bluuebunny/crossref_metadata_2025 huggingface dataset:
Scanning Electron Microscopic Study of Flowing Erythrocytes in Hepatic Sinusoids as Revealed by “in Vivo Cryotechnique”
Authors: Terada N , Fujii Y , Kato Y , Ueda H , Baba T , Ohno S
Published: July 2020

The flowing behavior of individual erythrocytes in blood vessels is usually determined by their deformability, which is controlled mainly by the nature of their interior constituents and the flexibility of their surface membrane. Moreover, the physical behavior of erythrocytes passing through capillaries has been examined in vivo by light microscopy. However, little has been known about ultrastructural changes of such erythrocyte shapes flowing in blood vessels in vivo. Recently, a new technique was developed for freezing cells and tissues in vivo without stopping the blood supply, which was referred to as “in vivo cryotechnique”.This method has been also suitable for obtaining informations about dynamic morphological changes.

Seven female Balb/c mice were anesthetized peritoneally with sodium pentobarbital (100μg/g body weight), and their abdomen was opened through a pararectus incision. For artificial cardiac arrest, some mice were anesthetized with an excessive dose of the anesthetic (500μg/g body weight), their respiration and heart-beat were completely stopped, and the following procedures were done within one minute. A liver was put on a plastic plate without disturbance of blood circulation, and the “in vivo cryotechnique” was performed. Briefly, a cryoknife was pushed into the liver as fast as possible and the tissue was immediately poured with liquid isopentane-propane mixture (-193°C) (Fig.la,b).
Development of a 3-Dimensional Internal Structure Microscope (3D-ISM) for the Observation to Biological Organisms.
Authors: Yokota Hideo , Kudoh Ken-ichi , Sato Kahei , Higuchi Toshiro
Published: July 2020

We developed a new type 3-Dimensional Internal Structure Microscope (3D-ISM) for the observation of internal structures of samples. The internal structure of a sample is obtained by cutting it into thin slices and observing the cutting side continuously while cutting. The position of the camera, as well as the sample position are fixed. Therefore there is no shift between the sections, and this system can obtain a true color image of the sample, which is a high resolution and a high-quality three-dimensional image compared with X-ray CT and MRI. After repeatedly slicing a sample, the digital image data from the sectional views is transferred to a computer, where 3-dimensional images of the internal structure of the sample are reconstructed. Using this system we analyzed many biological organisms. In this paper, a mouse specimen has been cut and the 3-dimensional images are shown.

This article presents the outline of the device and the principle of the observation. FIG.l shows a corresponding block diagram and Fig. 2 a schematic view of the 3D-ISM.
JSR and EJSR in the Finch Heart: Morphometry by Serial Sections.
Authors: Sommer JR , High T , Nassar R , Taylor I
Published: July 2020

“Coupling(s)” (Fig. 1) are structure complexes in cardiac muscle consisting of junctional SR (JSR) tightly apposed to the cytoplasmic side of the plasmalemma. They are either peripheral (PC; when on the cell surface) or interior (IC; when on transverse tubule(s), TT). The JSR is a store for calcium (Ca)-release through Ca channels/ryanodine receptors in junctional processe(s) (JP) on the JSR surfaces. Action potential(s) (AP) for excitation-contraction coupling translate, as mediated by L-type channels, into Ca-entry into a ˜ 20 nm junctional gap (JG) containing JP between plasmalemma and the JSR's junctional membrane proper. In mammalian cardiac myocyte(s) (CM), where IC are ubiquitously distributed along TT at Z-bands, an AP quickly effects almost simultaneous global contraction, even in the center of cells of large diameter, whence CICR can be viewed as a one-step event between Ca-entry and JSR Ca-release from couplings. Avian CM, however, have no TT and, thus, only PC.
Presence of Caveolae in the Granular Epithelial Cells of Rabbit Urinary Bladder
Authors: Mia AJ , Oakford LX , Yorio T
Published: July 2020

Previously, we reported the presence of caveolae, clathrin-coated pits and vesicles in toad urinary bladder granular epithelial cells, and in smooth muscle and endothelial cells. Since then we investigated the occurrence of caveolae in the rabbit urinary bladder cells. Here we report for the first time the presence of caveolae and clathrin-coated pits and vesicles in the granular epithelial, smooth muscle and endothelial cells of rabbit urinary bladders.

Urinary bladder sacs were surgically removed from doubly pithed toads and anaesthetized rabbits, and allowed fixation for 1 hr in 2% glutaraldehyde in PIPES buffer. These bladder tissues were divided for scanning (SEM) and transmission electron microscopy (TEM) for comparative analysis. A postfixation using 1% osmium tetroxide was carried out for 1 hr. Tissues were appropriately processed for SEM and TEM observations. Ultrathin sections, made with a diamond knife, were taken on bare nickel grids and then exposed to uranyl acetate and lead citrate for viewing in the TEM.
Immunohistochemical Evidence for Subclasses of Interstitial Cells of Cajal Associated with the Deep Muscular Plexus in Rat Small Intestine
Authors: Yunker AMR , Roth KA
Published: July 2020

Interstitial cells of Cajal (ICCs) were first described as primitive neurons found within organs innervated by autonomie nerves. In the gut, ICCs are juxtaposed among enteric nerve fibers and smooth muscle cells, suggesting they may modulate enteric neurotransmission and affect motility. The recent discovery of neurokinin-1 receptor (NKrl)-like immunoreactivity (ir) on ICCs has strengthened this hypothesis. This study compared the distribution of NKlr-ir to the staining patterns of other reported markers of ICCs including cholera toxin subunit b (CTB), neuron specific enolase (NSE), NADH diaphorase, NADPH diaphorase, nitric oxide synthase (NOS), and vimentin. Albino male rats were anesthetized and whole mount preparations of myenteric plexus-longitudinal muscle with attached circular muscle were dissected. ICCs were stained using multi-label techniques and cells were visualized with a Sarastro 2000 confocal laser scanning microscope equipped with Image Space software.

