bluuebunny/crossref_metadata_2025_split huggingface dataset:
Green fluorescent protein: Use of GFP-chimeras in the analysis of microtubule-associated protein 4 domains and microtubule dynamics
Authors: Olmsted J.B. , Olson K.R. , Gonzalez-Garay M.L. , Cabral F. 
Published: June 2020

Green Fluorescent Protein (GFP) is an endogenous 27 kDa fluorophore of the jellyfish, Aequorea victoria. Chalfie et al., first described the exogenous expression of this molecule in bacteria, and its utility as a reporter in higher eukaryotes. Potential applications of GFP have been expanded through the construction of variants with enhanced brightness and/or different spectral properties.We have explored using GFP for the analysis of the real-time behaviors of microtubules and their associated proteins. Constructs of microtubule-associated protein 4 (MAP 4) or β-tubulin were generated in pRC/CMV vectors and used in either transient or stable transfection assays in a variety of cultured cell lines (3T3, PtKl, BHK, CHO, Cos). The GFP-chimeras were visualized using conventional fluorescence microscopy and confocal laser scanning microscopy. Unusual features of the GFP reporter are that fluorescence intensity increased 2-10 fold upon illumination, and that phototoxicity was low.
Localization of Calcium in Porcine Epidermis Using Electron Energy Loss Spectroscopy (EELS)
Authors: Misra Manoj , Siangchaew Krisda , Libera Matthew 
Published: June 2020

The properties of biological systems are often governed by variations in the distribution of trace (0.01-0.1%) constituents. The importance of calcium ions for differentiation and proliferation of keratinocytes in vitro is well known. The situation in vivo warrants study. A major contribution to that study involves the determination of calcium ion distribution in the stratified layers of the epidermis. We describe here the first study to localize and verify calcium distribution in chemically fixed porcine skin using EELS.Freshly dissected porcine skin is processed according to Menon and Elias. Briefly, tissue fixed in a buffer containing 90 mM potassium oxalate and formaldehyde/glutaraldehyde is post fixed in 1% osmium tetroxide containing 2% potassium pyroantimonate. Control specimens are prepared without pyroantimonate treatment. Tissue dried in a graded series of acetone is embedded in Spurr resin and 30-40 nm sections are cut. Stained and unstained sections are examined using a Philips CM 20 FEG TEM equipped with parallel electron energy loss spectrometer (Gatan EELS) and a spectrum imaging system (EMiSpec). The microscope is used in STEM mode for the EELS work and second difference spectra are acquired.
Comparative ultrastructural study of camel, cow, and horse neutrophil
Authors: Hailat N. , Lafi S. , Bataineh Z. , Al-Rawashdeh O. , Al-Bagdadi Fakhri 
Published: June 2020

The ultrastructure of Arabian camel, cow, and horse neutrophils in peripheral blood was studied in order to provide detailed description of its constituents and to determine the similarities and the differences between these three species. Peripheral blood was taken from the three animals, centrifuged and the pellets were fixed in glutaraldehyde, dehydrated and embedded in Spurr‘s resin. Silver sections were mounted on copper grids and viewed by Zeiss electron microscope and microphotographs were taken.Camel's neutrophils were found to have smoother cell surface with small number of short pseudopodia (Fig. 1). The horse's neutrophil had longer pseudopodia while the cow's neutrophil had a large number of small ridges instead. The size of the cell in camel was larger than that in cow and horse. The nucleus of camel's neutrophil appeared less irregular compared with that of cow's and horse's, with marginated non-patchy chromatin, and with abundant heterochromatin.
Fungiform and circumvallate papillae volumes in the pig
Authors: Singh A. , Mack A. , Ireland W. P. 
Published: June 2020

Taste buds on the porcine tongue have not been quantitated. The morphometric information would be useful in investigation of nutrition-related issues both in human beings and animals.The tongue specimens were obtained from three, 6-month-old, castrated, male pigs immediately after they were killed in a local abbatoir. The organs were fixed in 10% buffred formalin, and the fungiform and circumvallate papillae were mapped and counted. Mapping was done by drawing an outline of the dorsal aspect of the specimens and marking the approximate positions of fungiform (dorsal and lateral) and circumvallate papillae. Samples from each tongue were harvested from dorsal, lateral and dorsal root regions. These samples were processed in a tissue processor [Model 166MP; Fischer Scientific]. Paraffin-embedded samples were serially sectioned at 15 μm, and stained with hematoxylin and eosin in a stainer [Varistain; Shandon]. The samples were embedded in such a manner that mucosal surface was cut in a plane transverse to long axis of the tongue.
Observation of passivated Al-1%Cu lines using environmental scanning electron microscopy (ESEM)
Authors: Carpenter D.T. , Smith D.A. , Lloyd J.R. 
Published: June 2020

