bluuebunny/crossref_metadata_2025_split huggingface dataset:
Energy filtering and correction for the contrast transfer function (CTF) of frozen hydrated objects as illustrated by a reconstruction of Dictyostelium S1 decorated actin
Authors: Schröder Rasmus R. , Manstein Dietmar , Jahn Werner , Spudich James A. 
Published: June 2020

Ice embedded samples can not be simply described as weak phase objects, but as strong interacting specimens with inelastically dominated multiple scattering. Removal of the inelastically scattered electrons allows closer to focus imaging and improves the structural signal to noise ratio, both leading to a higher structural information content. Recent work demonstrated, that for radial density distributions the CTF correction of energy filtered images was necessary and sufficient to obtain good agreement with X-ray data. Here we present the fully CTF-corrected three dimensional structure of F-actin decorated with Dictyostelium myosin head fragment (MHF). The results show aggreement to the known protein structure after CTF correction. In addition to the radial density profile also amplitudes of Bessel terms in the Fourier Transform are changing.Actin from rabbit muscle was decorated with Dictyostelium myosin head fragment under the usual buffer conditions. Images were taken with a Zeiss EFTEM EM902. Magnification was 50000x, electron energy 80kV, electron energy loss δE=0eV (zero-loss mode), energy width ΔE=20eV, defocus values were in the range of 500-900nm. Vitrified samples were observed at a temperature of 90K. All images were taken at an specimen electron dose of 500 e−/nm2, negative material was Kodak SO163 developed for 12 min in undiluted Kodak D19. For image processing and the helical reconstruction a new set of computer programs was written.
3-D imaging of freeze-dried actin filaments in fibroblast using high-resolution cryo-SEM
Authors: Chen Ya , Verkhovsky Alexander B. , Borisy Gary G. 
Published: June 2020

A new approach to image the macromolecular structure of actin filaments from detergent-extracted cultured cells using high resolution low-temperature scanning electron microscopy (cryo-SEM) is described. An advantage of studying the cytoskeleton by SEM is its large depth of focus which makes it easy to reveal the three-dimensional relationship between the cytoskeleton filaments. Recently, procedures have been investigated for high resolution visualization of biological samples by SEM. These studies have demonstrated the importance of cryo preparation techniques for preserving the morphology of the specimen and advanced metal coating techniques for revealing its topography.Coating for HRSEM requires a metal layer that will not obscure fine details while still yielding adequate SE signal. Magnetron-sputter coating of the samples with an ultrathin layer of metal at low temperature provides finer grain size than coating at ambient temperature. Chromium has many properties that make it suitable as coating metal for cytoskeleton: it has fine grain size, low BSE yield due to its low Z number, and it provides adequate SE signal.
Confocal microscopy, image restoration, and nuclear structure
Authors: Shaw Peter , Beven Alison , Rawlins David , Highett Martin 
Published: June 2020

Fluorescentin situhybridization and 3-D confocal microscopy have been used to study the arrangement of genes and transcripts in interphase plant nuclei. Wide-field fluorescence microscopy coupled with computer deconvolution has also been used on the same specimens and gives comparable results to confocal imaging. When image deconvolution is applied to confocal images, these show a significant improvement. A comparison of confocal and wide-field (CCD) images of the same specimen (a plant nucleolus labelled with probe to the rRNA genes) is shown in Figure 1. The deconvoluted wide-field data (Fig 1b) and the raw confocal data (Fig 1c) are remarkably similar; the former is somewhat clearer. The deconvoluted confocal data (Fig 1d) is clearest of all.Double-stranded DNA and single-stranded RNA probes to the 45S and 5S RNA genes have been used to investigate the functional organization of the nucleolus in pea root tissue.In situhybridization was carried out on vibratome slices approximately 50μm thick (about 3 cells).
3-D image restoration of fluorescence microscope images by regularization
Authors: Carrington Walter A. 
Published: June 2020

