , Howell D
Published: July 2020

Immunohistochemistry (IH) has revolutionized tumor diagnosis in recent years. The antigenic/molecular information now available is extraordinarily helpful not only in clinical diagnostic work, but also in improving our understanding of the cell biology of neoplasia. As a result, there is now less need for diagnostic conventional transmission electron microscopic (CTEM) studies on neoplasms. For example, lymphomas and leukemias are now largely defined by IH and related techniques such as flow cytometry, not CTEM. A postive S-100 stain in the right setting usually obviates the need to search for melanosomes.

However, CTEM is still a useful tool for surgical pathologists. One important advantage CTEM has over IH is that - like gross observations and the classic H&E section - CTEM can detect features not suspected in advance. This property of CTEM makes it particularly valuable in studying complex and unusual patients/tumors.
Cardiomyocyte Apoptosis in the Outflow Tract in Normal and Abnormal Cardiogenesis
Authors: Watanabe Michiko , Hitomi Midori , Sallee Denver , Jafri Anjum , Rothenberg Florence , der Wee Kathy van , Fisher Steven A
Published: July 2020

Apoptosis peaks within the cardiac outflow tract (OFT) myocardium during chick development (stage 30-31) and coincides with dramatic remodeling of the OFT 4. Apoptosis was assayed by TUNEL, Annexin V, transmission electron microscopy, and Lysotracker Red (Fig. 1-4). These assays reveal that as many as 92% of the apoptotic cells express cardiomyocyte markers. The timing of apoptosis suggests that it may occur to allow proper alignment of the great vessels over their respective ventricles. Our current aims are to determine the consequences of disturbing OFT cardiomyocyte cell death to heart morphogenesis and to elucidate the cellular and molecular components that initiate and regulate this high level of apoptosis.

To enhance apoptosis in the OFT myocardium, recombinant adenoviruses were injected into the pericardial space to deliver the gene for the death ligand Fas-L to the cardiomyocytes of chicken embryos prior to the stages when OFT remodeling occurs.
The Role of Apoptosis in Myocyte Death During Cardiac Hypertrophy
Authors: Goldsmith EC , Davis JG , Yan X , ᒫorell BH , Borg TK , Price RL
Published: July 2020

Apoptotic events can be the result of both cell anchorage-dependent and independent mechanisms. The term “anoikis” has been used to describe apoptotic events resulting from a lack of cell-ECM contact (Frisch and Francis, 1994). The integrin heterodimer α5β1 has been implicated in several systems as providing protection against apoptosis. Previous studies have demonstrated that the α5 integrin subunit is present only during cardiac development and conditions of cardiac hypertrophy and is not found in normal adult rat myocytes (Terracio et al., 1991). Cell anchorage-independent apoptosis can result from a variety of factors including biochemical and mechanical stimulus. Angiotensin II (Ang II) has been implicated as a modulator of myocyte apoptosis acting through the angiotensin II type I receptor (Kajstura et al., 1997). The role of a second Ang II receptor, angiotensin II type 2 receptor, has not been determined. The role of apoptosis in myocyte cell death during heart failure remains unclear.
Mammalian Apoptosis in Whole Neonatal Ovaries, Embryos and Fetal Limbs Using Confocal Microscopy
Authors: Zucker Robert M
Published: July 2020

The emergence of confocal laser scanning microscopy (CLSM) as a technique capable of optically generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure offers a viable alternative to traditional sectioning approaches. However, the imaging of such whole-mounts presents technical problems of its own. One of the major problems with using a confocal microscope to image whole organs and embryos is the depth of penetration of the laser light into the tissue. We have optimized the confocal microscope performance and developed a sample technique that increases the optical resolution of the system.

CLSM has been used to study cellular death (apoptosis) during GD 8-12 normal rat/mouse embryonic development, neonatal ovarian development (PD10-45) and fetal limb development (GD 11-15). LysoTracker Red (LT) was incorporated into the tissue prior to laser excitation. It is aldehyde fixable stain that concentrates into acidic structures or into cells that have high lysosomal activity.
Accelerated Mitochondrial Reactive Oxygen Species Formation Induces OnsẸt of the Mitochondrial Permeability Transition and Mitochondrial Swelling in Cultured Hepatocytes After TNFα Exposure
Authors: Qian Ting , Lemasters John J
Published: July 2020

INTRODUCTION: The mitochondrial permeability transition (MPT) is implicated in mediating TNFα-induced apoptosis in cultured hepatocytes. Opening of permeability transition (PT) pores in the mitochondrial inner membrane causes the MPT. After the MPT, mitochondria swell, the outer membrane bursts, and pro-apoptotic cytochrome c is released into the cytosol. in isolated mitochondria, ROS formation promotes onset of the MPT. However, how mitochondrial ROS formation regulates the MPT in intact cells in TNFα-induced apoptosis is unknown. AIM: The present study was designed to determine the role of mitochondrial ROS formation in TNFα-induced MPT and apoptosis in cultured rat hepatocytes.