Characterization of failure phenomena in passivated interconnects requires the ability to observe dynamic changes in buried lines (e.g. formation of electromigration voids or microstructural changes during an anneal). Conventional SEM cannot image passivated structures due to surface charging. HVSEM has been used with some success to image voids in metal lines under passivation, but it requires intensive specimen preparation and is not commercially available. The present study is intended to determine whether ESEM is suitable for such dynamic experiments on passivated structures.The lines were composed of Al-l%Cu and had a uniform thickness of 1.4μm. All lines also had both an underlayer and an anfi-reflective coating of Ti/TiN. The thickness of these layers varied slightly, but was typically around 600 Å. Observations were performed in an Electroscan model 2020 environmental scanning microscope with a LaB6 thermionic source using the gaseous secondary electron detector (GSED) and water vapor as an imaging gas.
SIMS depth profiling and direct ion imaging of a 16-megabit DRAM
Authors: McMahon G. , Phaneuf M. W. , Weaver L. 
Published: June 2020

The use of secondary ion mass spectrometry (SIMS) has long been established as a powerful tool forthe analysis of microelectronic devices. However, as newer fabrication technologies emerge, and device dimensions shrink, increasing demands are placed upon the SIMS analyst.The present work illustrates how the depth profiling and direct ion imaging capabilities of a Cameca ims 4f SIMS can be exploited to determine the structure and dopant chemistry of a state-of-the-art16 megabit DRAM (dynamic random access memory) device. The optical micrograph shown in Fig. 1 is an overview of a portion of the sense amplifiers located between two array blocks. In the centre of this area, p-type devices sitting in an n-well, surrounded on either side by n-type devices, all in a p-type substrate can be observed. The object of the study was to determine if a low concentration p-type implant is present below the n-type devices.
The scanning infra-red microscope (SIRM): Evaluating a new tool for mapping sub-100nm defects in semiconductors
Authors: Mule‘Stagno L. , Holzer J.C. , Falster R. , Fraundorf P. 
Published: June 2020

The manufacturing yield and reliability required of modern silicon micro-electronic devices has led to a need for wafer and device manufacturing processes which are immune to metallic contamination. To this end, various ‘gettering’ mechanisms have been developed in which unwanted metallic impurities are ‘gettered’ to regions of the wafer away from the active device layer. ‘Internal (or intrinsic) gettering’ is a method in which silicon-oxide defects are produced in the bulk of the wafer, with a near-surface defect-free zone (denuded zone). In the event ofmetallic contamination during high temperature device manufacturing steps, these bulk microdefects (BMDs) act as heterogeneous sites for metal-silicide precipitation during the cooling, and prevent precipitation of the metals in the device layer. For this reason it is essential to monitor the spatial distribution and density of bulk microdefects during device manufacturing.The standard method by which the density is measured is termed cleave and etch.
TEM study of precipitation in a Nial-3Ti-0.5Hf single-crystal alloy
Authors: Garg A. , Noebe R. D. , Howe J. M. , Wilson A. W. , Levit V. 
Published: June 2020

Three directionally solidified NiAl single-crystal alloys, NiAl-3Ti, NiAl-0.5Hf and NiAl-3Ti-0.5Hf (at.%), were grown by a Bridgeman technique using high purity alumina crucibles. The ingots were homogenized for 32h at 1644 K followed by aging for 6h at 1255 K and finally furnace cooled under an argon atmosphere. This heat treatment was found to be very effective in dissolving Hf-rich interdendritic particles that were present in the as-cast structure, and at the same time it produced fine second-phase precipitates in the alloy.Samples for transmission electron microscopy (TEM) were prepared from 3 mm diameter cylinders electro-discharge machined from the heat-treated ingots. Slices sectioned from the cylinders were mechanically ground and electrochemically thinned in a twin-jet Tenupol-3 polisher. Microstructural and energy-dispersive X-ray spectroscopy (EDXS) studies were conducted in a Philips 400T TEM equipped with a double tilt goniometer and a KEVEX Si/Li X-ray detector.
Anomalous backscattered electron behavior of MoB and Mo5SiB2(T2) phases in an as-cast Mo-B-Si alloy
Authors: Foumelle J. H. , Nunes C. A. , Perepezko J. H. 
Published: June 2020

