bluuebunny/crossref_metadata_2025_split huggingface dataset:
Developmental study of chorioretinal vasculature demonstrated by vascular casts
Authors: Matsunaga Nobuhiko , Amemiya Tsugio 
Published: June 2020

Introduction: Methylmethacrylate is a very useful material for the preparation of vascular casts, and many reports have described the vascular of ocular tissues. We attempted to study the development of the rat ocular vasculature.Material and Methods: 20-day-old fetuses, and 1-day-old, 7-day-old, 14-day-old and 11-week-old rats were anesthetized, and the chest was opened by splitting the sternum. Warm Ringer solution containing heparin sodium was perfused through a catheter inserted into the left ventricle. Blood was removed through an opening in the right atrium. After 2% glutaraldehyde in 0.1M phosphate buffer had been perfused, a mixture of methylmethacrylate resin and catalyst was injected through the same catheter. The eyes were enucleated carefully and macerated in a bath of concentrated potassium hydroxide and sodium hypochlorite solution. The vascular cast was rinsed in distilled water and dried at room temperature or with t-butyl alcohol. After electroconductive staining with osmium tetroxide gas, the cast was mounted on stubs with carbon adhesive tape, and then spattercoated with gold for viewing in a scanning electron microscope.
Immunoelectron microscopic localization of elastic tissue in archival material using an improved method
Authors: Fanning J. C. , White J. F. , Polewski R. , Cleary E. G. 
Published: June 2020

Elastic tissue is an important component of the walls of arteries and veins, of skin, of the lungs and in lesser amounts, of many other tissues. It is responsible for the rubber-like properties of the arteries and for the normal texture of young skin. It undergoes changes in a number of important diseases such as atherosclerosis and emphysema and on exposure of skin to sunlight.We have recently described methods for the localizationof elastic tissue components in normal animal and human tissues. In the study of developing and diseased tissues it is often not possible to obtain samples which have been optimally prepared for immuno-electron microscopy. Sometimes there is also a need to examine retrospectively samples collected some years previously. We have therefore developed modifications to our published methods to allow examination of human and animal tissue samples obtained at surgery or during post mortem which have subsequently been: 1. stored frozen at -35° or -70°C for biochemical examination; 2.
An improved technique for visualization of Neisseria gonorrhoeae pili by SEM
Authors: Dekker Nusi P. , Lammel Claudia J. , Brooks Geo. F. 
Published: June 2020

Piliated Neisseria gonorrhoeae (gc) are virulent and attach to human cells more readily than nonpiliated gc . Pili have been difficult to visualize in the scanning electron microscope (SEM), appearing as thick, branched structures, possibly because of artifacts or distortion associated with critical point drying (CPD) . The use of hexamethyldisilazane (HMDS), as an alternative to CPD with bacterial or tissue culture cells, as reported here, improved preservation and visualization of pili and other delicate surface structures.The methods were: Human endometrial adenocarcinoma (HEC-l-B, ATCC HTB 113) cells were grown in cell culture medium containing fetal calf serum on round thermanox coverslips. Suspensions of piliated and nonpiliated strains of gc (FA1090, F62-SF, and g1412) at dilutions of about 2 × 107 cfu were added to the cells and allowed to attach. Unattached gc were rinsed off and replaced with fresh media after 30 min. After 1 or 8h incubation, the cells were fixed in 2.5% gluteraldehyde in 0.1M cacodylate buffer for 4h; rinsed well in the buffer; post-fixed in 1% OsO4 in cacodylate buffer for lh; rinsed 3 × 10 min in buffer and 2 × 5 min in water; placed in 1% tannic acid in water for 30 min; rinsed 3 × 10 min in water; placed in 2% uranyl acetate for lh; rinsed 3 × 10 min in water; dehydrated in graded ethanols (50%, 70%, 95%, 2 × 5 min each; 100% 4 × 5 min; 100% 10 min); placed in HMDS for 10 min.
Gap junctions: A new level of detail revealed by shadowing at -170° to -182°C in a JEOL JFD-9000 equipped with cryopump and liquid nitrogen-cooled shrouds
Authors: Rash J. E. , Yasumura T. , Walrond J. P. , Steere R. L. , Okutomi S. 
Published: June 2020