METHODS: Hepatocytes expressing an IkB superrepressor were pretreated with 2 μM t-BuOOH 4 hours before TNFα exposure with and without CsA (2 μM, an inhibitor of the PT pore) and the antioxidants, desferal (0.5 mM) or diphenylphenylendiamine (DPPD, 10 μM). Cell viability was monitored by propidium iodide fluorometry.
Gently Down The Stream: Flow Cytometry As Microscopy
Authors: Shapiro Howard M , Shapiro Howard M
Published: July 2020

Flow cytometry is an analytical technique in which optical measurements are made of cells or other biological particles as the cells or particles flow, ideally in single file, in a fluid stream past one or more optical measurement stations. Modern optical flow cytometers typically measure light scattered at small (1-5°) and large (15°-135°) angles to an illuminating laser beam, and fluorescence emitted in three or more discrete spectral bands; the most complex instruments employ three or four spatially separated illuminating beams at different wavelengths and can measure twelve fluorescence signals from each cell or particle analyzed.

Flow cytometry was developed in the 1960's to speed up and automate microspectrophotometry and microfluorometry, which had previously been microscope-based, in order to facilitate development of observer-independent clinical tests which could replace the Papanicolaou smear and the differential leukocyte count. At that time, high-resolution scanning was an extremely slow process; high-intensity sources were restricted to arc lamps, and memory in which images could be stored was expensive.
Sizing Dna Fragments by Ultrasensitive Flow Cytometry
Authors: Jett James H , Habbersett Robert C , Yan Xiaomei , Yoshida Thomas M , Marrone Babetta L , Keller Richard A
Published: July 2020

As originally developed in the 1960's, flow cytometry was primarily a technique for the analysis of mammalian cells. Analysis of cellular constituents such as DNA or cell surface antigens made fluorescent by a variety of reagents has been the main stay of flow cytometric applications. Over the years, flow cytometric analysis techniques have been developed that range from multicellular spheroids containing a million or more cells down to single molecule detection. An outgrowth of single molecule detection capability is DNA fragment size analysis.

DNA fragment size analysis starts with a sample of naked DNA that can be derived from a variety of sources including PCR products, double stranded viral genomes, BAC/PAC clones, and bacterial genomes. For genomic or cloned DNA, restriction enzyme digests are analyzed to produce a fingerprint pattern. The fingerprint, i. e., the distribution of fragment sizes produced by the restriction enzyme digestion, is characteristic of the source of DNA and forms the basis for identifying the source.
Early Events During Invasion of J774 Cells by Listeria Monocytogenes
Authors: Webster Paul
Published: July 2020

The gram positive bacillus, Listeria monocytogenesis a contaminant of the food we eat and although it is a common pathogen in farm animals L. monocytogenesdoes not normally infect humans. However, immunocompromised individuals, infants and the elderly are susceptible to infection. The bacteria, taken into membrane bound phagosomes, use a thiol-activated cytolysin listeriolysin O to disrupt the membrane (LLO) and gain access to the cell cytoplasm. Subsequent actin polymerizations on the bacterial surface induce motility in the cell and long cytoplasmic protrusions at the cell surface that spread bacteria to neighboring cells.

L. monocytogenesenters cells by phagocytosis. By using short infection times of 5 min or less, it was possible to examine early interactions between J774 cells and L. monocytogenes.This study shows that during the early stages of internalization the bacteria are able to modify the protein composition of the forming phagosome membrane.
Analysis for Parent Drug and Drug Metabolite in Rat Liver, Bile Stones, and Bile Using Microscopy, Laser Capture Microdissection and Analytical Chemistry Techniques
Authors: Neilly J , Gagne G , Vogt A , Fagerland J , Miesbauer L , Burton R , Shen J
Published: July 2020

Microscopy, microdissection, and analytical chemistry techniques were used to localize and identify drug and drug metabolites in specimens from drug-treated rats. During a toxicology study in rats of a drug candidate, concretions (stones) were found in the common bile duct, and pigmented material was found in hepatocytes and liver macrophages after long-term exposure to the drug. Because a recent study showed the effective use of microscopy and mass spectrometry (MS) to identify drug metabolite in tissue sections, we used similar methods to confirm that pigmented material in liver cells was drug metabolite. Energy dispersive x-ray spectroscopy (EDS) suggested that drug and/or metabolite were also present in stones.

Pigmented hepatocytes and macrophages in frozen sections of unfixed liver exhibited fluorescence similar to that of parent drug. When excited by UV illumination, these cells fluoresced the same color blue as the bulk drug (Fig. 1).
Ultrastructure of Striatal Dopamine Synapses in Rats with Striatal Dopamine Depletion
Authors: Gray (Jay) Jerome W , Montine Thomas J , Deutch Ariel Y
Published: July 2020

Parkinson's disease (PD) is characterized by rigidity, tremor, bradykinesia, and postural instability. The proximate cause of these symptoms is striatal dopamine (DA) insufficiency. The motor symptoms of PD can be alleviated by DA replacement therapy. However, late in the course of the disease patients appear to become less responsive to DA replacement. This therapeutic change suggests the possibility of structural and/or functional defects in striatal medium spiny neurons, which receive convergent DA and cortical (glutamate) inputs.