It is well established that backscattered electron (BSE) image contrast in SEM is primarily associated with differences in mean atomic number (Z) of the phases. The BSE coefficient increases with higher Z with slight dependence on beam energy (Eo), except for some materials at < 5 keV.We report here the anomalous BSE behavior of MoB and T2(Mo5SiB2) phases observed imaging a 60Mo-30B-10Si (at%) alloy with a CAMECA SX-50. Specifics of the material and EMPA are reported in a companion communication. Mo-ss and Mo3Si are also present. The drastic differences in BSE behavior are shown in Figures 1 (7 keV), 2(15 keV) and 3 (25 keV). The material has not been coated; addition of 100 Å carbon does not change the observed behavior.At 25 keV (Fig 3), MoB dendrites are the darkest phases present, the expected behavior based on their mean Z (Table 1). The T2matrix is gray, whereas smaller regions containing Mo+Mo3Si have the greatest brightness.
Perfect TEM Membranes by focused ion beams: A stress reduction technique
Authors: Walker John F. , Odum James K. , Carleson Peter D. 
Published: June 2020

With the realisation that the critical dimensions in integrated circuits are shrinking to the point where scanning electron microscopy (SEM) techniques are not sufficiently accurate for many applications, advanced semiconductor fabs are looking to the increased resolution and analytical functionality of transmission electron microscopy (TEM) in failure and process analysis. TEM sample preparation is traditionally labour-intensive and needs skilled technical support but, with the acceptance of focused ion beam (FIB) workstations, this preparation and subsequent analysis is now becoming more routine. The reasons are: more reliable preparation with less risk of catastrophic breaking on unique specimens, highly site-specific preparation capable of viewing individual, sub-100 nm features, thin and uniform membranes even with tungsten plugs, and fast and easy preparation techniques.The initial stages of sample preparation involves preparing a sub-100 um sliver mounted on a TEM grid. When mounting this sliver on the grid, care must be taken to prevent any strain from being transferred to the silicon.
FIB system for TEM specimen preparation and its application
Authors: Yaguchi T. , Kamino T. , Koike H. , Ishitani T. , Kitano Y. 
Published: June 2020

The transmission electron microscope(TEM) is one of the most powerful instrument in materials characterization, and various TEM specimen preparation methods have been developed. It is known that Ar-etching method is most widely applied in the studies of high technology materials. However, it is time-consuming when a specific area is desired for examination. Because it is necessary to iterate through Ar-ion etching and TEM examination until the desired information is obtained.There is a great demand on a new specimen preparation technique that has high positional accuracy, reliability, and throughput.Focused ion beam (FIB) milling has been proposed as a solution to the above requirements. We have developed the FIB system (FB-2000) for SEM/TEM specimen preparation. An external view of the system is shown in Fig.l. The system is designed to use a compatible specimen holder (Fig.2) which allows both FIB milling and TEM observation without re-mounting the specimen. The instrument has maximum accelerating voltage of 30kV and a minimum beam diameter of lOnm. The FIB current density of 15A/cm2 is available.
The role of field-emission SEM in biology in the 21st century
Authors: Erlandsen S. L. 
Published: June 2020

To predict how field emission SEM (FESEM) will be applied to biological investigation in the transition to the 21st century requires some recapitulation of how dramatically changes have occurred in the last half of the 20th century. Although, initially developed in the 1930s by Knoll and later, von Ardenne, the commercial production of instruments in the 1960s was brought about chiefly due to contributions in secondary (SE) and backscatter (BS) electron detector design from the Cambridge group led by Oatley. Continued instrumentation development over the next three decades led to numerous improvements, i.e. new lanthanum hexaboride emitters, advances in specimen preparation, and was culminated in the late 1980s, by the commercial development of a high resolution SEM possessing short focal length lenses together with a cold field emission electron gun, resulting in electron probes with diameters of 3nm at 1.5 keV and 0.5nm at 30 keV.From the perspective of a biologist using FESEM for both structural and immunocytochemical studies, I would like to suggest changes in both instrumentation and methodology that would be of great practical value today, and would hopefully be surpassed by standard features in new instruments available at the turn of the century.
On the nature of linkage between retinal rod outer-segment disk and cellular membrane
Authors: Petrosian A. M. , Haroutounian J. E. 
Published: June 2020

