bluuebunny/crossref_metadata_2025 huggingface dataset:
Three- Dmensi0nal(3d) Relationships Between Tumor Cell Proliferation and its Anigiogensis in Human Breast Cancer by Confocal Laser Scanning Miroscopy(CLSM) with Triple-Labeling Immunohistochemistry.
Authors: Itoh J , Yasumura K , Ogawa K , Kawai K , Serizawa A , Osamura R Y
Published: July 2020

There have been considerable interests in the relationship between tumor cell proliferation and angiogenesis. In human breast carcinomas, it has been reported that angiogenesis is an independent prognostic factor and that proliferating factors are closely related with lymph node status. Therefore, the demonstration of proliferating and angiogenetic factors as well as the tumor blood vessels in human breast carcinoma simultaneously is important for clinical evaluation of breast cancer patients.

CLSM has been an exciting new apparatus because of it's more powerful resolution than conventional light microscopy and the high-performance on 3D optical sectioning. Images taken with CLSM can be digitized and made variable for image analysis manipulation and computer-assisted 3D reconstructions.

In the present study, we employed immunohistochemical comparative 3D visualization of proliferating and angiogenstic factors with microvessels in human breast carcinomas by CLSM.

The tissues from the surgically resected breast carcinoma were fixed in cold 4 % paraformaldehyde(PFA) and routinely processed in paraffin wax.
Analysis of Mechanisms of Cellular Injury on the Micrometer Scale
Authors: Burghardt Robert C , Barhoumi Rola
Published: July 2020

Non-invasive imaging tools using biosensors and biomarkers for defining the function of living cells and tissues are among rapidly emerging technologies being developed to monitor cellular toxicity mechanisms. Commercially available fluorescence probes and naturally occurring fluorescent molecules can be used to quantitatively monitor a number of functional endpoints in cultured cells. Innovative approaches to exploit the sensitivity, spectroscopy and temporal/spatial resolution properties of fluorescent probes may provide sensitive approaches for analysis of the molecular mechanisms of toxicity in cells exposed to a variety of toxicants. Kinetic analysis of multiple cellular parameters such as intracellular glutathione (GSH) and Ca2+ content, reactive oxygen species (ROS) generation, mitochondrial and plasma membrane potentials, intracellular pH, and gap junction-mediated intercellular communication (GJIC) with cellular component-specific fluorescent probes permits rapid identification of changes in these parameters and the chronology of injury in cells caused by acute toxicant exposure. Cellular signal transduction mechanisms are also likely to be vulnerable to toxic insults.
Intraneuronal Neurofibrillary Tangles Isolated from Alzheimer’ Disease Affected Brains Visualized by Vertical Platinum-Carbon Replication for TEM
Authors: Ruben GC
Published: July 2020

Neurofibrillary tangles (NFT) occur with high frequency in brains of patients with Alzheimer' disease (AD). The frequency of NFT directly correlates with the degree of dementia. Extra cellular plaque core amyloid also occurs in high frequently in AD brains when few intraneuronal NFT are present, these individuals are not demented.

The microtubule associated protein (MAP) tau is recognized as the principal constituent in paired helical filaments (PHF) and NFT. NFT contain PHF, amorphous structures containing tau, and a triple-stranded left-hand helical ˜2.1 nm filament similar to tau polymer which has been identified in isolated tangles. Tau is located in nerve cell axons and associates with and stabilizes the microtubules required for axonal transport. Sequestration of tau into tangles and its removal from microtubules not only compromises axonal transport but cripples synaptic communication between nerve cells.

NFT were isolated in the absence of detergent so these gel-like structures (Fig. 1) could be studied at the molecular level.
Liver Toxicity in Rats Fed A Mixture of Pcb Congeners and 2,3,7,8-Tcdd
Authors: Bailey GW , Dimlich RVW , Alexander KB , McCarthy JJ , Pretlow TP , Cornell BJ , Singh A , Chu I
Published: July 2020

Polyhalogenated aromatic compounds such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins (PCDDs) continue to be environmental contaminants because of their bioaccumulation in the food chain and high resistance to biodegradation. The current study was undertaken to determine if a mixture of PCB congeners (WHO-IPCS) were interactive with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in producing morphological changes in the rat liver. Both compounds are known to produce a broad range of biochemical and morphological alterations including enzyme induction.

Groups (N=5) of female Sprague-Dawley rats were administered TCDD (0, 2.5, 25, 250, 1000 ng/kg bw/day) or PCB (0, 2, 20 μg/kg bw/day) alone, or in combination with each concentration of both compounds. Incorrect concentrations were published in a previous abstract. The test substance was mixed with corn oil and given by gavage at 2 ml/kg daily for 28 days. At the end of the experiment, the rats were killed and liver samples were prepared for transmission electron microscopy.
A TEM and SEM Study of the Effects of Exogenous Calcium Oxalate Crystals on Renal Epithelial Cells
Authors: Vaughn KL , Hackett RL , Kahn SR , Shevock-Scott PN
Published: July 2020