To understand the neuronal reorganization occurring in Parkinson's disease, we used ultrastructural methods to examine the striatum of rats with striatal dopaminergic deafferentation induced by unilateral intranigral injection of 6-hydroxydopamine. After a six month survival, rats were deeply anesthetized with pentobarbital and perfused with 4% paraformaldehyde-1 % glutaraldehdyde solution in 0.1M Sorenson's phosphate buffer (pH 7.4). The brains were removed, post-fixed for 12 hours, embedded in paraffin, and coronal sections cut through the striatum and midbrain.
The Effect of Charging on Electron Diffusion in Solids
Authors: Demers Hendrix , Gauvm Raynald
Published: July 2020

The studies of insulating specimen by using scanning electron microscopy (SEM) or associated microanalytical techniques such as electron probe microanalysis (EPMA), Auger electron spectroscopy (AES), etc., is limited by the charging phenomena. Different techniques have been found to minimize this problem: coating the specimen with a conductor, working at low energy [1], etc. But for a better knowledge of this effect, we have started a study of the mechanisms of charging as well as its effect on the electrons trajectories in the case of an insulating specimen. with the success, in past years, of Monte Carlo (MC) simulation of electron scattering in solid specimens [2], we have been developing a new Monte Carlo program for the simulation of the electron trajectory in insulators.

With this program, we want to understand the effect of the trapping charge on a insulating specimen. The new MC will be constructed by adding a succession of refined model. in each step, the model goes deeper in the mechanisms for the charging phenomena.
SRM 482: Revisited After 30 Years
Authors: Windsor Eric , Carlton Robert , Wightal Scott , Lyman Charles
Published: July 2020

The National Institute of Standards and Technology’s (NIST) Standard Reference Material (SRM) 482 was issued by The National Bureau of Standards (NBS) in 1969 i has been continuously available to the public for over 30 years [1]. The standard cons of a set of 6 wires. Each wire is approximately 0.5 mm in diameter and 5 cm long. Tl wires represent different compositions within the binary copper-gold alloy system. Included in the set are the two end member compositions, pure copper and pure gold, along with 4 alloys of nominal composition: Au80-Cu20, Au60-Cu40, Au40-Cu60 an< Au20-Cu80. SRM 482 was produced specifically for the purpose of microanalysis usi the electron probe microanalyzer (EPMA).

Recent metallographic preparation of these wires revealed the presence of surface blemishes on some of the prepared wires (Figure la). Two of the authors from differei affiliations recently prepared cross-sections of SRM wires from separately purchased sets. Each was unaware of the procedures used by the other.
Quantitative X-Rays Microanalysis of Rough Surfaces
Authors: Gauvin Raynald , Lifshin Eric
Published: July 2020

The classical schemes to convert the x-ray intensity into concentration, using the ZAF or the φ(ρ z ) methods, are valid for specimens having homogeneous composition and flat surfaces. Quantitative schemes have also been developed for x-ray microanalysis of multi-layered specimens. More recently, a quantitative method has been proposed for the microanalysis of spherical inclusions embedded in a matrix.

For the case of specimens having a non-planar surface, a quantitative method based on the peak to background ratio, using photons of the same energy, has been proposed. in order to test this correction method, a Monte Carlo program has been developed in order to compute the complete x-ray spectrum, characteristic lines plus bremstrallung, emitted from materials having rough surfaces. Figure [1] shows the geometry of the rough surfaces simulated. The parameters of these surfaces are the summit semi-angle, θ, and the height of the roughness, h.
Edx Spectrum Imaging of Bulk Specimens: Its Role in Quantitative X-Ray Microanalysis
Authors: Anderson IM
Published: July 2020

Energy-dispersive X-ray (EDX) spectrum imaging, where a full spectrum is acquired at each pixel in a rastered image, combines the advantages of imaging and analysis for quantitative compositional mapping. This method has great advantages for the analysis of compositional variations, because the contrast inherent in EDX spectrum images arises from the distinctiveness of the characteristic X-ray spectra in various regions of the surveyed microstructure. EDX spectrum imaging provides for simultaneous spatial, spectral and temporal resolution of the X-rays emitted from the specimen. Many EDX manufacturers have incorporated a spectrum imaging capability within their acquisition and analysis software, and ongoing improvements in computing power will enhance the practicality of spectrum imaging methods, in particular the acquisition, manipulation and analysis of the large raw data files that are generated with this method. This paper surveys the potential role of spectrum imaging in tasks that support true quantitative X-ray microanalysis of bulk specimens: robust qualitative analysis, the construction of representative spectra that describe the specimen, and the extraction of elemental concentrations from the representative spectra.
X-Ray Microanalysis in the Environmental SEM Using Mapping and Fourier Deconvolution Techniques
Authors: Phillips Matthew R , Griffin Brendan , Drouin Dominque , Nockolds Clive , Remond Guy
Published: July 2020