In the retina of most vertebrates the cone outer segment disk are in continuity with the cell membrane, thus intradisk space is part of the extracellular space. The rod disk membranes, except for some basal ones, appear to be separated from the cell membrane. On the other hand it has been shown the existence of filament-like structures connecting retinal rod disks to one another and with the cell membrane. However due to the lack of observation of either electron-dense or fluorescent extracellular tracer penetration into rod outer segments, it is believed that rod intradisk space is isolated from the extracellular space. This investigation elucidates upon the nature of filament-like linkage between retinal rod outer segment disk and cell membrane. An incubation solution with 10 times lower than normal osmolarity was used to osmotically explode isolated dark adapted frog retina.Membranes under this condition were observed by transmission electron microscopy (TEM) using the freeze-fracture replica technique. Regularly spaced filaments were observed that were continuous between outer segment disks and the cell membrane in the retinal rods (Fig. 1). Probably these connections have a supporting function providing mechanical stability of the rod outer segment structure.
Hormonal regulation of adult hippocampal dendritic spine density
Authors: Woolley Catherine S. , McEwen Bruce S. 
Published: June 2020

Dendritic spines cover the surface of a wide variety of neuronal types and are the postsynaptic sites of approximately 90% of the excitatory synapses formed in the central nervous system. Interestingly, changes in the morphology and/or density of dendritic spines have been shown to occur naturally, implying that they are a normal part of brain function. Even in the adult, dendritic spines are remarkably plastic. The hormonal state of an animal has been shown to be an important factor in regulation of dendritic spine density, both during development and in the adult.In the adult female rat, hippocampal CA1 pyramidal cells are particularly sensitive to variation in the circulating levels of the ovarian steroids, estradiol and progesterone. Removal of estradiol and progesterone by ovariectomy results in an approximately 50% decrease in the density of dendritic spines on the lateral branches of the apical dendritic tree. Treatment with estradiol can either protect against or reverse this decrease; subsequent progesterone treatment for as few as 5 hours significantly augments the effect of estradiol. By 18-24 hours following progesterone treatment, spine density returns to low values.
Molecular dosimetry: Using slide-based methods to measure DNA damage and cancer risk from the environment
Authors: Law J. M. 
Published: June 2020

A need exists for fish sentinel species for monitoring environmental changes. Ideally, the specieswould be widely distributed, easily cultured in the laboratory, and develop measurable biomarker alterations following contaminant exposure. We have recently begun to examine the suitability of westernmosquitofish (Gambusia affinis) as a sentinel species. Mosquitofish are widely distributed in warm waters and have a limited home range. In the laboratory, mosquitofish are easily cultured using techniques developed for other small fish species such as the guppy (Poecilia reticulata). Theybreed prolifically and appear resistant to infectious diseases.Molecular methods are increasingly being used to measure extent of exposure to environmental carcinogens and to monitor patients receiving chemotherapy with alkylating agents. DNA adducts are currently detected with such methods as 32P-postlabeling, immunoassays, HPLC with fluorescence detection, and gas chromatography/mass spectrometry. Biomarkers may be assessed with slide-based immunohistochemical methods or in situ hybridization, allowing pathologists to localize pre-neoplastic changes in cells such as mutations in the p53 tumor suppressor gene or inappropriate cell proliferation.
Automatic particle picking from electron micrographs
Authors: Lata K. Ramani , Penczek P. , Frank J. 
Published: June 2020