When confluent monolayers of Madin-Darby Canine Kidney (MDCK) renal epithelial cells are exposed to calcium oxalate monohydrate (COM) crystals suspended in the nutrient media, crystals settle onto the cell surface. Here, they become entangled with the specialized membrane structures of cilia and microvilli (Figures 2). The cell membrane surface is structurally altered as patches of membrane devoid of microvilli are found adjacent to the crystal entrapped on the surface (Figure 4). Many COM crystals disappear from the cell surface and appear to be internalized. This phenomenon is seemingly common to all cultured renal epithelial cells. The uptake of crystals appear to increase with time and crystal concentration and stimulates a series of complex reactions. Morphologically, crystal aggregates are contained with intracellular vesicles which appear to be membrane lined (Figure 2,3). In addition, cells exocytose the internalized crystals and in MDCK cells, crystal-cytoplasm complexes can be observed leaving cells at the intercellular junctions.
Chemically Induced Accumulation of Phospholipids in Canine Organ Systems. II. Induction by Amiodarone and Chloroquine
Authors: Maleeff Beverly E , Handler Jeffrey A , Louden Calvert S , Hart Timothy K
Published: July 2020

Cationic amphiphilic drugs (CAD) are structurally characterized by their hydrophobic ring structure and hydrophilic side chain. While there is little known about the effect of CAD in dogs, studies in animal models have shown that over 30 CAD with various pharmacological activities have the ability to induce phospholipid-like inclusions in peripheral blood cells and in organs such as lung, liver, eye and spleen. Two CAD used for treatment of human disease are amiodarone, prescribed for arrythmia, and chloroquine, an antimalarial. The purpose of this study was to determine if these compounds induce cytoplasmic phospholipid inclusions in peripheral blood mononuclear cells (PBMC), polymorphonuclear lymphocytes (PMN), alveloar macrophages, liver and retina in dogs.

Samples were collected from male beagle dogs after 6 weeks of oral dosing with amiodarone (75 mg/kg/day) or chloroquine (75 mg/kg/day). Buffy coats were prepared from peripheral blood and fixed with 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. Alveolar macrophages were recovered from bronchoalveolar lavage fluid by centrifugation and the pellets fixed with buffered 2.5% glutaraldehyde-2% formaldehyde.
Gastrin, Nitric Oxide Synthase and their Role in Ulceration and Distention of Stomach after Cisplatin and Taxol Treatment
Authors: Wang Ying , Aggarwal Surinder K
Published: July 2020

Cisplatin, a potent broad spectrum anti-cancer agent, has been proven effective in the treatment of different kinds of cancer, such as bladder, lung, ovarian, head, neck, etc.. Drawbacks of this chemotherapeutic drug are its toxic side-effects, which include severe nausea, vomiting, stomach distention and peptic ulcer.

Taxol is another effective chemotherapeutic agent which is usually used in conjection with cisplatin. It has demonstrated impressive activity in breast, ovarian, lung, and head and neck cancers. The toxic side-effects include nausea, vomiting, nephrotoxicity. The mechanism of these gastrointestinal side-effects are still unclear. Because of the role of NOS and gastrin on stomach smooth muscle contractility and gastroprotection, these were studied in the rats after cisplatin and taxol administration.

Wistar rats(100-150 g) were injected with cisplatin(9 mg/kg) and taxol(20 mg/kg) in five divided dosages over a period of 5 days. Rats were killed one day after the last injection. Rat stomach tissues were fixed in Bouin’s solution and processed for light microscopy.
Prohead Perestroika: Bacteriophage T7 Capsid Before and After Maturation
Authors: Cerritelli Mario E , Conway James F , Cheng Naiqian , Steven Alasdair C
Published: July 2020

Bacteriophage T7 provides a well defined system to study the assembly of a moderately complex virus. The mature virion consists of a 60 nm icosahedral capsid containing the linear 40 kb genome and an internal protein “core”, and a short tail. The capsid originates as a DNA-free prohead, whose assembly requires both the major capsid protein (gpl0, ∼ 420 copies) and the scaffolding protein (gp9, ∼180 copies). Native proheads incorporate an oligomeric ring of the connector/portal protein at one vertex and the inner core, but prohead-like particles assemble when gp10 and gp9 alone are expressed. In maturation -as with other dsDNA phages - the T7 prohead undergoes a major conformational change, in which the shell structure alters dramatically, and the scaffolding protein is expelled. We have used cryo-EM in combination with other techniques to study this transition and to investigate the interactions between scaffold and surface shell in the T7 prohead.

Highly purified preparations of proheads were generated from the co-expression of head and scaffolding proteins from the cloned gene in E. coli. Tail-less mature capsids as well as DNA-filled heads were prepared from infection of a T7 strain that completely lacks the tail genes.
Transport Through the Secretory Pathway of VSVG Tagged With Green Fluorescent Protein: Role of Tubulovesicular Carriers and Microtubules
Authors: Presley John , Hirschberg Koret , Cole Nelson
Published: July 2020