In conventional freeze-fracture replicas, vertebrate gap junctions (GJ) are visualized as patches of 9 nm P-face particles with center-to-center spacings of 9-11 nm. Occasionally IMPs have a central “dimple”. On E-faces, GJ are represented by 4-6 nm diameter conical pits separated by 5-7 nm wide membrane ridges. In addition to the disparity of sizes of IMPs and pits, there is no discernable substructure in the pits corresponding to the dimples in the IMPs. In this report, we present replicas revealing a new level of detail for gap junctions, including evidence for channels in both P-face IMPs and E-face pits.Mice were fixed by perfusion with 2.5% glutaraldehyde in phosphate buffer. Samples were gradually infiltrated to 30% glycerol, frozen by plunging into liquid propane, fractured at -105° to -182°C (measured by thermocouple in sample stage), and shadowed at an angle of 45° in a JEOL JFD-9000 freeze-etch machine equipped with electron beam guns. The JFD-9000 was modified by the addition of interlocking liquid nitrogen-cooled shrouds for specimen and knife and a CTI-8F cryopump.
Effects of isoproterenol treatment on the parathyroid glands in golden hamsters of different ages, with special reference to the frequency of lipid droplets
Authors: Emura Shoichi , Shoumura Shizuko , Yamahira Tomo , Kawada Tomoo , Oda Hiroshi , Arakawa Michio , Isono Hideo 
Published: June 2020

Recently, we reported that there is an increase in lipid droplets in the parathyroid glands of isoproterenol-treated adult golden hamsters in which the synthesis and release of parathyroid hormone are stimulated. This investigation was undertaken to study the effects of different ages on lipid droplets in the parathyroid glands of the golden hamsters after administration of isoproterenol.The percent area occupied by lipid droplets in the parathyroid glands of 15, 20 and 25-day-old golden hamsters after 1 h of treatment with isoproterenol and the frequency of lipid droplets of 1-monthold (young), 3-month-old(adult) and 18-month-old(senile) animals after 0.5, 1 and 3 h of treatment with isoproterenol were investigated. The parathyroid glands of the control and isoproterenol-treated animals were removed and immersed in a mixture of 2.5% glutaraldehyde and 2% OsO4 in Millonig's buffer at pH7.4 for 1 h, dehydrated through increasing concentrations of acetone and embedded in Epon 812.
The interfacial behavior of hard tissue replacement as revealed by SEM
Authors: Pearsall Allan D. , Spears Robert , Sarubbi Kimberly 
Published: June 2020

INTRODUCTION:Hard Tissue Replacement (HTR) has been postulated to be an effective implant material for bone reconstruction and maintenance. HTR consists of a polymethylmethacrylate core coated with polyhydroxyethylmethacrylate and has been demonstrated in light microscopic studies to attach directly to bone. The present study was performed to add to the available information by investigating the interfacial bonding of this polymer using scanning electron microscopy.MATERIALS AND METHODS: Twenty Sprague-Dawley rats were anesthetized, a midline scalp incision was made to expose the calvaria and the periosteum reflected. Trephine defects were prepared by excising a 1 x 2-mm section of bone and the HTR polymer (24 mesh) inserted. The skin and periosteum were reapproximated and the incision closed. Pursuant to vascular perfusion with a mixture of aldehydes, the implants and surrounding tissue were extirpated in situ after 7-, 14-, 28-, and 56-day observation periods. Tissues were then washed in buffer, dehydrated, fractured in liquid nitrogen and dried in Freon. The samples were viewed in a JEOL JSM35CF SEM with x-ray energy dispersive analysis (EDXA).
A quantitative immunoscanning EM analysis of CD16 (FeRIII) antigen on peripheral blood leukocytes
Authors: Fernández-Segura Eduardo , Manuel Garcia-Lopez O. , Gutierrez Antonio , Campos Antonio 
Published: June 2020

