bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of directly frozen macromolecules and tissues in the field‐emission STEM
Authors: Leapman Richard D. , Andrews S. Brian 
Published: August 2011

SUMMARYA VG Microscopes HB501 field‐emission high‐resolution scanning transmission electron microscope (STEM) was used to image and analyse rapidly frozen, isolated macromolecules and small organelles in tissue cryosections. Dark‐field images were obtained from frozen‐hydrated microtubules demonstrating that sufficient contrast is available to reveal structural information. The samples were subsequently freeze‐dried in the STEM and low‐dose (≅ 103 e/nm2) dark‐field mass maps were recorded with single electron sensitivity. Elemental analysis of individual macromolecules was achievable at high dose using parallel‐detection electron energy‐loss spectroscopy, albeit with some structural degradation. Detection of copper (320 atoms) in di‐decameric haemocyanin molecules was easily within the limits of sensitivity. Elemental analysis of hydrated cryosections is limited by radiation damage to a resolution of approximately 1 μm2. For freeze‐dried sections, however, the high probe current and stable cold stage of the HB501 STEM allow energy‐dispersive X‐ray (EDX) microanalysis of low elemental concentrations in highly localized subcellular volumes. EDX spectra from cryosections of cerebellar cortex show that a 100‐s analysis time is sufficient to quantify the calcium content of 400‐nm2 regions within Purkinje cell dendrites with an uncertainity of ± 2 mmol/kg dry weight, equivalent to ± 12 atoms.
Freeze‐fracturing for conventional and field emission low‐temperature scanning electron microscopy: the scanning cryo unit SCU 020
Authors: Müller T. , Guggenheim R. , Düggelin M. , Scheidegger Ch. 
Published: August 2011

SUMMARYA dedicated cryopreparation system, the SCU 020 (Balzers), is introduced and described in detail for use in low‐temperature scanning electron microscopy (LTSEM). The basic unit consists of two parts: (i) a high‐vacuum preparation chamber equipped with a cold‐stage, motor‐driven fracturing microtome, planar magnetron (PM) sputter source, quartz‐crystal thin‐film monitor, Meissner cold trap, and turbo molecular pump stand; and (ii) a second part (separated from the first by a sliding, high‐vacuum valve) residing in the SEM chamber. This is equipped with an anti‐contamination cold trap, a fully movable goniometer cold stage (having motor drives for x, y, and rotation) and replaces the SEM's original stage (Raith). The SCU 020 is entirely self contained allowing independence from, and synchroneity with, the SEM of choice.LTSEM micrographs of specimen (that are fully frozen hydrated or partially freeze‐dried) surfaces or fracture faces, without or with various metal coatings, can be examined over a broad temperature range (‐150 to +50°C). This is made possible by the combined application of the two, independently controlled, cold stages and the on‐line, high‐vacuum, specimen cryo transfer between them. In‐situ etching is simple and straightforward. Intramembranous particles and membrane fracture steps, typically imaged in transmission electron microscopy (TEM), are resolved by PM sputtering with platinum at low specimen temperature and high‐resolution LTSEM in a field emission microscope.
Freeze‐fracture cytochemistry: a simplified guide and update on developments
Authors: Severs N.J. 
Published: August 2011

SUMMARYA wide variety of methods by which cytochemistry and freeze‐fracture can be successfully combined have recently become available. All these techniques are designed to provide information on the chemical nature of structural components revealed by freeze‐fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied. Colloidal gold labelling is the most widely used cytochemical technique in freeze‐fracture cytochemistry, and for many of the methods it is indispensable. In principle, there are four points in which the cytochemical labelling step may be integrated into the standard freeze‐fracture procedure: (i) before the specimen has been frozen, (ii) after it has been fractured and thawed, (iii) after platinum shadowing or (iv) after completion of the full replication sequence. Retention of the gold label so that it can be viewed with replicas can be achieved by depositing platinum and/or carbon upon the labelled surface, thereby partially entrapping the marker particles within the replica, or by retaining, attached to the replica, fragments of fractured membrane (or other cellular components) that would normally have been lost during the replica cleaning step. Another approach to visualizing the label is to use sections, either with portions of a replica included face‐on, or for examining the fracture path through the sample (without replica). Recent developments have centred on the use of replicas to stabilize half‐membrane leaflets; not only may these and associated attached components be retained for labelling just before mounting, but they provide a means for manipulating the specimen— specifically, turning it over during processing—so that additional structural information can be obtained.This article aims to explain how modern freeze‐fracture cytochemisty works, and how the various techniques differ in what they can tell us about membranes and other cellular structures. With the effectiveness of many of the techniques now demonstrated, freeze‐fracture cytochemistry is firmly established, alongside a range of related labelling techniques, for increasing application in cell and membrane biology in the 1990s.
Ice crystal damage and radiation effects in relation to microscopy and analysis at low temperatures
Authors: Echlin Patrick 
Published: August 2011