The ts045 mutant of VSV G protein has been used in numerous studies to identify biochemical and morphological properties of membrane transport, due to its reversible misfolding and retention in the ER at 40°C and ability to traffic out of the ER and into the Golgi complex upon temperature reduction to 32oC. The dynamic properties of membrane transport intermediates of the secretory pathway, including their lifetime and fate within cells, have not until now been explored due to the inability to follow transport in single living cells. Here, we attached green fluorescent protein to the cytoplasmic tail of VSV G protein in order to visualize ER-to-Golgi and Golgi-to-plasma membrane transport in living cells. VSVG-GFP expressed in Cos cells accumulated in the ER at 40°C and translocated to the Golgi complex when shifted to 32oC. Translocation of the protein was followed using time-lapse imaging of live cells on a confocal microscope. VSVG-GFP accumulated in tubulovesicular structures scattered throughout the cell upon shift from 40°C to 15°C for three hours.
Presence of P53 and Transforming Growth Factor Beta-1 in Human Cardiac Tissue With Congestive Heart Failure
Authors: Song Hong , Shuxun Ren , Fen Xia , Ruiwen Zhang , Chi-Ming Wei
Published: July 2020

p53 is a tumor suppressor gene which involves apoptosis and cell cycle arrest under certain stress stimulate. The expression of p53 is associated with ischemia-reperfussion induced myocardial apoptosis in rat and with the development of coronary artery restenosis after angioplasty in humans. However, the status of p53 in human cardiomyocytes and fibroblasts in congestive heart failure (CHF) remains controversial.

Transforming growth factor-beta (TGF-β) is a growth-regulating peptide that has been shown to enhance collagen production both in vivo and in vitro in cultured cells. Both p53 and TGF-β may inhibit the cell growth and induce cellular apoptosis. While the previous studies demonstrated that TGF-β1 is present in the animal myocardium, the presentation and localization of TGF-β1 in human myocardium remain unclear.

Therefore, the present study was designed to investigate the p53 and TGF-β1 in human ventricular myocardium in normal subjects and in patients with heart failure. Five normal subjects and five end-stage failing human ventricular myocardium tissue were obtained from cardiac transplantation.
Ultrastructural Observations and Heat Shock Protein 70 in Induced Unilateral Abdominal Cryptorchh) Testis in Rats
Authors: Al-Bagdadi F , Farouqi S , Stout R , Crawford P , Chandler J , Eilts B , Paccamonti D , Hosgood G , Kearny M , Duffield D , O'Donnell J
Published: July 2020

Infertility has been reported to be a significant consequence of cryptorchidism. Testicular cancer has been increasing since the beginning of the century (2). It has been reported in men with unilateral cryptorchidism who develop cancer that 75% of the cases are in the undescended testis. In earlier reports, investigators found that almost half of the cryptorchid testis that developed testicular cancer were abdominal (3). The extent of the degenerative changes are more extensive in the abdominal testis than the inguinal testis (1). Heat shock proteins have been detected in normal as well as in heat treated testes (4, 7). It has been stated that unchecked heat shock protein leads to irreversible damage and ultimately cell death (6). Heat shock protein is used in this study as a marker. The long range objective of this study is to determine the sequence of the ultrastructural degenerative damages of the testis over specific periods in rats as a model and relate these changes to a comparable human age.
Immunofluorescent Localization of Myosin on the Sperm Cells of Plumbago Zeylanica
Authors: Zhang Zhaojie , D. Russell Scott
Published: July 2020

Sperm cells in flowering plants are non-motile and are passive participants in their movement to the female reproductive cells. It is believed that actomyosin interaction may play a key role for sperm cell transmission in the pollen tube as well as in the embryo sac. However, indirect evidence has shown that the surface of sperm cells lacks amounts of myosin sufficient to support movement. Immunofluorescence microscopy was used in this study to further assess the presence of myosin on the surface of sperm cells of Plumbago Zeylanica.

Sperm cells of Plumbago Zeylanica were isolated according to published methods. Isolated sperm cells were blocked 20 min with 1% bovine serum albumin and 2% normal goat serum in phosphate buffered saline (PBS) (pH 7.3, in 15% sucrose), incubated 2 hrs in anti-myosin antibody (M-7648, Sigma Chemical Co., St. Louis, MO) diluted 1:10 with blocking solution, washed three times (5 min) with blocking solution and incubated 1 hr in 1:30 FITC-conjugated anti-rabbit IgG as secondary antibody (EY Labs, Inc., San Mateo, CA) in blocking solution. Samples were rinsed in PBS and mounted in an anti-fading solution with 1:1 PBS:glycerol with 3% n-propyl gallate.
Epidermal Keratinocyte Differentiation is Disrupted in Mice Lacking The Full Length Extracellular Calcium-Sensing Receptor
Authors: Bailey GW , Dimlich RVW , Alexander KB , McCarthy JJ , Pretlow TP , G. Kömüves László , D. Harris Jonathan , Ho Chrystal , D. Bikle Daniel
Published: July 2020

The importance of the extracellular calcium-sensing receptor (CaR) in the stringent control of extracellular Ca2+ concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis suggests a role for CaR in local regulation of cellular functions. Although extracellular Ca2+ regulates the differentiation of keratinocytes, the role of CaR in the epidermis is not established. In this work using knockout mice lacking full length CaR, we sought to determine the role of CaR in epidermal differentiation.