A common feature of many cell types which mediate major histocompatibility complex (MCH)-unrestricted lysis is the presence of receptors for the Fc region of IgG (FcR) on the surface. Monoclonal antibodies (MAbs) against these receptors have identified three distinct FcR, i.e., FcRI, FcRII, FcRIII. These receptors are expressed on different but overlapping cells populations, the level of FcR expression being a characteristc of each populations. FcRIII (CD16 antigen) is the low-affinity IgG Fc-receptor whLch is predominantly expressed on human natural killer (NK) cells and granulocytes. With the recent application of immunogold labelling for scanning electron microscopy (immuno-SEM) in the backscattered electron imaging (BEI) mode allow us to correlate antigenic expression with cell surface morphology. In this report we analize the expression and distribution of the CD16 (FcRIII) antigen on peripheral blood leukocytes as observed with immuno-SEM.Material and Methods.Mononuclear cells (MC) were isolated from normal peripheral blood using Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden).
Guanosine triphosphatase activity demonstrated by freeze substitution enzyme histochemistry
Authors: Takizawa Toshihiro , Saito Takuma 
Published: June 2020

During the past three decades, the freeze substitution fixation technique has been developed to observe fine morphology in close to the living state. Recently, this freeze substitution fixation has been successfully applied to enzyme histochemistry demonstrating the active sites of enzymes as well as their active state at the time of quenching. in this study, we have undertaken to study guanosine triphosphatase (GTPase), one of the key enzymes in the cGMP cascade, on the rod outer segment after freeze substitution fixation and have compared the results with those obtained from conventional immersion fixation enzyme histochemistry at the ultrastructural level.Freeze substitution fixation: After Nembutal anesthesia, frog, Xenopus laevis, eyes were excised and the neural retinae were gently separated from the pigment epithelium. The retinae were then rapid frozen using a quick freezing device, the E7200 Slammer (Polaron, Equipment Ltd. England), contacting on an ultrapure copper block at -196°C of liquid nitrogen.
High-sensitivity detection of gold labels by high-angle dark-field STEM imaging
Authors: Otten Max T. 
Published: June 2020

Labelling of antibodies with small gold probes is a highly sensitive technique for detecting specific molecules in biological tissue. Larger gold probes are usually well visible in TEM or STEM Bright-Field images of unstained specimens. In stained specimens, however, the contrast of the stain is frequently the same as that of the gold labels, making it virtually impossible to identify the labels, especially when smaller gold labels are used to increase the sensitivity of the immunolabelling technique. TEM or STEM Dark-Field images fare no better (Figs. 1a and 2a), again because of the absence of a clear contrast difference between gold labels and stain.Potentially much more useful is backscattered-electron imaging, since this will show differences in average atomic number which are sufficiently large between the metallic gold and the stains normally used. However, for the thin specimens and at high accelerating voltages of the STEM, the yield of backscattered electrons is very small, resulting in a very weak signal. Consequently, the backscattered-electron signal is often too noisy for detecting small labels, even for large spot sizes.
The use of 1nm silver enhanced gold probes for the detection of skin antigens
Authors: Cassella J.P , Shimizu H. , Ishida-Yamamoto A. , Eady R.A.J. 
Published: June 2020

1nm colloidal gold with silver enhancement has been used in conjunction with a low-temperature post-embedding (post-E) technique for the demonstration of skin antigens at both the light microscopic (LM) and electron microscopic (EM) levels.Keratin filaments and basement membrane zone (BMZ) associated antigens in normal human skin (NHS) were immunolabelled using antibodies against keratin 14, 10, and 1, the carboxy-terminus and collagenous portion of type VII collagen, type IV collagen and bullous pemphigoid antigen (BP-Ag).Fresh samples of NHS were cryoprotected in 15% glycerol, cryofixed in propane at -190°C, subjected to freeze substitution in methanol at -80°C and embedded in Lowicryl K11M at -60°C. Polymerisation of the resin was initiated under UVR at - 60°C for 48 hours and continued at room temperature for a further 48 hours. Semith in sections were air dried onto slides coated with 3-aminopropyltriethoxysilane. The following immunolabelling protocol was adopted: Primary antibody was applied for 2 hours at 37°C or overnight at 4°C. Following washing in Dulbecco’s phosphate buffered saline (PBSA) a biotinylated secondary antibody was applied for 2 hours at 37°C. The sections were further washed in PBSA and 1nm gold avidin was applied. Sections were finally washed in PBSA and silver enhanced.
The 215 KD mannose-6-phosphate receptor is involved in crinophagy but not in autophagy
Authors: Noda Toru , Bronson Mary L. , Yu Shang-Ming , Farquhar Marilyn G. 
Published: June 2020