X-ray microanalysis of any type of specimen in its natural state without the use of conventional SEM specimen preparation techniques has immense potential in a wide range of scientific and industrial applications. This capability would be particularly useful in microanalysis applications where it is highly desirable to preserve the integrity of the specimen, for example in semiconductor failure analysis and forensic investigations. in principle, this X-ray microanalysis goal can be achieved in an environmental or variable pressure scanning electron microscope (VPSEM) because specimen charging and vacuum stability problems are negated by the presence of a gas in the specimen chamber. However, the accuracy and spatial resolution of X-ray microanalysis in the VPSEM is significantly degraded by the chamber gas as it scatters primary beam electrons, generating spurious X-rays far from the analysis point. to date, two different X-ray measurement strategies have been developed to facilitate X-ray microanalysis at high chamber pressure in the VPSEM.
Modifications Needed for Performing High-Resolution Cryosem in a Hitachi S-4700 Fesem Using an Emitech K1250 Cryo-Preparation System
Authors: Chen Ya , Frethem Chris , Erlandsen Stanley L
Published: July 2020

Cryo-techniques have been successfully used in scanning electron microscopy (SEM). They are especially promising for high-resolution SEM to improve specimen preservation and reduce radiation damage [1, 2]. A number of cryo-preparation systems are commercially available for SEM, however, our experience has shown that modifications are needed to perform highresolution imaging (>50,000x).

Emitech K1250 system consists of a sample preparation chamber, control unit, and cryo-stage. Magnetron sputter coating is standard and electron-beam evaporation is optional. A vacuum transfer device facilitates the sample transfer between the preparation chamber and the SEM to prevent contamination. The Emitech cryo-stage replaces the Hitachi S-4700 standard stage and the cryo-stage temperature is monitored and controlled by the Emitech control unit.

The specimen is mounted on a sample holder that mounts to the cryo-stage. Therefore, the distance from the specimen to cryo-stage, the thermal capacity of the sample holder, and the thermal contact between them will affect the actual temperature of the specimen.
Direct Imaging of Sodium Stearate Crystals Dispersed in Waterpropylene Glycol Mixtures by Cryo-Electron Microscopy
Authors: Ma Yue , Liang J , Zheng Y , Erlandsen S L , Scriven L E , Davis H T
Published: July 2020

Cryo-scanning electron microscopy (cryo-SEM) and cryo-transmission electron microscopy (cryo- TEM), in conjunctions with rheological measurements, light and confocal microscopy, x-ray scattering, and solid state NMR, are used to characterize sodium stearate (NaSt) crystals dispersed in waterpropylene glycol (PG) mixtures at macroscopic, microscopic, molecular, and atomic levels. NaSt is a surface-active, structural agent in household and personal cleaning products, including deodorant sticks and soap bars. A better structural characterization of NaSt/PG/water systems has practical importance in personal care and cosmetic industries. NaSt crystals and other soap crystal morphologies have been studied by the TEM/replica technique. However, the replicas were made of the residue after the original sample or its aqueous dilution were dried, and the original structure may have been lost during drying. Cryo-SEM was not used to study NaSt crystals because of its lower resolution and because the crystals are highly susceptible to radiation damage by electron beam.
A STEM-EDX Study of the Sequestration of Heavy Metal in Different Cell Components by Pseudomonas Aeruginosa
Authors: Goldberg JJ , Jensen TE
Published: July 2020

Cells of Pseudomonas aeruginosa were exposed to six different heavy metals, Al, Cd, Cu, Mn, Pb, Zn, exposure was done at the same concentrations of 20 ppm, except for the Cd which was done at 5 ppm. The cells were then analyzed using the STEM mode of a transmission electron microscope in conjunction with a PGT IMIX energy dispersive xray spectrometer. The data was then analyzed using a bulk sample analysis program (ZAF method) in standardless mode (w/w). We analyzed the cell wall the cytoplasm and polyphosphate bodies. Approximately 20 cells were analyzed per treatment and averages of the elements present were calculated. The cells of Pseudomonas aeruginosa were grown in nutrient broth and then embedded in Epon according to Luft's procedure. Quantitative analysis of the three different parts of the cell revealed that in cells exposed to Al, the wall contained on average 0(55%), C(20%) P( 8.0%) K(10%) Cl(2%) S(5.0%),Al(2.0%), the cytoplasm contained on average, 0(53%) C(20%) P(5.0%) Ca(4.0%) K(12%) Cl(4.0%) Al(2.0%).
Biogenic Mineral Formation by Iron Reducing Bacteria
Authors: Dohnalkova Alice C , Gorby Yuri A , McLean Jeff , Fredrickson Jim K , Kennedy David W
Published: July 2020

Dissimilatory iron reducing bacteria have been extensively studied for their ability to reduce ferric iron Fe(III) to ferrous iron Fe(II), as well as several multivalent heavy metals and radionuclides as a mode of energy-yielding respiration. Shewanella putrefaciens strain CN32 was used to investigate the mechanism of biogenic metal reduction in systems simulating conditions of natural anaerobic iron reducing environments in the subsurface contaminated with U and Tc as a possible strategy for bioremediation of soils containing these contaminants. As previously reviewed, U(VI) is soluble in most groundwaters, while U(VI) generally precipitates as the insoluble mineral uraninite. Formation of bioreduced minerals can lead to immobilization of these contaminants in the subsurface, which might be a very useful strategy for in situ bioremediation.