Dorsal skin of Casr−/− knockout mice lacking full length CaR, and Casr+/+ (wild type) control mice, aged 4 to 7 days after birth was fixed in 4% formaldehyde in PBS, and in 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M cacodylate buffer. The samples were embedded in paraffin (for immunohistochemistry and for in situ hybridization) or in Spurr’s or LR White resins. Digoxigenin labeled antisense and sense RNA probes for loricrin and filaggrin were used for in situ hybridization.
Analysis of DNA Levels During Gonomery in Early Cleavage Divisions of the Freshwater Copepod Mesocyclops Edax
Authors: Rasch EM , Wyngaard GA
Published: July 2020

Gonomery, the separate grouping of maternal and paternal chromosomes during the first few mitoses following fertilization (Fig. 1) and chromatin diminution, the fragmentation and elimination of whole chromosome regions during specific stages of early cleavage in all primordial cells of the soma, occur in several species of copepods, ascarid nematodes, ciliated protozoa, Japanese hagfish 9 and a few other invertebrates. Because of the marked loss of DNA from all presumptive somatic cells by selective chromatin diminution during the 4th cleavage division in M. edax, it was of interest to determine DNA levels in chromosomes during gonomery in anaphases preceding diminution (Fig. 1). It was also important to estimate the DNA content of sperm as an index of the relative contribution by the male genome at fertilization in this species.

Mature males and egg-carrying females were fixed in 3:1 methanol/acetic acid for 3-5 mins, swollen in 45% acetic acid for 2-3 mins, squashed, frozen in liquid N2 for coverslip removal, thawed in 2 changes of absolute ETOH and air dried.
Haplotype-Specific Alphoid Oligonucleotides Can Distinguish Normal Homologous Chromosomes by Fish
Authors: Gregory Matera A , L. O'Keefe Christine
Published: July 2020

The field of molecular cytogenetics is presently hampered by the need for allele- and homologue-specific hybridization probes. Vast stretches of the human genome display a considerable amount of polymorphic variation between individuals. Alpha satellite DNA is a tandemly repeated sequence located at the centromeres of all primate chromosomes and is a rich source of polymorphisms. These DNA variants are phenotypically silent; they are also stable and heritable in Mendelian fashion. We have shown that we can use this genetic diversity to create homologue-specific probes using fluorescence in situ hybridization (FISH) However unlike most classical heteromorphisms, which are detected by banding techniques, our approach does not depend upon detection of gross alterations in chromosome structure, but on the basis of sequence composition. We have constructed highly-specific oligonucleotide probes that are the first reported FISH probes to discriminate between the cytogenetically indistinguishable chromosomes of normal individuals.

These sequence-specific probes have been used to follow the transmission of a single chromosome 17 through three generation families, similar to a typical polymorphic marker.
Multi-Mode Light Microscopy of Microtubule and Endoplasmic Reticulum Dynamics in Migrating Newt Epithelial Cells.
Authors: Waterman-Storer C M , Salmon E D
Published: July 2020

We have developed a multi-mode digital imaging system (1-3) which acquires images with a 12 bit cooled CCD camera. A multiple band pass dichromatic mirror and robotically controlled excitation filter wheels provide rapid wavelength selection for epi-fluorescence with DAPI, fluorescein or GFP and X-rhodamine fluorophores while maintaining image registration on the cooled CCD detector. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. A robotically controlled emission filter wheel in front of the CCD camera inserts an analyzer in the light path for DIC imaging. To maximize fluorescence light intensity, the analyzer is removed and an optical flat of equivalent optical thickness is inserted for fluorescence imaging. A slider is inserted at the field diaphragm position of the fluorescence epi-illuminator to provide in-focus slit and spot targets for 360 nm photoactivation of “caged” fluorophores. The microscope system is robotically controlled and image acquisition and analysis is performed using MetaMorph™ digital imaging software.
Three-Dimensional Analysis of Tranverse Tubules in Normal and Failing Heart: A Combined Confocal and High Voltage Electron Microscope Study
Authors: Martone M E , Edelman V M , Thor A , Young S J , Lamont S P , Ross J , Ellisman M H
Published: July 2020

Early electron microscopic studies documented that significant changes in the membrane systems of cardiac cells occur in both ischemic and non-ischemic heart failure. These studies relied on analysis of two-dimensional sections and although quantitative changes were observed, the overall organization of the tranverse tubules (T-tubules) and the sarcoplasmic reticulum could not be assessed. In a 3-dimensional study using high voltage electron microscopy (EM) of the T-tubules in spontaneously hypertensive rats, Nakamura and Hama (1991) observed that concomitant with an increase in surface area, the T-tubule system becomes progressively more disorganized and exhibits structural irregularities such as increased numbers of longitudinal tubules, numerous short dead end branches and complex tubular aggregates. These authors suggested that this disorganization may interfere with synchronous contraction over the entire cell.

In the present study, we examined the 3-dimensional organization of T-tubules in the left ventricle of explanted human hearts using confocal microscopy and EM tomography.
The Interactions of Fibrin and Endothelial Cells within a Fibrin Clot
Authors: Jerome W G , Hantgan RR , Handt S
Published: July 2020

A life threatening blood clot is the major cause of heart attacks. Thrombolytic therapy attempts to restore blood flow by activating the body's own fibrinolytic system at the site of the occlusive thrombus. However, for unknown reasons, therapy is unsuccessful in greater than 20% of patients. We have previously shown that the endothelial cells lining the wall of the vessel can play a substantial role in inhibiting clot lysis. This is due chiefly to the secretion of inhibitory molecules by endothelial cells. However, endothelial cells also have receptors for fibrin and little is known about how the direct interaction of fibrin with cells may influence lysis. To investigate this we have undertaken a series of microscopic studies to analyse the influence of endothelial cells on clot structure. We report here that endothelial cells can organize clot fibers into tight assemblies. We also show that, at least in culture, fibrin can act in concert with antithrombotic molecules to dramatically affect endothelial structure
Resources for the Study of Cellular Structure by High Voltage Electron Tomography, Serial Thin Sectioning, Specific Labeling, and Image Analysis
Authors: Mastronarde David , Kremer James , O’Toole Eileen , Morphew Mary , Ladinsky Mark , McIntosh Richard
Published: July 2020

We are working to improve methods for the study of cellular fine structure. Our approach is to advance each of the key steps in the preparation of specimens for EM: high quality fixation that will preserve both structure and antigenicity; methods for specific labeling; efficient acquisition of 3-D electron microscopic data; and software for 3-D reconstruction and display.