In the small intestine, ICCs associated with circular muscle, but not longitudinal muscle, expressed NKlr-ir.
Microcysys in the Posteroventral Cochlear Nucleus of the Gerbil by Scanning Electron Microscopy
Authors: Yu SM , Lee CI
Published: July 2020

Microcysts are most evident in the posteroventral and anteroventral cochlear nucleus (PVCN & AVCN) of the gerbil. They are reported as a neurodegenerative disorder or spongioform degeneration or a dynamic process related to the degree of auditory stimulation in the cochlear nucleus of the gerbil. Little information is avalable on the origin and formation of microcysts in the cochlear nucleus of the gerbil. The aim of this study was to investigate the internal morphologic changes of microcysts in the gerbil PVCN during postnatal development by scanning electron microscopy.

The mongolian gerbil, Meriones unguiculatus, were sacrificed by cardiac perfusion of a saline nitrite flush, followed by a 3% glutaraldehyde in 0.1M phosphate buffer. After fixation the brain were washed in 0.1M phosphate buffer,embedded in 5% agar, vibratome-sliced through the cochlear nucleus, and then postfixed in the 1% osmium tetroxide in 0.1M phosphate buffer for one hour. The sectioned tissues were dehydrated in a graded ethanol series to absolute ethanol and transferred to liquid carbon dioxide for critical point drying.
Morphometry of Synapse and Granule Cell Changes in the Hippocampus of Aging Fischer 344 Rats
Authors: White S L , Gatlin C L , Horn J W , Shannon H E , Smith W C , Jordan W H
Published: July 2020

Morphologic changes in Alzheimer’s disease (AD) include a decrease in synapse number while the surviving synapses are increased in size by a process known as compensatory synaptogenesis. There is disagreement among investigators as to whether the synapse changes observed in AD in humans also occurs in the aging rat. The hippocampus, plays a significant role in learning and memory in both humans and rats and is known to be particularly susceptible to cell loss associated with aging. s In the rat hippocampus, age-related synaptic density declines as much as 27% have been reported by some authors, while other investigators have failed to detect a change in synapse number with age. A secondary factor in the total number of synapses in the dentate gyrus is the number of granule cells. Proliferation or regeneration (neurogenesis) of hippocampal granule cells is known to occur well into the adult life of the rat. The goal of the current study was to further explore age-related changes in rat hippocampal synapses and granule cells using the disector morphometry method.
An Integrated Biological Imaging Facility: Capabilities of the Biological Microscopy and Image Reconstruction Resource
Authors: Frank J , Mannella CA , Rieder C
Published: July 2020

The Biological Microscopy and Image Reconstruction Resource (BMIRR) is operated by the Wadsworth Center as a national biotechnology resource, with funding through the NIH Center for Research Resources and from NSF. This biological imaging resource has evolved continuously over the past two decades. Early development focussed on correlative, same-cell light and electron microscopic techniques, combining the capabilities of video-enhanced light microscopy and high-voltage electron microscopy. A current area of development is electron microscopic tomography, whereby the full 3D capabilities of higher voltage (400-1200 KV) electron microscopy is brought to bear on biological problems. In particular, the recent development of techniques for merging projection data from two mutually perpendicular tilt series has permitted significantly improved resolution, reducing the missing wedge of information to a missing pyramid. Attention is now turning to optimization of conditions for applying tomography to frozen-hydrated specimens, using automated data collection on our cryo-IVEM. Combined with parallel advances in same-cell manipulation and viewing, the BMIRR provides biologists with a unique combination of imaging and computational tools for research into the 3D structure and dynamics that underly cellular processes (see figures and refs. 2-4).
Exploring Macromolecular Interactions at High Resolution Using Electron Cryomicroscopy at NCMI
Authors: Schmid MF , Sherman M B , Brink J , Jakana J , Kelsey M , Dougherty M , Chiu W
Published: July 2020

The National Center for Macromolecular Imaging (NCMI) is a resource supported by the National Center for Research Resources (NCRR) of NIH. Its mission is to advance electron imaging of macromolecular assemblies to near atomic resolution. We have focused on biological assemblies which are too large or too complex to be studied by conventional x-ray crystallography or NMR spectroscopy techniques. As is the case with all the NCRR-supported facilities, we also conduct methodology development, collaboration, service, technology dissemination and training.