To determine the metal reduction and the formation of biogenic Fe(II), U(IV) and Tc(IV) minerals, experiments with CN32 exposed to well-defined aqueous solutions were conducted. Metal reduction was measured with time, and the resulting solids were analyzed by X-ray diffraction, scanning electron microscopy (SEM) and energydispersive X-ray spectroscopy (EDS).
Seeing the Forest for the Trees: Selective Staining and Contrast Enhancement Methods for Biological Electron Microscopy
Authors: Ellisman M H
Published: July 2020

Electron tomography has proven to be an invaluable tool for studying the 3-dimensional organization of a wide range of structures, from large cellular processes down to individual macromolecular complexes. An important requirement for electron tomography of thick specimens is the need for selective staining to delineate the structure of interest from other cellular constituents. of particular usefulness in this regard is the method of fluorescence photooxidation, whereby the reactive oxygen generated by a fluorescent compound is used to oxidize diaminobenzidine into a reaction product that can then be visualized with the electron microscope. The main advantages of this method are that not only does it allow for excellent correlated light and electron microscopy, but also the relatively small size of the oxidizing agent used allows for excellent 3-D labeling with high resolution.

This method has proven to be a particularly versatile technique.
The Si/SiO2 Interface: Atomic Structures, Composition, Strain and Energetics
Authors: Pennycook S J , Duscher G , Buczko R , Pantelides S T
Published: July 2020

A number of recent studies of grain boundaries and heterophase interfaces have demonstrated the power of combining Z-contrast STEM imaging, EELS and first-principles theoretical modeling to give an essentially complete atomic scale description of structure, bonding and energetics. Impurity sites and valence can be determined experimentally and configurations determined through calculations.

Here we present an investigation of the Si/SiO2 interface. The Z-contrast image in Fig. la, taken with the VG Microscopes HB603U STEM, shows that the atomic structure of Si is maintained up to the last layers visible. The decrease in intensity near the interface could originate from interfacial roughness of around one unit cell (∼0.5 nm), or may represent dechanneling in the slightly buckled columns induced by the oxide. Fig. lb, taken from a sample with ∼1 nm interface roughness, shows a band of bright contrast near the interface. This is not due to impurities or thickness variation since it disappears on increasing the detector angle from 25 mrad to 45 mrad (Fig. lc), and is therefore due to induced strain.
Things That Go “Bump” in the VPSEM - and How We Image with Them
Authors: Griffin Brendan J
Published: July 2020

Variable pressure scanning electron microscopes (VPSEM) differ from conventional SEM by operating at pressures ranging from the ‘high vacuum’ SEM levels of 10-6 torr up to typically around 2 torr. The environmental SEM or ESEM is a commercial variant which employs an unique multistage pressure-limiting aperture (PLA) system to attain specimen chamber operating pressures of up to 50 torr. Early instruments used air or argon as the imaging gas but more commonly today water vapour is used. A wide range of gases have been employed, including potentially explosive hydrogen-methane mixtures. The choice of gas is operator-based and can be varied during the imaging session.

Early VPESM were restricted to backscattered electron imaging (BSE) until the development of the gaseous secondary electron detector in the ESEM. Gaseous secondary electron detectors are now available for all models of VPSEM and together with compatible cathodoluminescence and EDS XRay detectors, the full range of SEM-based imaging options is present.

The principal distinguishing feature of VPSEM is, of course, that samples can be examined uncoated. Gas-electron interaction generates a positive ion supply that can minimise conventional charging artefacts, in a simple imaging model.
FIB/SEM Dual Beam Instrumentation: Slicing, Dicing, Imaging, and More
Authors: Giannuzzi Lucille A
Published: July 2020

In a focused ion beam (FIB) instrument, ions (typically Ga+) obtained from a liquid metal ion source are accelerated down a column at energies up to ∽ 50 keV. The beam of ions is focused by electrostatic and octopole lens systems and the ion dose (and beam diameter) is controlled using real and/or virtual apertures. Beam sizes in FIB instruments on the order of 5-7 nm may be achieved.

The versatility of the FIB instrument enables large regions of material (e.g., 500 μm3) to be removed at high beam currents in just a couple of minutes. Lower beam currents (i.e., beam diameters) are usually used to remove smaller amounts of material within the same time frame (e.g., ∽ 5μm3). The introduction of an organometallic gas in close proximity to the target allows for the deposition of metals, SiO2, and other materials, by an ion beam assisted chemical vapor deposition process.
The Evolution of Accessibility: The San Diego State University Electron Microscope Facility
Authors: Barlow Steven B
Published: July 2020

A microscope facility at teaching institutions needs to be more than an isolated refuge of researchers. Students must be given easier access to the equipment and an increased awareness of the power of these tools for understanding the scientific principles taught in their lecture classes. The San Diego State College of Sciences supports a shared instrumentation Electron Microscope Facility, a suite of seven rooms that occupies 1500 square feet. The laboratory is fully equipped for all aspects of sample preparation for examination by confocal scanning light microscopy, scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Historically, specialists with many years training in equipment operation, sample preparation, and research protocols have had exclusive access to EM facilities. At SDSU, however, every effort is made to encourage inclusivity via familiarity with and hands-on access to equipment in the EM Facility. in all, over 1000 SDSU students and several hundred community members participate each year in opportunities to access the SDSU EM Facility.
Inside Adobe Photoshop and Acrobat
Authors: Kost Julieanne
Published: July 2020

Adobe Photoshop has been the defacto standard for imaging professionals for many years. Most scientists have adopted this program because of the ease of use and the quality of the output. It is obvious to most who use this program that most users only master a limited subset of this programs' capabilities.