Our work on high quality structure preservation has focused on methods for fast freezing and freeze substitution. Both plunge freezing of specimens grown on coated gold grids and high pressure freezing of either cultured cells or tissue specimens have yielded well preserved material. These samples are suitable for freeze substitution fixation with either anhydrous aldehydes in acetone at -90°C, for the preservation of antigens, or aldehydes, tannic acid, OsO4, and uranyl acetate for optimal preservation the structure.

We have used a JEOL JEM-1,000 high voltage microscope to image sections about 250nm thick, employing a goniometer stage to perform dual axis tomography for 3-D reconstruction with approximately isotropic resolution at ∼7nm.
The BNL STEM Facility
Authors: Wall Joseph S , Simon Martha N , Hainfeld James F
Published: July 2020

The STEM facility at Brookhaven National Laboratory has been in operation since Oct. ‘77, using a custom-built instrument (STEM1) with cold field emission source, 2.5Å probe, -150°C cold stage, efficient dark field detectors and computer control & data acquisition system. A specimen changing air lock and several portable vacuum chambers permit vacuum transfer of specimens from a separate vacuum system where they were freeze dried overnight.

The large angle dark-field signal produced by the STEM is directly proportional to the total mass within the probed area. STEM mass mapping is based on this linear relationship and the fact that only specimen-specific atoms remain on the substrate after washing with volatile buffer and freeze drying. All images are digital and available via Internet. PC software can be provided for analysis.

STEM mass accuracy ranges from a fraction of a percent on well-defined individual particles such as viruses in the 50 MDa to 10 GDa range, to ∼1% around 1 MDa and ∼10% in the 50 kDa range.
The Biological Electron Microscope Facility, University of Hawai‘I at Mānoa: a Shared Microscopy Resource in the Pacific
Authors: Carvalho TM , Dunlap MF , Allen RD
Published: July 2020

The Biological Electron Microscope Facility (BEMF) at the University of Hawai‘i at Mānoa (UHM) is located 2400 miles over water from the next nearest research university. BEMF is a multi-user core facility, administered by the Pacific Biomedical Research Center (PBRC), an organized research unit at the UHM. The mission of the BEMF is to provide state-of-the-art instrumentation, services and training for electron microscopy to the biomedical and biological researchers in Hawai‘i and the Pacific region. The BEMF was established in 1984 under the direction of Dr. Richard D. Allen, and has since grown steadily in its instrumentation, expertise, and use. In the past 5 years it has served researchers from over 50 laboratories in PBRC and the colleges of Natural Sciences, Tropical Agriculture and Human Resources, Engineering, Medicine, and Ocean and Earth Sciences and Technology, as well as visiting investigators from other Hawai‘i, mainland and foreign institutions.

The BEMF has a full line of instrumentation for conventional transmission and field emission scanning electron microscopy as well as a complete line of instruments for cryoelectron microscopy.
Multi-Photon Microscopy, High Resolution Imaging Deep in Strongly Scattering Specimens
Authors: Denk Winfried
Published: July 2020

Imaging small structures substantially below the tissue surface in living specimens poses special challenges mainly because light is scattered by ever present refractive index inhomogeneities. Confocal microscoy removes the blurring caused by scattered and out-of-focus light but does so only at the expense of photodynamic damage that is often unacceptable when observing live specimens.

Multi-photon absorption microscopy[l] solves these problems because excitation is virtually limited to the focal plane. Out-of-focus photobleaching and photodamage are therefore eliminated. In scattering samples substantial improvements accrue even for the focal plane because, different from confocal microscopy, where only ballistic fluorescenc photons can be used, in the multi-photon microscope scattered photons can be utilized in addition [2-4], provided whole-field detection is used[5].

Many questions in the study of the nervous system require the investigation of intact portions of neural tissue in order to preserve the multiply branched processes of neurons, often extending over hundreds of microns, together with the local nervous circuitry.
Two-Photon Excitation Imaging of Glucose Metabolism in Living Tissue
Authors: W. Piston David
Published: July 2020

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. It provides three-dimensional resolution and eliminates background equivalent to an ideal confocal microscope without requiring a confocal spatial filter, whose absence enhances fluorescence collection efficiency. This results in inherent submicron optical sectioning by excitation alone. In practice, TPEM is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 limits the average input power to less than 10 mW, only slightly greater than the power normally used in confocal microscopy. Because of the intensity-squared dependence of the two-photon absorption, the excitation is limited to the focal volume.
Novel Non-Injection Approaches for Revealing and for Microscopic Investigation of the Microcirculatory Bed in Different Organs and Tissues
Authors: Chilingaryan N , Chilingaryan A , Chilingaryan M
Published: July 2020

Certain disadvantages of traditional injection methods and difficulties arising during investigation of experimental and pathological material, limit the possibilities for extensive study of the microcirculatory bed (MCB). This has prompted the authors to develop new non-injection methods for revealing the MCB on histological sections by means of direct staining of the structural elements of the vessel wall. These methods are based on selective precipitation of the extra- or intracellular orthophosphates with ions of Zn, Cd, Co, Ca, Sr, and Pb in the vascular endothelium and smooth muscle cells. These methods (which we have called “histoangiological”) provide clear-cut contrast dyeing of the MCB in different organs and tissues as well as tumors in rat, cat,pig, dog, and in humans.