The objectives of this Research Resource are to develop the technology of high resolution electron cryomicroscopy of biological macromolecules, assemblies and crystals in an integrated approach that includes specimen preparation, hardware and software development. A current methodology development includes the characterization of the 1k x 1k slow-scan CCD camera for both electron diffraction and imaging of ice-embedded specimens. These studies have defined the advantages and limitations of the CCD camera for electron crystallographic analysis. Another core research project deals with the problems of specimen movement due to the electron beam and/or charging and seeks to minimize these phenomena. Our efforts have resulted in a better understanding of the physics of charging. We have modified a Gatan ion-beam coater so that we can apply a thin layer of carbon onto ice-embedded specimens.
Tissue Compatibility of Silicon Microfabricated Probes Inserted into the Brain
Authors: Turner J N , Shain William , Szarowski D H , Anderson M , Martins S , Davis R , Craighead H G , Isaacson M
Published: July 2020

The application of nano- and microfabricated devices based on silicon electronics technology is an emerging interdisciplinary area combining engineering and biology. The placement of electrically active probes in damaged or diseased tissues of the central nervous system could have enormous impact on the health and quality of life of large numbers of individuals by restoring lost function, or by treating or controlling disease states. Such probes have been fabricated at a high level of engineering sophistication. Unfortunately, when inserted into the brain a tissue reaction is initiated forming a scar that surrounds and electrically isolates the probe within a few weeks. This reaction is thought to primarily involve glial cells, and is undoubtedly dominated by the bulk surface of the probes which have a silicon oxide layer on top of single crystal silicon.

Model probes (Fig. 1) were microfabricated by photolithography with a 1×1mm tab used for gripping and inserting probes.
Neurological Research and Shared Imaging Resources at the National Institutes in Okazaki, Japan
Authors: Hama Kiyoshi
Published: July 2020

The Okazaki National Research Institutes, consisting of three cooperative research Institutes, the Inst. of Molecular Science (NIMS), the Inst. of Basic Biology (NIBB) and the Inst. of Physiological Sciences (NIPS), employs about 700 researchers and 200 full time technical staff. To support cooperative research projects, ONRI has several special shared research facilities in addition to those conventionally used for bioscience laboratories: a 1000 kV high voltage electron microscope designed for three dimensional morphometry and computer tomography of biological structures, a 300 kV ultra cryoelectron microscope equipped with an energy filtering system, which is now being set up, and a two photon excitation laser confocal microscope. In addition, there is an MRI, a rapid MRI, SQUID with 37 + 37 channels, and micro SQUID for non-invasive examination of brain function in human and experimental animals. A UVSOR (UV synchrotron orbital radiation) lab., a laser lab., and an ultra-low temperature lab. are available for neuroscience collaboration with Japanese and international scientists.
Neurocartography: Geometry and Biology of the Central Nervous System
Authors: Tretiak O J , Nissanov J
Published: July 2020

The central nervous system of a vertebrate organism exhibits a very complex spatial organization structure and function. These relationships are the subject of intense study for over a century, and recent developments in imaging have attracted ever increasing effort devoted to the understanding of brain function. One can produce any number of quantitative images that provide maps of the anatomy and function of nerve tissues. For example, autoradiography can yield functional images (2-deoxy glucose), maps of neurotransmitters receptors (over 100 know types), and gene expression labeled with complementary mRNA (in-situ hybridization). Immunohistochemistry produces maps of a large variety of neuroactive components, such as transmitters.

To illustrate a typical procedure, we describe the mapping of brain function with 2-deoxy glucose (2DG). A rat performing some task is injected with a solution of 2DG radiolabeled with 14C¨ Subsequently, the animal is sacrificed, the brain is cryosectioned (ca. 20 μm), and contact autoradio-grams of these sections are made on X-ray film.
Three Dimensional Optical Data Storage Using Two-Photon Induced Photobleaching in a Polymer Matrix
Authors: Bailey GW , Dimlich RVW , Alexander KB , McCarthy JJ , Pretlow TP , Pan S J , Shih A , Park M S , Liou W S , Bhawalkar J , Prasard P , Cheng P C
Published: July 2020

The intensity of two-photon induced fluorescent shows quadratic dependency to the excitation intensity, as a result, one can achieve optical sectioning in fluorescent microscopy without the use of a confocal aperture. When the excitation power is properly adjusted, the fluorescent and photobleached volume is restricted to the vicinity of the focal points; therefore, it is possible to use this mechanism to achieve 3D data storage and microstructure fabrication (stereolithography).

The recording medium used in this experiment was poly-HEMA block doped with a high efficient two-photon fluorophore, APSS, (4-[N-(2-hydroxyethyl)-N-methyl) amino phenyl]-4’-(6-hydroxyhexyl sulfonyl) stilbene). The block was made by dissolving APSS in HEMA with an initiator, 2,2’-azobisisobutyronitril (AiBN) (1% mole ratio) and polymerized at 60° C for 48 hours. The polymer was then trimmed into a small rectangular block (3×5×1 mm3) and surfaced by using a ultramicrotome with a glass knife. A modified Biorad MC500 LSM equipped with a home-made computer controlled stage scanner was used for both writing (stage scanning) and read-back (beam scanning).
Using Multiphoton Microscopy for the Study of Embryogenesis
Authors: White John , Centonze Victoria , Wokosin David , Mohler William
Published: July 2020