This session is an attempt to introduce more of this imaging applications' capabilites from one of Adbobe's Photoshop Evangelists. I will try to show you how to get the most out of Adobe Photoshop by revealing the most productive and efficient digital imaging workflows. I will focus on developing a greater understanding of the current technology within this powerful imaging application program as well as explain the most efficient methods of manipulating, printing, and posting images on the web. I will demonstrate quick and easy ways to crop and resize images as well as make tonal changes and apply custom color tables (Fig. 1).
Single Molecule Force Spectroscopy Maps to Study Receptors Clustering
Authors: Bhatia R , Almqvist N , Banerjee S , Primbs G , Desai N , Lai R
Published: July 2020

An atomic force microscope (AFM) allows molecular resolution imaging of hydrated specimens. However, it is often limited in providing identity of the imaged structures, especially in a complex system such as a cellular membrane. Cell surface macromolecules such as ion channels and receptors serve as the interface between the cytoplasm and the extracellular region and toward which many regulatory signals are directed. Their density, distribution and clustering are key spatial features influencing effective and proper physiological responses. We used a method that uses AFM “force-volume maps” to identify and map regional distribution as well as ligand-, or antibody-induced real-time clustering of receptors on the cell surface. This technique also allows simultaneous imaging of the resultant changes in cellular micromechanical properties, such as elasticity and cytoskeletal reorganization of the cell. As an appropriate physiological sample, we have examined spatial distribution and real-time clustering of VEGFR, the receptor for vascular endothelial growth factor which is an important angiogenic factor in human and animal tissues.

We have used AFM probes conjugated with anti-VEGFR-antibody (anti-Flk-1 antibody) to examine binding (or unbinding) forces between VEGF-R2 (Flk-1) in both in vitro as well as in live endothelial cells. A quantal set of binding and unbinding forces was measured between the antibody conjugated to the AFM tip and purified VEGFRs adsorbed on to a mica surface (Fig 1). The unbinding force varied between 60 and 240 pN and was a multiple of discrete quantized strength of approximately 60 pN (Figure 1B).
Hitachi S-4700 ExB Filter Design and Applications
Authors: Joens Steve
Published: July 2020

Electron beam - specimen interactions and SEM signals have been well understood and documented for many years. These interactions result in a variety of electron signals including the most common, secondary and backscattered electron. Each electron signal produces unique characteristic information about the sample surface, subsurface, and elemental composition. Important information can be gained by controlling and filtering electron signals collected by the electron detector system.

The S-4700 Cold Field Emission SEM incorporates a set of electrodes and plates positioned in the objective lens upper pole piece in close proximity to the upper secondary detector (figure 1). When a positive voltage is applied to the electrode plates, a high yield of secondary and backscattered electrons spiral up the column of the objective lens. The backscattered electrons are filtered with the ExB producing a SE rich signal. The information from this type of signal provides absolute detail from the sample surface, but can be prone to charging with some highly nonconductive samples. Figure 2a shows the effect of charging while observing uncoated Teflon ™. The image becomes distorted with bright intermittent horizontal lines. Surface detail is enhanced due to the high contribution of SEs from the sample surface.

When the electrode voltage is set negative, through the instrument GUI, the low energy secondary electrons are repelled providing a signal rich in backscattered electrons. The information from this type of signal provides compositional information and inherently reduces charging. The uncoated Teflon ™ sample in figure 2b shows all charging affects have been eliminated.
Development of a Field Emission VP-SEM and Some Initial Applications
Authors: Nishimura Masako , Itoh Sukehiro , Joens Steve
Published: July 2020

The use of variable pressure SEMs (VP-SEMs) is increasing in various fields of science and industry, allowing microscopy in a variable pressure environment of 1 ∼ 270 Pa utilizing backscattered electrons for imaging. The VP-SEM allows microscopy of insulated samples without the need for sample preparation. Charging artifacts can be minimized as well. When the VP-SEM is operated with a cooling stage, which allows cooling of samples at −20° and above, vaporization of water from samples is reduced. This permits microscopy of wet samples at close to the natural state for extended periods of time.

Poor S/N ratio and deterioration of resolution, both of which are due to collisions among residual gas molecules and primary/backscattered electrons, have limited the performance of VP-SEMs. For resolving these limitations, we have completed the development of a new field emission VP-SEM which operates with a stable Schottky field emission source, a new environmental secondary electron detector (ESED), and a multi-stage differential pumping system. Fig. 1 shows a sectional view of the column with the differential pumping system. This design allows stable gun vacuum conditions with variable specimen chamber pressure 10 through 3,000 Pa, permitting a pressure difference from the gun by 1011 Pa without problems.
Evactron™ Cleaning of SEM Specimens using an In-Situ RF Plasma on the SEM Chamber
Authors: Vane R , Strossman G
Published: July 2020

The EVACTRON is a new device (US Patent 6,105,589) designed to remove hydrocarbons from SEM specimens and SEM chambers to prevent contamination artifacts. The device uses a lowpowered RF plasma to make oxygen radicals from air. These radicals oxidize hydrocarbons to make CO, H2O, and CO2 that can be removed by the vacuum pump. The use of air as an oxygen source is convenient to the SEM operator but limits the cleaning effectiveness of the system to easily oxidized carbon species. The system does remove high percentages of the usual contamination problems of vacuum pump oil and skin oil hydrocarbons from surfaces in a relatively short time.