In these methods, the reaction end product is lead sulfide which provides a high degree of contrast. Vessels and capillaries are detected due to precipitation of the deposit in the vascular endothelium.
Multiple Approaches to Visualizing Fibrin Clot Structure and Assembly
Authors: Weisel John W
Published: July 2020

Fibrin clot formation is necessary for maintaining the integrity of the vasculature via physiological processes of hemostasis and wound healing and is also involved in pathological processes, such as thrombosis and atherosclerosis. A variety of structural and biophysical approaches has been used to examine intermediates in the formation of clots and to visualize in vitro clots and ex vivo thrombi.

Structures at all stages of polymerization have been examined to learn about molecular mechanisms of assembly. Fibrinogen is a polyfunctional, multi-domain protein that is essential for platelet aggregation and for the formation of the three-dimensional network of fibrin fibers which is the structural basis of the clot. Distinctive functions for several of fibrinogen's domains in the fibrin assembly process have been elucidated. Enzymatic removal of the fibrinopeptides exposes binding sites in the central region which then interact with complementary sites at the ends of a neighboring molecule to yield fibrin oligomers.
Freeze Fracture at the Turn of the Century: Techniques for Visualizing and Labeling Tissues, Cells, and Molecules
Authors: Yasumura Thomas , Rash John E
Published: July 2020

Freeze-fracture techniques have contributed much to our understanding of membrane composition and macromolecular architecture. However, further substantive contributions from freeze fracture have seemed unlikely because: a) replicas typically fragmented, destroying histological orientation within samples; b) histochemical staining techniques were thought to be incompatible with bleach- or acid-cleaned freeze-fracture replicas; c) the limit of resolution was presumed to be 3-5 nm; and d) there were no methods for directing the fracture plane to specific components within a tissue. Recent developments in “grid-mapped freeze fracture” and freeze-fracture immunocytochemistry have addressed each of these limitations.

Using grid-mapped freeze fracture, samples are mapped in three dimensions using confocal microscopy before freeze-fracture (Fig. A), as well as after replication and stabilization in Lexan plastic on a gold “Finder” grid (Fig. B). After replica cleaning, areas of interest are correlated in confocal and TEM images —— for example, cilia of the ependymal layer of rat spinal cord (Fig. C).
A Technique for In Vivo Morphological Evaluation of Chronically Implanted Neuronal Silicon Substrate Electrodes for Confocal Microscopy
Authors: Edwards CA , Finger PA , Anderson DJ , Wiler JA , Hetke JF , Altschuler RA
Published: July 2020

Various multichannel silicon electrodes have been developed for stimulating and recording in the central nervous system of experimental animals. These electrodes can be used in their planar form or can be assembled into 3-D structures for volume interaction with tissue. However, bio-compatibility issues arise concerning the introduction of any electrode into living tissue. From earlier observations, we noticed that the neuronal cells had a tendency to adhere to the chronically implanted silicon substrate. Routine microtechnique became an obstacle when attempting to section the implanted and embedded electrode. Most often, this was attempted by passing a stainless steel blade through paraffin, or plastic embedded tissue, resulting in disrupting the tissue material interface. We needed to develop a reproducible procedure which preserved the integrity of this interface for future LM and confocal microscopic studies.

In order to preserve the electrode/tissue interface, we used the Exakt microgrinding/micropolishing technique, where the electrode/brain block was embedded in Technovit 7200 resin and cut with a diamond impregnated band saw.
Principles of Practical Advantages and Limitations of Light Microscopic 3d Deconvolution
Authors: Holmes TJ , O’Connor NJ , Wang M , Wang C , Turner JN
Published: July 2020

There has been much provocative discussion over the past few years about the advantages and limitations of deconvolution methods. In this tutorial we will provide a reasonable understanding of the underlying theoretical principles of deconvolution, with an aim at understanding expected advantages and limitations. These principles will be presented for a mixed audience composed of biologists, neuroscientists, engineers, physicists and other scientists and graduate students. These principles will be presented in the context of both widefield and confocal microscopy deconvolution.