Autophagy and crinophagy represent the two major pathways for digestion of intracellular material via lysosomes have been described. Though both phenomena involve in corporation of cell organelles into lysosomes and thus degradation by lysosomal enzymes, the process by which autophagic and crinophagic vacuoles acquire lysosomal enzymes remains to be clarified. The aim of this work is to find out if mannose-6-phosphate (M6P) receptors are involved in this process. As a typical working model, we used hepatocytes of leupeptin-treated rats for autophagy (Fig. 1) and mammotrophs of female rats treated with estradiol for 6 days, for crinophagy. To localize the M6P receptor and lysosomal proteins, we carried out immunocytochemistry, using a immunoperoxidase pre-embedding technique and immunogold labeling on ultrathin frozen sections.Many autophagosomes induced by leupeptin treatment contained recognizable mitochondria and rough ER. The configuration and fine structure of these organelles are gradually lost and the contents of the vacuoles become more homogeneous and electron-dense, suggesting that partial digestion of the contents of autophagosomes still occurs after leupept in treatment.
STM of nucleic acids
Authors: Bloomfield Victor A. , Arscott Patricia G. 
Published: June 2020

Scanning tunneling microscopy (STM) enables remarkable visualization of surface topography with a vertical resolution of 0.2 Å and horizontal resolution of 2-3 Å. The possibility of visualizing nucleic acids at this high resolution has major significance, since the binding of proteins and other aspects of gene regulation depend on structural variations at the Å level. Conventional EM has the advantage over fiber diffraction or solution techniques that local structure of individual molecules can be seen, rather than averaging over entire molecules and populations of molecules. An added advantage of STM is that contrast is achieved by detection of sample height and work function variations, rather than by chemical treatments such as shadowing or staining.We have used STM to measure the helical pitch, half-period oscillations interpreted as alternating major and minor grooves, and molecular diameters of poly(rA)-poly(rU) and DNA. Average pitches are measured by two-dimensional Fourier transforms and by topographic profile peak count vs.
Localization of myocin in Micrasterias cells by immunoelectron microscopy
Authors: Lehtonen Juhani , Suoranta Ulla-Maija 
Published: June 2020

The unicellular green alga Micrasterias has a very differentiated form and is an excellent object for morphogenetic studies. The complicated cytoplasmic streaming system during development is an essential part of the morphogenetic process. The streamings are supported by the acto-myosin system. The presence of actin has been verified by fluorescence microscopy and both the act in-inhibitor cytochalasin B and the myosin inhibitor N-ethylmaleimide clearly inhibit the streamings.This work deals with the localization of myosin in the cell. The methods used were immunoelectronmicroscopy, polyclonal antibody/gold probe, and cryosections. In old semicells, the gold-label localized only in vesicle structures. In growing semicells, the label was abundant in growing lobes, in all levels. In control samples, without antibody, there was no label.According to the results, myosin seems to be part of the dictyosome vesicle membranes. This is clear in the case of old semicells. In growing lobes, the inner structure of the lobes was not preserved very well in cryosections, in any preparation, but there is good evidence that in new, growing parts of the cell there is generally very little material other than cell wall material vesicles. This idea is important for later studies on plant cell growth and cytoplasmic transport.
Quantitative HREM using non-linear least-squares methods
Authors: King Wayne E. , Campbell Geoffrey H. 
Published: June 2020

Non-linear least-squares methods have been coupled with high resolution image simulation to determine the critical electron microscopic imaging parameters, such as thickness and defocus, from experimental high resolution electron optical images of yttrium aluminum garnet (YAG). For a quantitative fit between experimental and simulated images we seek to minimize the residual image fi(x) at each pixel,(1)where is the intensity value of the ith pixel in the experimental image, is the intensity value of the ith pixel in the simulated image based on the image model x, and W is the image that represents the uncertainty associated with measurement of the ith pixel in the experimental image.
Quantitative χn analysis of HREM images with applications to planar defects
Authors: Zhang H. , Marks L. D. 
Published: June 2020