By combining optical sections of a fluorescently labelled cell with quantitative calibration of the microscope’s blurring, we are able to computationally reverse the blurring of the microscope. We apply this approach to images taken on a conventional wide field microscope using a cooled CCD camera. Our algorithm is flexible in its use of data, which enables us to obtain good results with a small number of optical sections. We use this flexibility to minimize photodamage and photobleaching, so that we are able to obtain 3-D movies of living cells with high spatial and temporal resolution. We obtain rejection of out of focus light, resolution and accuracy of quantitative fluorescent measurement superior to confocal microscopes with substantially less photobleaching and photodamage to the sample. Recent algorithmic advances have enabled us to achieve transverse resolution as good as 100nm and axial resolution as good as 400nm.The wide variety of cells and labels examined in 3-D in our lab illustrate the robustness and effectiveness of our image restoration approach. These applications range from samples as sparsely labelled as single copy genes in lymphocytes and fibroblasts labelled by in situ hybridization to densely labelled cells with fluorescently labelled dextran or Fura-2 that completely fill the cell volume.
Microstructure of the avian eggshell
Authors: Xiao S.Q. , Agarwal M. , Heuer A.H. 
Published: June 2020

Fresh white and dry brown eggshells were studied by TEM and SEM. For TEM, thin sections of the eggshell were prepared by both ultramicrotomy and ion-beam thinning.The various regions from the inner to the outer part of the eggshell can be summarized as follows : the shell membrane, the mammillary layer, the palisade layer and the cuticle. The shell membrane is about 80 μm thick and is composed of collagen fibers with a mantle and core structure. The mammillary layer is a mixture of calcite crystallites, organic matrix and collagen fibers; it starts at the outer shell membrane and merges gradually into the palisade layer. The collagen fibers present near the palisade are surrounded by tiny calcite crystals (Fig. 1). In this region, the periphery of fibers are calcified. The SEM study of the etched cross section revealed clusters of fine organic sheets at the junction of the membrane and the palisade layer (Fig. 2). Further etching results in the collapse of these sheets, to form knob-like features on the surface of the membrane.
Morphogenetic machines revealed: Microscopy of living cells in the embryo of the frog,Xenopus laevis
Authors: Keller Ray 
Published: June 2020

The amphibian embryo offers advantages of size, availability, and ease of use with both microsurgical and molecular methods in the analysis of fundamental developmental and cell biological problems. However, conventional wisdom holds that the opacity of this embryo limits the use of methods in optical microscopy to resolve the cell motility underlying the major shape-generating processes in early development.These difficulties have been circumvented by refining and adapting several methods. First, methods of explanting and culturing tissues were developed that expose the deep, nonepithelial cells, as well as the superficial epithelial cells, to the view of the microscope. Second, low angle epi-illumination with video image processing and recording was used to follow patterns of cell movement in large populations of cells. Lastly, cells were labeled with vital, fluorescent dyes, and their behavior recorded, using low-light, fluorescence microscopy and image processing. Using these methods, the details of the cellular protrusive activity that drives the powerful convergence (narrowing)
Applications of ultramicrotomy for materials characterization
Authors: Blackham R.T. , Haugh J.J. , Hughes C.W. , Burke M.G. 
Published: June 2020

Essential to the characterization of materials using analytical electron microscopy (AEM) techniques is the specimen itself. Without suitable samples, detailed microstructural analysis is not possible. Ultramicrotomy, or diamond knife sectioning, is a well-known mechanical specimen preparation technique which has been gaining attention in the materials science area. Malis and co-workers and Glanvill have demonstrated the usefulness and applicability of this technique to the study of a wide variety of materials including Al alloys, composites, and semiconductors. Ultramicrotomed specimens have uniform thickness with relatively large electron-transparent areas which are suitable for AEM anaysis.Interface Analysis in Type 316 Austenitic Stainless Steel: STEM-EDS microanalysis of grain boundaries in austenitic stainless steels provides important information concerning the development of Cr-depleted zones which accompany M23C6precipitation, and documentation of radiation induced segregation (RIS). Conventional methods of TEM sample preparation are suitable for the evaluation of thermally induced segregation, but neutron irradiated samples present a variety of problems in both the preparation and in the AEM analysis, in addition to the handling hazard.
Feature enhancement by non-uniform ion-milling for SEM of integrated circuits
Authors: Shaapur F. 
Published: June 2020