The basic principles of Fourier optics will be presented. From these principles, we can explain why it is that the confocal microscope provides a superior in-plane and axial (z-axis) resolving power, compared to the widefield microscope. In particular, for example, the Fourier transform of a 3D widefield image contains a missing cone region (to be explained in the tutorial), and this missing cone region effectively spoils the axial resolution of unprocessed widefield data.
All Models are Wrong an Overview of 3D Deconvolution Methods
Authors: Conchello José-Angel , Markham Joanne , McNally James G
Published: July 2020

Three dimensional (3D)microscopy is a powerful toll for the visualization of biological specimens and processes. In 3D microscopy, a 3D image is collected by recording a series of two-dimensional (2D) images focusing the microscope at different planes through the specimen. Each 2D optical slice in this through focus series contains the in-focus information plus contributions from out-of-focus structures that obscure the image and reduce its contrast. There are two complementary approaches to reduce or ameliorate the effects of the out-of-focus contributions, optical and computational. In the optical approach a microscope is used that avoids collecting out-of-focus light, such as a confocal microscope (see and references therein), a two-photon or three-photon fluorescence excitation microscope, or atwo-sided microscope. In the computational approach, the through-focus series is processed in a computer using any of a number of debluring algorithms to reduce or ameliorate the out-of-focus contributions. In the past two decades, several methods for debluring microscopic images have been developed whose common aim is to undo the degradations introduced by the process of image formation and recording
All You Need to Know About Electron Backscatter Diffraction: Orientation is Only the Tip of the Iceberg
Authors: Michael J R
Published: July 2020

This tutorial will describe the technique of electron backscattered diffraction (EBSD) in the scanning electron microscope (SEM). To properly exploit EBSD in the SEM it is important to understand how these patterns are formed. This discussion will be followed by a description of the hardware required for the collection of electron backscatter patterns (EBSP). We will then discuss the methods used to extract the appropriate crystallographic information from the patterns for orientation determination and phase identification and how these operations can be automated. Following this, a number of applications of the technique for both orientation studies and phase identification will be discussed.

EBSD in the SEM is a phenomenon that has been known for many years. EBSD in the SEM is a technique that permits the crystallography of sub-micron sized regions to be studied from a bulk specimen. These patterns were first observed over 40 years ago, before the development of the SEM and were recorded using a special chamber and photographic film.
In-Situ Transmission Elecron Microscopy (TEM) Study of the Sintering of Sputtered Copper Nanoparticles on (001) Copper
Authors: Yeadon M , Yang JC , Averback RS , Bullard JW , Olynick DL , Gibson JM
Published: July 2020

The large surface area: volume ratios and fine grain size of nanophase materials give rise to novel and exciting structural and electrical properties that are of considerable scientific and technological interest. Using copper as a model system we have investigated the sintering of sputtered copper nanoparticles (4-20nm diameter) with a copper substrate in a novel UHV in-situ TEM.

The nanoparticles were generated in a UHV chamber built into the side of the column by sputtering in 1.5Torr Ar. They were transported into the microscope in the gas phase and deposited on an electron transparent (001) copper foil mounted on a heated support. A typical bright-field (BF) image of the sample immediately after deposition at room temperature is shown in Fig. 1. The particles have assumed a random orientation on the substrate and remain stable for many hours at room temperature. The presence of both single particles and agglomerates of particles is evident in this image and examples are labelled ‘P’ and ‘A’, respectively
Electrode Potential-Induced Morphology of Au, Pt and Au-Cu Alloy Particles Formed in Solution
Authors: Lu Da-ling , Tanaka Ken-ichi
Published: July 2020

It has been well known that Au single crystal undergoes reconstruction in vacuum as well as in acidic solutions at negative electrode potentials. The reconstruction is lifted when electrode potential sweeps to positive values. Taking into account of these facts we considered when Au particles grow on an electrode in solution at different electrode potentials, morphology of the particles may be different. Based on this idea, we investigated formation of Au, Pt and Au-Cu alloy particles grown in solutions and observed icosahedral and decahedral particles of Au and Pt formed at negative potentials. These particles are identical with multiply twinned particles (MTPs) observed in vacuum.

Particles were electrodeposited on an amorphous carbon film which was evaporated on a collodion film supported by a Au mesh as required for viewing in a transmission electron microscope (TEM). A three-compartment electrochemical cell was employed at room temperature with Pt wire counter electrode
Synthesis and Characterization of Tin Oxide Nanoparticles Heavily Doped with Antimony
Authors: Liu J , Coleman J P , Madhukar P
Published: July 2020

Antimony-doped tin oxide (ATO) materials have many interesting and useful properties for industrial applications. Important uses of ATO include anti-static pigments, catalysts, electrochromic devices, solar cells, gas sensors, etc. Although the electro-optical and the structural properties of ATO materials with low dopant levels have been extensively investigated,the fundamental structural/chemical properties of heavily doped ATO materials have not been well understood. We report here some preliminary results of a systematic study of the structural evolution of tin oxide nanopaiticles heavily doped with antimony.

The ATO powders were prepared by adding a mixture of antimony(V) chloride and tin(IV) chloride to deionised water with concurrent addition of sodium hydroxide. The resulting white precipitates were filtered, washed and dried at room temperature. A series of Sb-doped SnO2 samples was obtained by annealing the precipitates at various temperatures. Samples suitable for high resolution transmission electron microscopy (HRTEM) were prepared by dry-dispersing ATO powders onto holey carbon films.
Size-Controlled Synthesis of Cds Nanocrystals in Vesicles Confirmed in Situby Cryo-Transmission Electron Microscopy
Authors: Kennedy M T , Korgel B A , Zasadzinski J A , Monbouquette H G
Published: July 2020

Size-quantized CdS nanocrystals, synthesized within phosphatidylcholine (PC) vesicles by reaction compartmentalization, were observed in situ to confirm the proposed method of formation and to investigate the concept of membrane-mediated crystal growth. Vesicle solutions representing three different CdS nanocrystal sizes, and a solution containing unreacted CdCl2 inside vesicles, were analyzed using cryo-transmission electron microscopy (cryo-TEM). Cryo-TEM has proved capable of resolving both the vesicle bilayer and the encapsulated nanocrystal, and yields previously unknown positional information for the crystal in relation to the bilayer.