Multiphoton fluorescence excitation imaging is an optical sectioning technique for fluorescence microscopy. At very high photon densities, two or more photons may coordinately excite an energy transition in a fluorophore that corresponds to the sum of the energies of the individual photons. by this means, a fluorophore may be excited by a wavelength that is considerably longer than its single photon excitation wavelength. Ultra-fast pulsed (femtosecond) lasers can produce the peak power densities in the focal volume of an objective lens needed to provide sufficient 2- or 3- photon excitation events for imaging. The use of short-pulse lasers provides the high peak powers necessary for imaging yet with modest mean power levels that do not thermally damage biological specimens. Production of multiphoton events depends on the square of photon density for 2-photon excitation and the cube of photon density for 3-photon excitation. The power density therefore rapidly falls off away from the focal volume of an objective lens, thereby confining fluorescence excitation to the focal volume.
Coronary Intravascular Ultrasound
Authors: E. Nissen Steven , Murat Tuzcu E
Published: July 2020

For several decades, contrast angiography has served as the principal imaging modality used to assess the anatomic severity of coronary artery disease. Recent technical advances in acoustics and microelectonics have permitted development and refinement of miniaturized ultrasound devices capable of real-time tomographic coronary imaging. This approach, intravascular ultrasound, represents an emerging alternative to angiography for direct visualization of coronary anatomy during diagnostic and interventional catheterization. Clinical studies comparing angiography to intravascular ultrasound have demonstrated frequent differences in quantitative or qualitative findings. Accordingly, intraluminal ultrasound imaging is increasingly utilized to confirm, refute, or supplement angiographie data in patients with coronary disease.

The discrepancies between angiography and intravascular ultrasound can be explained by the characteristics of these two different imaging techniques. Angiography, which portrays the vessel as a silhouette of the lumen, whereas ultrasound depicts the coronary from a tomographic perspective, providing cross-sectional images that portray not only the lumen, but also the deeper intramural structures.
Rapid, Automated, and Robust Tracing of Vasculature, and Quantification of Visual Features from Angiographic Images
Authors: Can Ali , Shen Hong , Al-Kofahi Khalid , Turner James N , Tanenbaum Howard L , McCandless Brian , Roysam Badrinath
Published: July 2020

The rapid tracing of vasculature, and quantification of visual landmarks such as those corresponding to the branching and crossover points from images is extremely useful in a variety of fields including ophthalmology, radiology, and neurobiology. A novel algorithm that enables rapid, automatic, robust, adaptive, and accurate tracing of vasculature has been developed. Given an image (Fig 1), it automatically generates a labeled graph representation of the vasculature (Fig 2), and a table listing the locations of visual landmarks such as crossover and branching points (Fig 3). For each such point, it also lists the intersection angles, and the local thickness of the intersecting vasculature segments. This data is often useful by itself. They can also be used for image registration and montage building.

This algorithm greatly improves upon previous work in several ways. First, it is fully automatic, and free of parameters that need to be adjusted carefully (just needs optional sensitivity setting).
Three Dimensional Molecular Scale Imaging of Plasma Proteins Under Aqueous Conditions
Authors: Marchant Roger E , Holland Nolan B , Siedlecki Christopher A
Published: July 2020

Atomic force microscopy (AFM) provides unique opportunities to study cell-surface and molecular scale interactions in three dimensions under aqueous conditions. In AFM, a small probe attached to cantilever (fig. 1) is used for interacting with a sample to obtain sensitive measurements of molecular structure, intermolecular forces (sub-nN) and interfacial properties. The data ouput can be presented in the form of a topographical image, force map or viscoelastic response (e.g., Figs. 2-4). Thus, AFM combines high resolution imaging with the ability to measure surface-dependent intermolecular forces and properties. AFM also is amenable for coupling with optical imaging methods including immunofluoresence and gold bead labeling.

This presentation will focus primarily on the use of AFM for molecular level imaging of plasma proteins, von Willebrand Factor (vWF) and fibrinogen. Both proteins play central roles in the regulation of hemostasis and thrombosis by participating in coagulation or by facilitating adhesion, spreading and aggregation of activated platelets.
Cryoelectron Microscopy of Red Cell Skeletons Reveals that Spectrin has a Helical Conformation
Authors: Yang Li , Josephs Robert
Published: July 2020

In vivo the red blood cell reversibly and rapidly deforms as it passes through the small capillaries. Although the physiochemical basis of the red cell's deformability is unknown, it is regarded as being a property of the membrane protein spectrin. We have used cryoelectron microscopy to study the structure of spectrin in membrane skeletons to determine how its molecular structure confers elastic properties on the cell membrane. Cryomicroscopy has the virtue of minimally perturbing easily deformable structures such as spectrin.

Figure 1 is an electron micrograph of a portion of a frozen-hydrated red cell skeleton. The spectrin molecules are indicated by the arrow heads. They cross link the skeletal network by interacting with short rod like segments F-actin (indicated by the arrows) containing about 13 actin molecules. In such micrographs the spectrin appears to have a sinusoidal shape whereas in negatively stained preparations spectrin appears to be straight.

We have used the procedure of Margalef in order to determine the three dimensional trajectory of the spectrin molecule.
Practical Methods for TEM, Part II.
Authors: Chien K , Gonzalez R , Heusser RC , Shiroishi H , Heathershaw ML
Published: July 2020

This is an extension of our presentation from last year. We intend to continue the discussion and review several practical procedures developed in our laboratory for routine and immuno-electron microscopy.