A number of different methods have been suggested in the literature for using HREM in a quantitative fashion, including R factors and cross-correlation analyses. The problem with many of these is that it is difficult to realistically gauge the errors involved when they are applied to real systems. For instance, R-factors defined by:(1)(where n=1 or 2 and Ic is the calculated image, Ie the experimental data) assume a signal independent error. Furthermore, the absolute value of R is strongly dependent upon background levels which is misleading.
Ultra-low voltage scanning electron microscopy
Authors: Joy David C , Joy Carolyn S 
Published: June 2020

Although the benefits of operating the scanning electron microscope at low beam energies have been evident since the earliest days of the instrument, the successful utilization of the SEM under these conditions has required the development of high brightness field emission electron source, advanced lenses, and clean vacuums. As these technologies became available the level at which imaging became regarded as “low energy” has fallen from 10keV, first to 5keV, and more recently to 1keV. At this energy state of the art instruments can now provide an excellent balance between resolution – which becomes worse with decreasing energy – and desirable goals such as the minimization of sample charging and the reduction of macroscopic radiation damage – which tend to become more challenging as the energy is increased.An interesting new opportunity is to perform imaging in the ultra-low energy region between leV and 500eV. Over this energy range significant changes in the details of electron-solid interactions take place offering the chance of novel contrast modes, and the rapid fall in the electron beam range leads to the condition where the penetration of the incident beam into the sample is effectively limited to 1 or 2 nanometers. The practical problem is that of achieving useful levels of resolution and acceptable signal to noise ratios in the image. At energies below IkeV chromatic aberration dominates the probe formation in conventional instruments even when using an FEG source. However, the use of optimized retarding field optics essentially maintains chromatic aberration independent of landing energy down to very low values. Figure (1) shows an example of the performance that can be achieved on a commercial instrument – an Hitachi S-4500 – modified to operate in this mode, in this case at 50eV landing energy. The resolution of the image is judged from edge sharpness and detail to be significantly better than 0.1µm and, from experimental observation, this performance is apparently limited by residual astigmatism caused by uncorrected sample charging rather than by fundamental aberrations in the probe forming optics. Comparable, if somewhat lower resolution, images have been achieved on this, and other FEG SEMs, at energies as low as leV.
The influence of surface topography in the adhesion of polystyrene to aluminum
Authors: Shaffer O. L. , Yankovskaya S. , Namkanisom A. , Chaudhury M. , El-Aasser M. S. 
Published: June 2020

Polymer/metal adhesion has been studied by scanning(SEM) and transmission electron microscopy(TEM). Now with atomic force microscopy(AFM) we can scan the metal's surface and correlate the roughness with adhesion energy. We have chosen aluminum as the metal substrate and polystyrene(PS) as the nonreactive polymer adhesive. A great deal of research has been done on primed metal surfaces which are used to improve adhesion between the metal and the polymer. Our interest is to study the mechanical interlocking of the polymer to the metal without a primer coupling agent.Aluminum alloy 6061 was cut into plates 25x60x1 mm, cleaned with an alkali cleaner, rinsed with water, etched and anodized. The plates were etched in a solution of Na2Cr2O7·2H2O, H2SO4, and water in a 1:10:30 ratio by weight at 50-52°C for 12-20 min. After etching , they were rinsed, dried and anodized in 10% phosphoric acid solution with 15 V for 20 min, then were rinsed and dried again.A 1% PS solution in toluene was deposited on the prepared aluminum surface and air dried.
Etching of LLDPE and LLDPE/HDPE blends
Authors: Bischel M. S. , Schultz J. M. 
Published: June 2020

Despite its rapidly growing use in commercial applications, the morphology of LLDPE and its blends has not been thoroughly studied by microscopy techniques. As part of a study to examine the morphology of a LLDPE narrow fraction and its blends with HDPE via SEM, TEM and AFM, an appropriate etchant is required. However, no satisfactory recipes could be found in the literature. Mirabella used n-heptane, a solvent for LLDPE, as an etchant to reveal certain morphological features in the SEM, including faint banding in spherulites. A 1992 paper by Bassett included a TEM micrograph of an axialite of LLDPE, etched in a potassium permanganate solution, but no details were given.Attempts to use n-heptane, at 60°C, as an etchant were unsuccessful: depending upon thickness, samples swelled and increased in diameter by 5-10% or more within 15 minutes. Attempts to use the standard 3.5% potassium permanganate solution for HDPE were also unsuccessful: the LLDPE was severely overetched. Weaker solutions were also too severe.