Egg-PC vesicles containing Cd2+ were formed by detergent depletion, yielding vesicles of uniform size and a defined internal Cd2+ concentration. External Cd2+ was removed from the solution by cation exchange. Ammonium sulfide was added to the vesicle dispersion to produce nanocrystals via diffusion of S2- across the vesicle membrane. Particle size was predicted based upon the vesicle size, as determined from light scattering, and the encapsulated Cd2+ concentration.
Interfacial Film Induced Fails in Titanium Salicide Interconnections with Tungsten Vias in High Density Dram Devices
Authors: Kane Terence , Anderson Ron
Published: July 2020

The scalability requirements of higher and higher density DRAM and logic CMOS devices has popularized the salicide process for source to drain connections using TiSi2 and ( more recently ) CoSi2.

The desirability of these salicide processes is well documented with low sheet resistivity properties offering improved access time in DRAM and faster microprocessor speeds.

Besides the obvious drawback in temperature stability of these salicides, the irregular reaction of titanium or cobalt with silicon during RTA processing can result in irregular or “rough” surface features in the salicide. Submicron contaminants (especially metallic species) introduced during in-line electrical test of Kerf structures with high wettability are not easily dislodged by conventional aqueous/ultrasonic cleaning steps. Solvent process cleaning steps can leave residues in the irregular “rough” salicide surface leading to interfacial films between the salicide and the subsequent via connection to overlying bit lines ( or metal one lines ).
Silicide Identification in Rta-Processed Ti Salicide by Analytical Electron Microscopy
Authors: Quintero A , Libera M , Cabrai C , Lavoie C , Harper JME
Published: July 2020

Titanium suicides have low resistivity, low contact resistance, and are widely used as interconnects in electronic devices. The most desirable structure is the C54 variant of titanium disilicide (TiSi2). It is typically formed during thermal annealing by a polymorphic transformation from the C49 TiSi2 structure. The C49 to C54 transformation has been studied extensively and there has been substantial effort to devise ways in which to lower the temperature associated with this transformation. This research uses high-resolution imaging (HREM), convergent-beam diffraction (CBED), and energy-dispersive X-ray microanalysis (EDS) to study the development of suicide morphology in response to rapid thermal annealing (RTA). Two sets of specimens have been studied: (i) 32nm Ti thin films on undoped single-crystal Si substrates [Ti/Si] and (ii) 32nm Ti films separated from an undoped single-crystal Si substrate by a 0.12nm thick Mo interlayer [Ti/Mo/Si]. This paper shows structures formed after RTA at a ramp rate 3 °C/sec to 750 °C with a hold of 1 sec
Characterization of Sil-ycy Alloy Layers Incorporating Si4c Building Blocks
Authors: Chandrasekhar D , Smith David J , Kouvetakis J
Published: July 2020

Group IV based alloys have received considerable attention in recent years, because of the possibility to tailor the band gap of the material system with respect to that of Si. Significant results have already been achieved with Si-Ge system, where the band gap of pseudomorphic Si1-xGex alloys is smaller than that of Si. Introduction of C onto substitutional lattice sites in Si has been proposed as a possible alternate method for tailoring the electronic properties of Si. Carbon incorporation into Si substitutionally could result in alloys whose band gap would be a function of carbon concentration and lie between the values for silicon (E =1.1 eV) and β-SiC (Eg = 2.3 eV). However, due to the low-equilibrium solubility limit of C in Si, 3.5x1017cm−3 at the eutectic temperature, highly supersaturated and metastable layers are essential to significantly alter strain and electrical properties of the alloys.

In our present study, we have synthesized and characterized Si1-yCy (0.04 < y < 0.20) films grown on (001) Si substrates by ultra-high vacuum chemical vapor deposition at 625°C.
Interfaces and Defects in Opto-Electronic Semiconductor Films Studied by Atomic Resolution STEM
Authors: Xin Y , Wallis DJ , Browning ND , Sivananthan S , Pennycook SJ , Humphreys CJ
Published: July 2020

The growth of thin films on dissimilar substrates is of great technological importance for modern optoelectronic devices. However, device applications are currently limited by lattice mismatches between the film and substrate that invariably lead to defects detrimental to device performance. It is therefore of key importance that the mechanisms leading to the formation of these defects are understood on the fundamental atomic level. Correlated atomic resolution Z-contrast imaging and EELS in the STEM is a unique methodology by which this information can be obtained. In this paper, the application of this methodology to determine a novel graphoepitaxial growth mechanism for CdTe on (001)Si is demonstrated, and its potential for the study of GaN is discussed.

Fig.la shows a high resolution Z-contrast image of a cross sectional view of the CdTe/Si interface showing clearly the CdTe and Si dumbbells. Due to the differences in atomic number, the location of the interface is clear.