TEM Vs IM-LM In the last decade, the use of diagnostic TEM in the medical field has experienced a drastic decline. The main reason is due to the advances of immuno-labeling techniques for light microscopy and also the decreased coverage of medical insurance for EM. Currently, immuno-histochemistry or in situ hybridization staining can be performed on large paraffin or frozen sections. These procedures can be performed quickly and much less expensively than TEM. However, TEM is still needed when PAP staining is not specific or minimum numbers of cells are stained. It is also possible to process immuno-labeled paraffin slides for TEM examination to confirm a diagnosis. In addition, when only a few microorganisms such as pneumocystic carinii or protothecosis are stained by Grocott’s methenamine silver in paraffin sections, a re-examination and confirmation by TEM can be performed without the use of uranium or lead staining.
The Development and Application of the Tripod Polishing Technique
Authors: Benedict J , Klepeis SJ , Anderson R
Published: July 2020

The Tripod polishing technique is used to mechanically polish materials for further analysis by optical, SEM or TEM. The mechanical polishing is done using the Tripod Polisher (FIG. 1), which consists of three Delrin capped micrometers and an L-bracket. The micrometers provide accurate side-to-side and front-to-back adjustments of the polishing plane. The L-bracket is where the sample is attached to the polisher and it comes in a number of configurations, depending on the type of analysis needed. by attaching the sample at different locations on the L-bracket, either planar sections or cross sections can be made using the Tripod Polisher.

A sample to be cross sectioned is attached to the front of the L-bracket with wax. The micrometers are then adjusted so the bottom of the Delrin feet are aligned with the area of interest. The polisher is held on a rotating diamond wheel, with the Delrin feet and the sample in contact with the wheel.
Negative Staining: a Valuable Technique for Studying Subcellular Components
Authors: Miller Sara E
Published: July 2020

Negative staining is the most frequently used procedure for preparing particulate specimens, e.g., cell organelles, macromolecules, and viruses, for electron microscopy (Figs. 1-4). The main advantage is that it is rapid, requiring only minutes of preparation time. Another is that it avoids some of the harsh chemicals, e.g., organic solvents, used in thin sectioning. Also, it does not require advanced technical skill. It is widely used in virology, both in classification of viruses as well as diagnosis of viral diseases. Notwithstanding the necessity for fairly high particle counts, virus identification by negative staining is advantageous in not requiring specific reagents such as antibodies, nucleic acid probes, or protein standards which necessitate prior knowledge of potential pathogens for selection of the proper reagent. Furthermore, it does not require viable virions as does growth in tissue culture. Another procedure that uses negative contrasting is ultrathin cryosectioning (Fig. 5).

In 1954 Farrant was the first to publish negatively stained material, ferritin particles.
A New Preparation Technique for High Resolution Imaging of Protein Structure: Casein Micelles.
Authors: McManus William R , McMahon Donald J
Published: July 2020

Models for the structure of the bovine casein micelle have been proposed in the past (1,2,3,4). These models are based on the chemical and physical properties of the micelles and fall into two general catagories, framework and submicelle models. Electron Microscopy techniques were one of the tools used in the development of these models. However, no definative model has been established. We have developed a new electron microscopy method and it is being applied to the re-examination of the protein structure of the casein micelle, with the goal of establishing a definative model for the structure of the casein micelle.

High resolution scannning electron microscopy has proven unsuccessful in revealing the protein structure in the casein micelle (Fig 1). To create high resolution scanning electron microscopic images, fine metal coatings are required in the 5 to l0nm range. Following the application of these coatings, images of casein micelles resemble a melting lumpy sphere. This is due to the metal layer obscuring the proteins from view.
A Novel use of Fluorescence Microscopy in Evaluation of Renal Biopsies
Authors: McMahon JT , Myles JL , Tubbs RR
Published: July 2020

Light, immuno, and electron microscopy are standard for evaluation of renal biopsies. For light microscopy (LM), special stains including PAS and Jones silver methenamine (JSM) are frequently used to enhance recognition of renal anomalies related to disease. We recently discovered that fluorescence microscopy (FM) extends the usefulness of frozen and paraffin sections stained with hematoxylin and eosin and may presage the findings of immuno and electron microscopy. With UV illumination and reflected light fluorescence excitation, standard histologie sections reveal pathologic glomerular and other renal changes not evident by conventional bright field microscopy and may obviate the need for additional special stains.

For frozen sections, tissues mounted on gum tragacanth were snap frozen in isopentane chilled with liquid nitrogen and cryosectioned at a thickness of 4-6 μm. For paraffin sections, tissues were fixed in neutral buffered 10% formalin or in Hollande’s fixative (3.6% formalin, 40% picric acid, 25% copper acetate, 1.5% acetic acid) and sectioned at 2-4 μm.
A New ion Beam Based Etching/Coating System for Scanning Electron Microscopy (SEM) and Light Microscopy (LM)
Authors: Alani R , Mitro RJ , Tabatt CM , Malaszewski L
Published: July 2020