The present-day interactive manual selection of biological molecules from digitized micrographs for single particle averaging and reconstruction requires substantial effort and time. Thus a computer algorithm capable of recognition of structural content and selection of particles would be desirable.A few approaches have been proposed in the past. The method by Frank and Wagenknecht is based on the principle of correlation search. Van Heel's method is based on the computation of the local variance over a small area around each point of the image field. The method by Harauz and Fong-Lochovsky is based on edge-detection. The present work was focussed on the detection and classification of particlesby exploiting the standard statistical methods of discriminant analysis.The proposed technique is described in the block diagram (Fig.1). As illustrated in the figure, the program consists of three distinct segments devoted to, respectively, preparation of the data, training session and automatic selection based on a discriminant function set up in the training, hi the data preparation segment, the micrograph is (i) reduced four-fold in size, (ii) low-pass filtered and (iii) run through a peak search algorithm.
Fraunhofer in-line electron holography of frozen-hydrated DNA superhelices
Authors: Matsumoto Takao , Tanji Takayoshi , Tonomura Akira 
Published: June 2020

We have visualized DNA molecules in solution by Fraunhofer in-line electron holography. So far, applications of the technique at high resolution have been limited to gold particles. This report is the first application of Fraunhofer in-line electron holography to a nanometer-sized biological specimen.The phase shift of an electron beam due to DNA is so small, estimated as only 2π/33 for 100-keV electrons, that it has been only possible to visualize them with either heavy-atom staining or metal shadowing techniques. However, these methods give only low-resolution images of the molecules, and worst of all, preservation of specimens is poor. The frozen-hydrated specimen preparation improved the preservation of specimen, and images of naturally supercoiled DNA in solution have been visualized using underfocusing of 1-2 μm. Such a defocusing is large enough for such a small object as DNA (diameter ∽ 2 nm) to be in a Fresnel condition;z≈ a2/λ, wherezis the defocusing, a is the diameter of an object, andλis the wavelength.
Induction of chromatoid bodies in trophozoitesof Entamoeba histolyticagrowing in log phase at high CO2tension
Authors: Morales-Vallarta M. , Villareal-Treviño L. , Guerrero-Medrano L. , Ramírez-Bon E. , Said-Fernández S. , Mata Cárdenas B. 
Published: June 2020

Chromatoid bodies are typical citoplasmic structures found in ameba of genusEntamoebathey are arrays of helical ribonucleoproteic structures formed by the aggregation of ribosomes. Chromatoid bodies are found only in cystic forms ofEntamoeba, in trophozoites cultured in slow growing conditions, or in the presence of protein synthesis inhibitors. This paper reports the formation of chromatoid bodies in trophozoites ofEntamoeba histolyticaHM-1-IMSS in active growing phase with high CO2tension.Trophozoites ofE. histolyticaHM-1-IMSS strain were cultured with 103cell/ml in 16 × 150 nm screw - capped tubes, each containing 10 ml of TYI-S-33 medium without vitamine (3), and gassed with CO2- - - (0.2 L/Min/2 Min). The cultures were incubated at 37°C and sampled every 24 h for counting and morpho logic observations. For electron microscopy study, the specimens were fixed in 2.5% glutaraldehide in 0.2 M phosphate buffer, pH 7.2; postfixed in 1% osmium tetroxide in 0.2 M phosphate buffer, pH 7.2; — dehydrated in ethyl alcohol, and embedded in epoxy resin (Medcast).
Visualization of transcytosis in MDCK cells using laser scanning confocal microscopy
Authors: Martin Greg , Cariappa Rohit , Hubbard Ann L. 
Published: June 2020

The plasma membrane of polarized epithelial cells is composed of two structurally and functionally distinct domains -- the apical and basolateral -- that also differ in molecular composition. The routes followed by integral membrane proteins from their site of synthesis to their site of function varies between different kinds of epithelia. Madin-Darby canine kidney (MDCK) cells deliver plasma membrane proteins directly to the correct domain, while polarized hepatocytes deliver all newly synthesized plasma membrane proteins initially to the basolateral membrane, then retrieve and redirect the apical membrane proteins. We are studying the targeting signals and delivery routes of DPPIV, a single transmembrane protein whose destination is the apical domain in polarized epithelial cells.DPPIV transfected into MDCK cells is delivered to the basolateral plasma membrane after long (13hr) treatment with Brefeldin A (BFA). After BFA’s removal these molecules are retrieved from the basolateral membrane and transcytosed to the apical plasma membrane. This protocol provides a useful model for studies of the indirect route of protein sorting in polarized epithelial cells, since DPPIV at the basolateral surface can be labeled with specific antibody and then subsequently followed in living cells.