A variety of techniques has been developed to enhance the image contrast of EM specimens in both physical and biological sciences. In SEM of materials, feature enhancement is mainly based on augmentation of surface topography by non-uniform material removal from different structural elements and preferential erosion at adjoining interfaces. The enhanced surface topography, in turn, translates into better response to the probing beam resulting in higher image contrast.In cross-sectional imaging by SEM (XSEM) of ICs, feature enhancement has been carried out by chemical or mechanical, i.e., ion-milling, surface treatments. Ion-milling has been used to clean surfaces and to enhance their topography. The non-uniform material removal from the surface, in this case, merely relies on an inherent characteristic of the structural elements, i.e., their relative erosion rates during the ion-thinning. An additional factor, i.e., minimization of the “shadowing” effect by adjusting the orientation of ion incidence may also be used to elevate the non-uniform ion-milling even further.
A study of the microvasculature of the normal rabbit bladder with vascular corrosion casting, SEM and TEM
Authors: Hossler Fred E. , Monson Frederick C. 
Published: June 2020

The mammalian urinary bladder performs two functions, urine storage and expulsion. Bladder function is dependent upon the delivery of oxygen and nutrients via a rich blood supply, yet studies of the bladder vasculature during distension have concluded that blood flow is restricted during filling. Following surgically induced ischemia, regeneration of the wall and its vasculature have been reported but not described in detail. Beyond the gross level, the functional vascular anatomy of the bladder wall is poorly understood. Preliminary to studies of angiogenesis and mucosal regeneration following ischemia, and vascular accommodation during distension, the present study utilizes routine transmission (TEM) and scanning (SEM) electron microscopy, and vascular corrosion casting (VCC) to describe the normal microvasculature of the rabbit bladder.Bladders were perfuse fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, for routine TEM and SEM. Thin sections were cut from Araldyte and stained with lead and uranium for TEM, and samples were critical point dried from ethanol and CO2 for SEM.
Three-dimensional transmitted brightfield imaging: pragmatic data collection and preprocessing considerations
Authors: Cooper J.A. , Bhattacharyya S. , Turner J.N. , Holmes T.J. 
Published: June 2020

We have been developing algorithms for 3D image reconstruction of biological specimens with absorbing stains. This is important because there are many absorbing stains which are widely used in conjunction with transmitted light brightfield (TLB) microscopy, yet most of the 3D microscopic imaging research has been directed toward fluorescence microscopy. For instance, horseradish peroxidase (HRP) is used widely in the neurosciences for its many advantages as a tracer and intracellular marker. It is readily injected into individual neurons, transported long distances, and fills both the dendritic and axonal fields, while it may double as an electron microscopy stain for correlative analysis. With such advantages, it is clear that absorbing stains will continue to be widely used. Their utility will furthermore broaden with 3D visualization and quantitation.The main principles behind our methodology are the following. Standard optical serial sectioning data collection is used. The iterative, constrained image reconstruction algorithm is designed to reconstruct the 3D optical density distribution .
Use of a post-embedding, indirect, double-sided labeling procedure to identify cona binding sites within apical vesicles of a filamentous fungus
Authors: Bourett Timothy M. , Howard Richard J. 
Published: June 2020

Vesicles are thought to play an important role in the polarized apical extension of fungal hyphae. These vesicles are likely to contain cell wall precursors and other molecules destined for exocytosis at the hyphal apex, including polysaccharides and/or other glycosylated molecules which should be recognized by certain lectins, including Concanavalin A (ConA). Here we describe the presence of ConA binding sites (CABS) within the apical vesicles of Trichoderma viride. In addition, we compare single- and double-sided ConA labeling patterns to assess the impact of these different techniques on the interpretation of results.Somatic hyphae of T. viride were grown as a cell monolayer over pieces of cellophane on the surface of nutrient agar plates. The cellophane pieces and cells were plungefrozen in a liquid propane/ethane mixture, freeze-substituted in 2% OsO4 in acetone,and subsequently embedded in Quetol resin. Thin sections were picked up with empty, formvar-dipped, single-slot, gold grids.
Projections from the periaqueductal gray (PAG) to the periambigual area: Relation to cholinergic vago-cardiac neurons
Authors: Xu Shuang-jin , Rizvi Tilat A. , Jiang Maorong , Ennis Matthew , Shipley Michael T. 
Published: June 2020