The design and performance of a new instrument, based on improved Penning ion guns [1] for etching and coating samples for SEM and LM in a single vacuum chamber, are described. The instrument is based on an existing high resolution ion beam coating system, which is capable of producing high quality ultra-thin and amorphous conductive films, required for present high resolution electron microscopes. [2]. The fact that in this system both etching and coating processes are combined in one chamber, the specimen handling and specimen contamination are minimized. Furthermore, the system eliminates the traditional multiple mounting /dismounting of samples to various holders for mechanical polishing, etching, coating and microscopy purposes. The specimen can stay with the same holder throughout the entire process, increasing the sample through-put. Moreover, the system offers an alternative method to the traditional “wet chemical etching,” technique with its well known problems.
Video Light Microscopy
Authors: Spring K R , Herman B , Salmon E D
Published: July 2020

This tutorial emphasizes the fundamentals of the use of light microscopy in combination with electronic imaging. The goal of the tutorial is to enable the attendees to make informed choices both about the modes of imaging best suited to their application and about the most suitable components to be employed. The didactic portion of the program will consist of five presentations, each dealing with different aspects of modern imaging systems and their applications to problems of biological significance. A laboratory demonstration session, organized to complement the lectures will follow.

The morning session will begin will a brief review by Dr. Spring of the principles of light detection and the formation of the video signal. Dr. Salmon will then describe video-enhanced contrast microscopy. This transmitted light imaging technique, widely utilized to visualize low contrast specimens exploits the wide dynamic range and high gain of video cameras for the capture of images that are virtually invisible through the eyepieces because of high background light.
Environmental Scanning Electron Microscopy: - Advantages and Disadvantages
Authors: McKernan Stuart
Published: July 2020

The possibility of performing scanning electron microscopy under “environmental” conditions (ESEM); where the specimen does not have to be under high-vacuum conditions, or even coated to make it conductive, has existed for several years[l]. Several different flavors of environmental scanning electron microscope now exist, as well as a newer generation of microscope which is capable of imaging at very low accelerating voltages. Techniques for rapidly freezing hydrated specimens and obtaining very high resolution images from them in the SEM have also been improved recently. As a result of all this innovation, it is now possible to examine many classes of specimens in the SEM that were previously thought to be impossible to image (or at least not worthwhile frying).

Among the advantages of the environmental SEM are the fact that images of non-conductive surfaces can be obtained without the necessity for additional sample coating. This can be especially important in dynamic experiments, and hot-stage work, where the presence of a surface coating may substantially affect the samples behavior
Biological Applications of Environmental Scanning Electron Microscopy
Authors: Gilpin C J
Published: July 2020

The environmental scanning electron microscope (ESEM) first marketed by ElectroScan and now by FEI/Philips has been recognized as the only scanning electron microscope which allows liquid water to be present in the sample chamber at temperatures above freezing. As most biological material contains a high percentage of water it would seem essential to use this technology to examine specimens in their natural state. The purpose of this presentation is to describe in detail the way in which the microscope should be used to maximize the information obtainable from a variety of biological samples. This tutorial will also consider methods which can be applied to “difficult” samples whose structure becomes altered during what would be considered a normal imaging schedule.

The main features of the microscope are the use of the Peltier effect cooling stage and control of chamber water vapor pressure to enable wet specimens to be imaged.
Novel and Advanced Applications of the Low Vacuum and Environmental Scanning Electron Microscope (SEM)
Authors: Griffin Brendan J
Published: July 2020

The environmental SEM is an extremely adaptive instrument, allowing a range of materials to be examined under a wide variety of conditions. The limitations of the instrument lie mainly with the restrictions imposed by the need to maintain a moderate vacuum around the electron gun. The primary effect of this has been, in a practical sense, the limited low magnification available. Recently this has been overcome by modifications to the final pressure limiting aperture and secondary electron detector (Fig.l). The modifications are simple and users should be brave in this regard.

A variety of electron detectors now exist including various secondary, backscattered and cathodoluminescence systems (Figs 2-5). These provide an excellent range of options; the ESEM must be regarded as a conventional SEM in that a range of imaging options should be installed. In some cases, e.g. cathodoluminescence, the lack of coating provides an advantage unique to the low vacuum SEMs.
Field Emission TEM and STEM of Ultrafine Metal Particles
Authors: Disko M M , Orchowski A
Published: July 2020

Accurate descriptions of dispersed metals are of central importance in optimizing the activity, selec-tivity and stability of a diverse set of catalysts and chemistries. Improved methods for quantifying metal dispersion, alloying and support interactions help form mechanistic understandings of catalyst function at the atomic scale. In this paper we discuss ideal capabilities of a transmission electron microscope for catalysis, and give an update on the use of a next-generation field-emission TEM/STEM for structural and analytical studies of supported metal catalysts.

Characterizing nanometer-sized metal particles requires high performance imaging, high sensitivity analysis, and minimal disruption of the physical state of the particles. No single microscope combines all key performance factors with ease of use. Schottky field-emitter instruments provide high current-density probes with excellent stage stability (nm drifts in > 10 min. observed), < 0.7 eV energy reso-lution for EELS, and a turbo-pumped vacuum system for minimal carbon contamination while using nanometer probes and nanoamps of current. Scanning TEM performance is also key.
Mesoscopic Silica Thin Films Via Template-Assisted Self-Assembly
Authors: Yao N , Trau M , Manne S , Nakagawa N , Lee T , Honma I , Aksay I A
Published: July 2020

Synthesis of mesoscopic silicates has stimulated considerable interest as a biomimetic approach to the fabrication of organic/inorganic nanocomposites. However, for future applications of these materials, controlled shapes and patterns, particularly as continuous thin films, rather than the microscopic particulates, need to be processed. Recently, we have described a synthesis scheme, using the supramolecular assemblies of surfactant molecules as a template for the condensation of inorganic species, to prepare silica mesoscopic thin films at a solid interface from a bulk solution. Here, we concentrate on the growth of such thin films and the effect of the template on the nanostructure formation.