SUMMARYThere are several limitations to the low‐temperature techniques which are currently being used for the preparation, examination and analysis of biological and organic samples by means of high‐energy beam instrumentation. The low thermal conductivity of samples and the inadequacy of rapid cooling techniques means that, with the exception of thin‐film suspensions and the surface of impact‐cooled bulk specimens which may be vitrified, ice crystals of varying sizes will be present in nearly all samples which are quench cooled. Data are presented which indicate the depth to which adequate cryo‐fixation may be achieved for both morphological and analytical studies. Although dynamic processes may be time resolved in the outer parts of quench‐cooled samples, the decreased freezing rate below the surface makes resolution of these processes much less certain. The quality of information which may be obtained from quench‐cooled samples is limited by radiation damage. Low‐dose microscopy of vitrified thin‐film suspensions of macromolecules continues to provide valid structural information at the molecular level. The increased doses needed for X‐ray microanalysis present serious problems with the high spatial resolution analysis of thin frozen‐hydrated sections although much less damage is observed in dried samples. A case is presented for using the outer fracture faces of frozen‐hydrated bulk samples for low‐resolution analysis of cells and tissues.
The potential of cryofixation and freeze substitution: observations and theoretical considerations
Authors: Kellenberger E. 
Published: August 2011

SUMMARYThe theoretical and experimental evidence in favour of cryofixation and freeze‐substitution are critically reviewed. The solubility of macromolecules in water is due to the hydration shells. Their behaviour at different temperatures and the consequences of their removal during the processing for embedding are explained. Gelation prior to the transfer into solvents prevents macromolecules aggregating. During substitution at low temperatures, DNA is gelled, justifying the use of the term cryofixation. It is proposed that the preservation of hydration shells at the lowest temperature, and their transformation into minute gaps after a rise of temperature, facilitates the exhibition of epitopes.
Contrast analysis of cryo‐images of n‐paraffin recorded at 400***kV out to 2·1 Å resolution
Authors: Brink J. , Chiu W. 
Published: August 2011

SUMMARYN‐Paraffin was used as a test specimen for evaluating the relative merits of 400‐kV versus 100‐kV electron microscopy in recording data for electron crystallographic analysis of beam‐sensitive materials. The parameter used for comparison, the relative contrast R, is the ratio of amplitudes from the computed Fourier transform of images and amplitudes from an electron diffraction pattern from the same crystal. R will thus be a measure of the contrast from an experimental image relative to that of a perfect image. Electron diffraction patterns and bright‐field images were recorded at 400 kV at a specimen temperature of −167°C. Using the flood‐beam imaging technique the best R‐value is 0 08 for all reflections in the resolution zone from 4 to 3 Å. This value is equivalent to that found at 100 kV. In the resolution zone from 3 to 2 Å we have found R — 0 02. Using the spot‐scan imaging technique, on the other hand, R was measured to be 0·42 for the reflections between 4‐ and 3‐Å resolution. This amount of relative contrast is 1·7 times that observed at 100 kV. Reflections at 3–2 Å displayed an R‐value of 0 05. Besides obtaining higher R‐values when applying the spot‐scan imaging technique at 400 kV, we observe a higher yield of images with isotropic diffraction and/or higher resolution reflections. Various contrast‐attenuating factors, including the modulation transfer function of the photographic film and the cryo‐holder, envelope functions for spatial and temporal coherence and lens and high‐tension instabilities, the contrast transfer function and lastly the radiation damage effects, have been considered in interpreting the observed image contrast. Overall, use of 400 kV in combination with spot‐scan does offer important improvements in contrast levels, which can be very useful in determining the three‐dimensional structure from protein crystals.
Ergodicity and integral range
Authors: Lantuéjoul Ch. 
Published: August 2011