Activation of the midbrain periaqueductal gray (PAG) elicits species specific defense reactions and marked autonomic adjustments. In agreement with recent studies by Bandler and colleagues in the cat, we found that activation of rostrocaudally-oriented, longitudinally organized columns in PAG elicit differential cardiovascular and behavioral responses. Activation of dorsolateral/lateral PAG produces aggressive/flight behaviors accompanied by pressor responses while stimulation of ventrolateral PAG produces immobility and depressor responses. The pathways mediating these selective behavioral and autonomic responses are poorly understood.The projection from PAG to the sympathoexcitatory zone in the rostral ventrolateral medulla is a likely substrate for PAG-evoked pressor responses. However, circuits mediating PAG-evoked depressor responses are not known. The present studies have identified a robust, focal projection from PAG to the nucleus ambiguus (NA), a potent depressor area in the ventral medulla. In addition, we have used tract tracing techniques and immunocytochemistry to examine the organization of PAG projections in relation to cholinergic preganglionic parasympathetic NA neurons that innervate the heart.
Differentiation of malignant mesothelioma from adenocarcinoma
Authors: Garancis John , Cafaro John , Cafaro Anthony , Venezia Raymond 
Published: June 2020

Malignant mesothelioma (MM) is a rare tumor arising predominantly from either the visceral or parietal pleura or peritoneal surfaces. It primarily effects individuals between 45 and 75 years. of age and asbestos is felt to be the single most important etiologic agent. Studies suggest that the incidence of MM is increasing perhaps at a rate of as high as 13% per year. The diagnosis of MM can be difficult and is often complicated by the tumors similarity to metastatic adenocarcinoma, both in it's microscopic appearance and to some extent clinical course.In this particular study the immunohistochemical properties of 22 malignant mesotheliomas (20 pleural and two peritoneal) are compared with 20 pulmonary adenocarcinomas. Each tissue was examined using monoclonal antibodies to wide-spectrum cytokeratin, CEA, Leu-M1 and B72.3. Extensive asbestos exposure was identified in all but 1 of the mesothelioma cases. The cases of peritoneal MM were found to have a history of chrysctile exposure only (Fig.1). Distant metastases were present in four cases of MM.
Transport pathways of low-density lipoproteins by arterial endothelium of hypercholesterolemic rats
Authors: Kao C.H. , Yang V. C. , Chen J.K. , Kuo J.S. 
Published: June 2020

Atherosclerosis is more likely to occur at the branched regions of the large or mediumsized arteries. Physiological evidences show that changes in endothelial permeability at the branched regions can cause low density lipoproteins (LDL) to filtrate and accumulate in the intima. However, the LDL transport pathways across the arterial endothelium at the ultrastructural level still remain uncertain. The purpose of this experiment is utilizing fluorescein 1,1'	-dioctadecyl-3,3,3',3'	-tetramethylindocarbocyanine perchlorate (Dil) and colloidal gold as the tracer substances to investigate the transport pathways of LDL in the branched and the unbranched regions of arteries in the diet-induced hypereholesterolemic rats.Rat or human LDL was coupled to Dil or to 10-15nm colloidal gold particles according to the standard procedures. Male Srague-Dawley rats, weighing approximately 250 gm were fed high-cholesterol diets over a period of 12 months. At 6 and 12 months after feeding, animals were anesthetized by intraperitoneal injections of sodium pentobarbitol. The vascular bed was cleared of blood by perfusion with oxygenated PBS supplemented with 0.25% glucose and 1 mM CaCl2 at 37°C through the left ventricle. After the blood washed out, the right and the left femoral arteries and the left ventricle, the thoracic and the abdominal aorta were cannulated with polyethylene tubings. The tubings between the left ventricle and the thoracic aorta and the tubings between the abdominal aorta and the femoral arteries were connected with peristaltic pump to form two closed circuits.
Cell injury and calcium accumulation following sulfur mustard exposure
Authors: Petrali J.P. , Hamilton T.A. , Mills K.R. , Ray R. 
Published: June 2020