We employed tetraethoxy silane (TEOS), dissolved in acidic solution, as a silicate source and cetyltrimethyl ammonium chloride (CTAC) as the templating surfactant. Typical molar ratio was 1 TEOS : 1.2 CTAC : 9.2 HC1 : 1000 H2O. Concentrations of TEOS and HCl in the mixture were approximately l/7th of those for typical particle synthesis scheme, in order to sufficiently slow down the rate of TEOS hydration such that homogeneous nucleation is effectively switched off.
Microstructural Instability in Mosi2/Sic Nanolayers
Authors: Lu YC , Kung H , Hirvonen J-P , Jervis TR , Nastasi M , Ruck D , Mitchell T E
Published: July 2020

Thin film multilayers have been the focus of extensive studies recently due to the interesting properties they exhibit. Since the improvement in properties can be attributed directly to the unique nanoscale microstructures, it is essential to understand the factors affecting the microstructural stability in these nanolayer structures. The intermetallic compound, MoSi2, despite its superior oxidation resistance and high melting point, suffers from inadequate high temperature strength and low temperature ductility, properties which hinder its high temperature structural applications [1]. SiC is a potential second phase reinforcement due to its high temperature strength and thermal compatibility with MoSi2. The addition of SiC in a nanolayered configuration has been shown to exhibit significant increase in hardness after annealing [2]. It has also been shown that when annealed above 900°C, the layers break down and grain growth sets in, with a significant decrease in hardness and. Due to the lack of a thermochemical driving force, the two phases remain separate at all temperatures investigated. In this study, the stability of the MoSi2/SiC nanolayers structure under ion irradiation has been investigated.
FCC Superlattice Packing of Faceted Silver Nanocrystals
Authors: Harfenist SA , Wang ZL , Alvarez MM , Vezmar I , Whetten RL
Published: July 2020

The ordering of size selected nanocrystaline particles into large, well ordered supercrystals has been under recent study. Here we describe the ordering of silver nanocrystals, of core diameter ca. 4.5nm, passivated with alkythiolate self-assembled monolayers. The faceted nanocrystals are found to size selectively condense into ordered supercrystals with edge lengths on the order of 0.1 to 0.5 microns. Further, evidence has been found in High Resolution TEM micrographs of packed monolayers of these same nanocrystals implying an orientational ordering of the silver nanocrystals’ atomic lattices within and co-aligning with the supercrystal’s “superlattice”. A three dimensional model using a truncated octahedron for the nanocrystal core morphology is constructed from this evidence into an FCC superlattice which fits the remaining experimental observations.

Micrographs of the same region of a nanocrystal Ag supercrystal (NCASX) are shown in figure 1 under differing defocus conditions.
HRTEM Study of Sputtered Cu-6at% Ta Films
Authors: Liu Y , Wang H , Mazumder J , Rigsbee JM
Published: July 2020

Cu-refractory metal composites such as Cu-Ta alloys are attracting research interest because of their potential high electrical and thermal conductivity and high mechanical strength at elevated temperatures [1]. In the previous study, it has been shown that the hardness of Cu-Ta is three times of the Cu substrate [2] and the Ta was found to exist as discrete particle and aligned particles. In this presentation, we report the detailed study on the microstructure of the heat treated film and hot-pressed film.

The Cu-6at%Ta films were deposited on Cu foil at 100°C by RF co-sputtering. The films examined in this study was hot-pressed under 35 MPa pressure at Ar atmosphere for 1 hour. Cross-sectional TEM samples were examined using a Hitachi 9000 high resolution transmission electron microscope (HRTEM) at the Center for Microanalysis, Materials Research Laboratory, University of Illinois at Urbana-Champaign.
TEM Studies of Silicon Nitride-Silicon Carbide Nanocomposites
Authors: Wang E Y , Pan X , Mansfield J P , Kennedy T , Hampshire S
Published: July 2020

Si3N4/SiC nanocomposites have been shown to exhibit excellent strength and fracture toughness compared to monolithic Si3N4 materials. Recently, the microstructure and chemistry of Si3N4-based nanocomposites fabricated by hot-pressing amorphous Si-C-N precursor powders has been investigated. In the present work, our studies on the microstructure of Si3N4/SiC nanocomposites made by hot-pressing the mixture of Si3N4 and SiC commercial powders are reported.