The EVACTRON SEM-CLEAN™ system provides a safe method for plasma cleaning inside the SEM. Air as a diluted oxygen gas mixture is chosen as the plasma reactant gas. Air is passed through a low-power RF glow-discharge to create oxygen radicals inside a generator mounted on a specimen chamber port. The generator chamber is subject to the same vacuum as the specimen chamber, and the vacuum pressure and gas flow are controlled by the EVACTRON SEM-CLEAN controller. The radicals are carried out of the plasma into the whole of the specimen chamber by convection. in the chamber they react with all exposed surfaces including the specimen if present. The plasma itself is confined to the generator chamber, which prevents ion and electron bombardment damage to the instrument or specimen.
The ORNL Aberration-Corrected STEM/TEM Project
Authors: Allard L F , Voelkl E , Blom D A , Nolan T A , Kahl F , Pennycook S J , Kersker M
Published: July 2020

Field emission electron microscopes operating at 200kV or 300kV and incorporating aberration correctors for either the incident electron probe or for the primary aberrations of the objective lens (OL) are currently under development for several laboratories in the world. OL-corrected instruments require monochromators for the electron beam, built into the electron gun prior to the accelerating stages, in order to optimize the contrast transfer characteristics of the objective lens to push the instrumental resolution limit to well beyond 0.1nm. This will allow the point resolution limit as controlled by the correction of spherical aberration Cs to potentially extend to the instrumental limit of better than 0.1nm. Figure 1 shows the contrast transfer characteristics of a Cs-corrected 200kV TEM, both without and with a beam monochromator.

Dedicated STEM instruments such as the 300kV VG-603 and lOOkV VG-501 at Oak Ridge National Laboratory, and other VG instruments at Cornell University and IBM Co. are also being adapted (by Nion Co., Kirkland, WA) to incorporate aberration correctors for the incident probe. The aim is to improve the resolution of the VG-603 instrument in dark-field imaging mode, for example, from 0.13nm to 0.05nm. in another ORNL project, the High Temperature Materials Laboratory has contracted JEOL Ltd. to construct a STEM-TEM instrument with a probe corrector designed and built by CEOS GmbH (Heidelberg, Germany).
Quantitative In-Situ Nanoindentation of Thin Films in a Transmission Electron Microscope
Authors: Minorl AM , Stach EA , Morris JW
Published: July 2020

A unique in situ nanoindentation stage has been built and developed at the National Center for Electron Microscopy in Berkeley, CA. By using piezoceramic actuators to finely position a 3-sided, boron-doped diamond indenter, we are able to image in real time the nanoindentation induced deformation of thin films. Recent work has included the force-calibration of the indenter, using silicon cantilevers to establish a relationship between the voltage applied to the piezoactuators, the displacement of the diamond tip, and the force generated.

In this work, we present real time, in situ TEM observations of the plastic deformation of Al thin films grown on top of lithographically-prepared silicon substrates. The in situ nanoindentations require a unique sample geometry (see Figure 1) in which the indenter approaches the specimen normal to the electron beam. in order to meet this requirement, special wedge-shaped silicon samples were designed and microfabricated so that the tip of the wedge is sharp enough to be electron transparent.
Angled Plan View FIB Sample Preparation for Accurate via Sidewall Measurements
Authors: Conner J , Prabhu L , Anderson A , Bagchi S , Rai R , Beck J , Duncan L
Published: July 2020

Critical dimensions in advanced semiconductor devices are now such that a significant fraction of some structures is contained within the thickness of a TEM foil. An important example is the vias used in multilevel metallization to interconnect transistors and other structures. Current generation vias have a diameter of 300 nm, and vias in devices under development are targeted at 180 nm. These dimensions are to be compared to the thickness of a TEM foil, which may be ∼50 nm. Vias are typically fabricated by etching holes in a dielectric layer, then depositing into these holes a barrier metal, such as Ta, followed by a Cu seed layer and finally Cu fill. Critical to via processing reliability are the thicknesses of barrier and seed layers. A TEM cross section of such a via will contain sharply curved layers which, when seen in projection, will lead to inaccurate measurement of layer thicknesses. Ordinary plan view preparation of these vias avoids this projection problem, but uncertainty is introduced in the height at which the via is sectioned.