Cellular injury, irrespective of causative agents, is associated with a change in the transmembrane potential for calcium with subsequent accumulations of calcium to intracellular domains. At this time however, the association of sulfur mustard (HD)-induced cytotoxicity and calcium metabolism remains at controversy. This study correlates the ultrastructural cytopathology, cytochemical localization and x-ray microanalysis of calcium in cultured cells exposed to HD in vitro.NG108-15 and HeLa cells, exposed to 300 and 500 ¼M HD respectively and harvested at 1 to 24h post exposure, were fixed in 1.6% formaldehyde and 2.5% glutaraldehyde for 3 h at 4°C and processed for routine ultrastructural evaluation. Replicate populations were cytochemically processed for precipitation of calcium by incubating in a medium of 2% potassium pyroantimonate and 1% osmium tetroxide for 90 min in the dark at 20°C. Cells were then placed in a secondary medium of 0.5% tannic acid for 15 min, rinsed in 1% sodium sulfate, dehydrated in graded ethanols and embedded in epoxy resin.
Peanut agglutinin receptors in the epididymai spermatozoa of the palm squirrel (Funambulus penanti)
Authors: Bawa S. R. , Bawa R. , Bains H. K. 
Published: June 2020

Physiological ripening of the mammalian spermatozoa during their sojourn in the epididymal duct is also mirrored on the morphological plane by lectin-labeling studies. In this report peanut agglutinin (PNA), which specifically labels methyl α-D-galactopyranoside, has been employed to monitor the maturation process of the epididymal spermatozoa of the Indian palm squirrel (Funambulus penanti) employing fluorescence microscopy and transmission electror microscopy (TEM in toto).Spermatozoa were released from the epididymis by gently mincing the tissues in phosphate-buffered saline (pH 7.4). For fluorescence microscopy, sperm suspensions were smeared onto poly-L-lysine coated coverslips and labeled with 10 ¼l of fluorescein isothiocyanate-linked PNA (100 ¼l/L). For TEM in toto sperm suspensions were adsorbed onto poly-L-lysine and Formvar coated grids and subsequently labeled with colloidal gold-linked PNA. After washing in buffer the grids were examined with a JEOL 1200 EX transmission electror microscope. Controls were prepared by incubating sperm samples in 0.2 M α-D galactopyranoside.
A new cytochemical method: Combined enzyme cytochemistry and immunocytochemistry on ultrathin cryosections
Authors: Takizawa Toshihiro , Robinson John M. 
Published: June 2020

Cryo-ultramicrotomy has been a useful method for immunocytochemistry because it can preserve the ultrastructure of cells and also display antigenic sites in cells. Recently, cryo-ultramicrotomy has been successfully applied to enzyme cytochemistry for the demonstration of some phosphatases with lead as the capture metal. However, this application has some problems. The lead reaction product is unstable with uranyl acetate staining, thus leading to poor ultrastructural detail. The purpose of this study is to develop the new method of enzyme cytochemistry using cerium as the capture metal on ultrathin cryosections labeled with immunogold probes. This method should be of importance for demonstrating the ultrastructure localization of certain enzyme and the simultaneous localization of antigens in a variety of cell types.Freshly isolated human neutrophils were fixed with paraformaldehyde and/or glutaraldehyd in 0.1 M cacodylate buffer (pH 7.2) with 5 % sucrose, then pelleted into 10 % gelatin and subsequently infiltrated with 2.3 M sucrose.
Predation-induced E. coli filaments: Are they multicellular?
Authors: Gillott Marcelle A. , Holen Dale , Ekman Jonathan , Harry Michelle , Boraas Martin 
Published: June 2020

Escherichia coli can have a filamentous growth form due to mutation or, in the current study, selection of predator-resistance when preyed on by a bactivorus protozoan. When used as food for the mixotrophic flagellate, Poterioochromonas malhamensis, filamentous E. coli appeared in continuous and batch cultures. The filamentous morphology of E. coli is persistent in plate and liquid culture in the absence of the flagellate. Typically, filament length is long in exponential growth and short in stationary phase.A major question in the morphology of these filamentous E. coli (Fig. 1) is whether they are chains of cells that have failed to separate or whether they are a continuous filament without septa. Fluorescence light microscopy using DAPI, a DNA-specific stain, indicates that the filaments show both continuous fluorescence (Fig. 2a), indicating a continuous filament, and discrete units of fluorescence (Fig. 2b), indicating separate cells. Division patterns of living filaments observed with video microscopy show that cell division does occur, but it has been observed only at 10-20 μm from the ends of filaments.