Si3N4/SiC nanocomposites were prepared by hot-pressing at 1750 °C for 1 hour at 40 Mpa in a nitrogen atmosphere, with sintering aids of 5.5 wt% Y2O3 and 3 wt% Al2O3. The details of the processing procedure have been reported elsewhere. Two different materials were investigated in this work: specimen A consisting of 5 vol% β-SiC; and specimen B consisting of 5 vol% α-SiC. TEM specimens were prepared by conventional procedures. The microstructure and chemical composition were studied in the University of Michigan Electron Microbeam Analysis Laboratory on a JEOL 2000FX.
The Examination of Calcium ion Implanted Alumina with Energy Filtered Transmission Electron Microscopy
Authors: Hunt EM , Hampikian JM , Evans ND
Published: July 2020

Ion implantation can be used to alter the optical response of insulators through the formation of embedded nano-sized particles. Single crystal alumina has been implanted at ambient temperature with 50 keV Ca+ to a fluence of 5 x 1016 ions/cm2. Ion channeling, Knoop microhardness measurements, and transmission electron microscopy (TEM) indicate that the alumina surface layer was amorphized by the implant. TEM also revealed nano-sized crystals ≈7 - 8 nm in diameter as seen in Figure 1. These nanocrystals are randomly oriented, and exhibit a face-centered cubic structure (FCC) with a lattice parameter of 0.409 nm ± 0.002 nm. The similarity between this crystallography and that of pure aluminum (which is FCC with a lattice parameter of 0.404 nm) suggests that they are metallic aluminum nanocrystals with a slightly dilated lattice parameter, possibly due to the incorporation of a small amount of calcium.Energy-filtered transmission electron microscopy (EFTEM) provides an avenue by which to confirm the metallic nature of the aluminum involved in the nanocrystals.
Dynamic Observation of the Thinnest Gold Nano-Bridge by Newly Developed UHVTEM
Authors: kondo Yukihito , Takayanagi Kunio
Published: July 2020

The nano-structure science have become popular as the nano-fabrication technology has progress recently. Among the nano-structures, the nano-wires have been attracted attention because they show quantized conductance which would be used in quantum devices[l]. The quantized conductance has been observed even at room temperature with the nano-wires prepared by tip-indentation in scanning tunneling microscope [2-4]. However, the direct observation of the nano-wires has not been examined because of lack of their stability and difficulty of specimen preparation.

We have generated a gold thin wire with the thickness ranged 0.8-2 nm, referred to as a nano bridge (NB)[5], in an ultrahigh vacuum transmission electron microscope (UHVTEM) [6]. The NB is stable at room temperature and shows good reproducibility. The shape of NB is surprisingly uniform (see Fig. 2). The structure is stabilized by stable hexagonally-closed-pack (hcp) planes for the NB of 2 nm in thickness [5]. The well-defined structure provide a good opportunities for applied and theoretical studies.
Procedure to Determine the Structure of Three-Horned Multishell Fullerene
Authors: Kondo Yukihito , Takayanagi Kunio
Published: July 2020

Fullerenes have many families such as simple ball-shaped fullerenes, nanotubes, and onions. Additionally torus, Y-shaped particle, and graphitic network with periodic holes or dimples have been theoretically anticipated. The presence of various shapes in fullerenes is mainly attributed to the combination of five-membered and seven-membered rings in addition to six-membered rings composing ordinary graphite crystal. The five-membered rings are necessary for positive curvature of the fullerenes, while the seven-membered rings for negative curvature. Experimentally, the negative curvature has been restricted so far to the joint between cone and tube in nanotubes and the spiral tubes. We have found a new type of multishell fullerenes with negative curvature which consist of three horns protrude into three-fold symmetric directions, referred as three-horned multishell fullerene (THF) in this report, using a high resolution transmission electron microscope (TEM). Here, we first describe the formation briefly and secondly concerned with procedure to determine the structure of the THF.
Nanoscale Patterning of BaTiO3 on Block Copolymers
Authors: Lee T , Yao N , Aksay I A
Published: July 2020

Nanoscale pattering of inorganics can be facilitated by using complex fluids as templates. One such approach is to use block copolymeric nanodomains in solid films (< 50 nm) as the template for patterning ceramic clusters with desired shapes and orientations. For instance, we use a triblock copolymer of polystyrene (PS) -polybutadiene (PB) - polystyrene (PS) - Kraton Dl 102® (MW = 71,000 and PS/PB = 28%/78%) as a template material to pattern barium titanate for energy storage applications. The important features of our approach are that (i) the interdomains of PBs in the preformed solid thin films are chemically OH-functionalized in situ, and (ii) the barium titanate precursors are solely localized within and reacted to the interdomains of PBs in solid thin films by condensation.

Thin films were prepared by spin casting a 1% w/w Kraton D1102®/toluene solution on a coverglass. Thin films were transferred to gold TEM specimen grids by the water lift off method.
Characterization of Nanophase Particle Aggregates by Transmission Electron Microscopy
Authors: Gonzalez G , Freites A , Rojas C
Published: July 2020

In the last few years there has been a growing interest in nanoparticles and nanophase materials. Nanoparticles are considered to be a new state of the condensed matter due to the high relation of atoms in the surface respect to the number of atoms in the volume, and their properties are neither those of atoms nor those of bulk materials.

There are many interesting questions still to be answered and processes to be understood, from nucleation and growth processes to properties

In the present work we report the results of the fabrication of Mo, MoW and MoNi, particles by d.c. sputtering, under different pressures, from 0.2 torr to 0.8 torr. Their morphology, size distribution, aggregation and structure has been studied by Transmission Electron Microscopy.

We analyzed our results contrasting them with those of Chow 1,2 and Birringer 3 and try to explain with a new perspective the formation mechanism of the particles, and the dependence of particle size with Ar pressure.