We have developed an angled plan view (APV) FIB preparation technique that allows accurate measurement of barrier and seed thicknesses at discrete heights from bottom to top of a via.
XpertEze : Developing Embedded Intelligence for the Scanning Electron Microscope
Authors: Caldwell NHM , Breton BC , Holburri DM
Published: July 2020

The second generation of computer-controlled scanning electron microscopes (SEMs), i.e. instruments directed by software executing on conventional personal computers (PC-SEMs), has become the industry standard. Although limited automation has been incorporated by most manufacturers, these features do not significantly alleviate the requirement for operator expertise to attain optimal performance from the microscope. Our approach to the expertise deficit has been to pursue a knowledge-based approach to microscope control, encapsulating human knowledge in software to achieve expert performance. The task of SEM operation was decomposed into five phases - Specimen Preparation, SEM Preparation, SEM Initialisation, SEM Optimisation and External Analysis - and the central three phases were modelled in detail. A stand-alone prototype system (XpertEze) was developed to utilise this model for the LEO 400 series. A web-based version was implemented to support intelligent remote microscopy.

A stand-alone knowledge-based system has several disadvantages: its user interface competes for screen space with the conventional control software, its speed of execution is reduced as its instructions must be transferred via application programming interfaces to the microscope, and the operator must learn and manage two sets of software. Our ongoing work has been to remove these problems by incorporating the XpertEze system directly within the control system.
Implentation of Automated Image Acquisition and Image Analysis of Concrete in an Environmental Scanning Electron Microscope
Authors: Schalek Richard L , Elzafraney Mohamed , Soroushian Parviz , Drzal Lawrence T
Published: July 2020

The annual concrete consumption of two tons for every U.S. citizen makes it the most widely used construction material. Preserving this large concrete infrastructure requires an understanding of the deterioration and failure processes. Concrete exhibits composite behavior at several length scales (millimeter, micron, and submicron). in conjunction with this complex micro structure, damage processes such as crack formation and void collapse occurs at these various length scales. The diverse length scales combined with the large dimensions (meters) of concrete systems necessitates examination of macroscopic areas/volumes at microscopic magnifications to characterize damaged structures. This translates into examining large specimens, capturing a large number of images, and performing analysis on each image. Automating the image capture and image analysis greatly reduces the labor in evaluating damaged concrete structures.

Automated specimen positioning and image capture from an environmental scanning electron microscope (ESEM, ElectroScan 2020) and automated image analysis (Noesis Visilog 5.3 image analysis software) were synthesized with statistical and stereo logical principles into a comprehensive system for quantitative micro structural investigation of concrete.
Automated Filament Finding and Selection from Cryo Electron Micrographs
Authors: Zhu Y , Carragher B , Potter CS
Published: July 2020

For several years, we have been involved in developing a completely automated system for cryoelectron microscopy, which will allow a 3D electron density map of a macromolecular structure to be generated from a specimen inserted into the microscope with no manual intervention required by an operator. Towards this goal, we have developed a system, called Leginon, to automatically acquire images from the electron microscope. In order to integrate the 3D reconstruction process with the data acquisition step we need then to automatically identify and segment objects from the electron micrographs. This presents a challenge, particularly in the case when micrographs are acquired very close to focus, which results in an image with extremely low contrast. We present here the results of a two-stage automated process for detecting and selecting filamentous structures from images of this kind. Images are acquired in defocus pairs; a near-to-focus (NTF) image is acquired first, followed by a far-from-focus (FFF) image. In the first stage of the process, a threelevel perceptual organization algorithm is used to identify and segment filaments in the FFF image. In the second stage the NTF image is aligned to the FFF image using phase correlation techniques and the filaments are selected at the same coordinates as were identified in the FFF image.

In humans, perceptual organization is the ability to immediately detect structural relationships such as co-linearity, parallelism, and connectivity among image elements. Researchers in human vision perception have long recognized the importance of the common rules by which our visual system attempts to group information. Some of these rules include proximity, similarity, common fate, continuation and closure.
Elements and Organic Substances Analyzed with TOF-SIMS in the Photoreceptor Outer Segment Disc and Palpebral Conjunctiva of the Rat
Authors: Gong Huaqing , Amemiya Tsugio , Takaya kenichi , Tozu Miyako , Ohashi Yoshiharu
Published: July 2020

To make specimens for the biological materials, especially the retina and palpebral conjunctiva, and to detect the elements and organic substances.

Twelve-week-old male Wistar Kyoto rats were used. Immediately after the eyes were enucleated under pentobarbital anesthesia, the eyeballs and eyelids were put into liquid nitrogen without any fixation and covered by O.C.T. compound (TISSUE-TEK, Miles Inc.). Semi-thin sections (5 μm) were cut with a cryomicrotome at −30 °C (Zeiss, Germany). Sections of the retina and palpebral conjunctiva were placed on siliconwafer plates 10 × 7 × 1 mm and air-dried for 30 min or freeze-dried for 8 hrs. A metal microscope (Leica- DMRBE) was used to confirm the morphological features of the unstained conjunctiva and retina on the siliconwafer plate prior to analysis with a TOF-SIMS microscope. Without coating, the specimens were examined with a TOF-SIMS microscope (TRIFID) (Physical Electronics, USA). Positive and negative ion images were examined with a Ga+ source at an acceleration voltage of 15 kV. Secondary ion acceleration voltage of 4.5 keV was used. The amount of trace elements was analyzed by Wincadence (Physical Electronics, USA) software.