SUMMARYWhen attempting to estimate stereological parameters starting from measurements in a limited domain, the question has to be addressed of how large the domain size must be in order to ensure representative measurements. To answer that question, the concept of ‘integral range’ is introduced. It allows a comparison between the scale of the phenomenon under study and the scale of observation. The integral range can be assessed using curves of dispersion variance, which can also yield empirical laws of change of scale.
Direct visualization of colloidal gold‐bound molecules and a cell‐surface receptor by ultrahigh‐resolution scanning electron microscopy
Authors: Tanaka Keiichi , Mitsushima Akira , Yamagata Noboru , Kashima Yuzuru , Takayama Hisao 
Published: August 2011

SUMMARYThe structure of protein A‐coated colloidal gold particles, and of macrophage cell‐surface receptors conjugated with immunogold particles, was studied using an ultrahigh‐resolution scanning electron microscope. Protein A, when conjugated with 15‐nm gold, formed a coat completely surrounding the particle. Particles conjugated with both protein A and immunoglobulin G (IgG) were similar, but with additional protrusions formed by the IgG. IgG molecules directly bound to gold were resolved sometimes as complexes of three units, sometimes as more filamentous, V‐shaped structures.On the cell surface of a macrophage reacted with a monoclonal antibody to Mac‐1 antigen (the murine C3bi receptor) followed by protein A‐gold, gold particles were seen to be linked via the IgG to the receptor, visualized as a round granule.
The characterization of microstructures in the atom probe
Authors: Hetherington M. G. , Miller M. K. 
Published: August 2011

SUMMARYThe atom probe can be used to characterize fine‐scale microstructures. The results presented in this and a subsequent paper attempt to develop a methodology for interpreting information concerning 3‐D structures from the sequence of ions detected in the atom probe. Probability theory (Bernoulli trials and Markov processes) is used to build appropriate mathematical descriptions of different types of microstructure.
Estimation of section thickness and quantification of iron standards with EELS
Authors: Door R. , Frösch D. , Martin R. 
Published: August 2011

SUMMARYThe possibilities of using electron energy‐loss spectroscopy (EELS) for quantification of elemental concentrations in ultrathin sections are examined. Dynabeads, which are polystyrene beads with a known iron content, are proposed as internal iron standards. The quantity of an element present depends on the thickness of the specimen. A prerequisite for estimation of absolute section thicknesses with EELS is the knowledge of the mean free path λ of electrons in the specimen. This factor is determined for the embedding resins Epon and Nanoplast by comparing EELS data with directly observed thicknesses in re‐embedded sections. Dynabeads were found to include iron in a homogeneous distribution and to be stable in the electron beam.
Improved preservation of phospholipid‐rich multilamellar bodies in conventionally embedded mammalian lung tissue—an electron spectroscopic study
Authors: Fehrenbach H. , Richter J. , Schnabel Ph. A. 
Published: August 2011

SUMMARYDifferent conventional methods of tissue processing were studied to determine the extent to which phospholipid‐rich multilamellar bodies of pulmonary alveolar epithelial type II cells of the pig were preserved. Prolonged treatment with half‐saturated aqueous uranyl acetate yielded excellent results on the stabilization of the multilamellar substructure, irrespective of whether glutaraldehyde‐paraformaldehyde or glutaraldehydetannic acid was used as a primary fixative. The lamellar periodicities were observed to be 5·5–6·1 nm.Differences in the phosphorus distribution among several types of lipid bodies of alveolar epithelial type II cells were studied by means of electron spectroscopic imaging and electron energy‐loss spectroscopy. Multilamellar bodies gave phosphorus signals which were significantly higher than those obtained from granular regions of composite bodies, whereas homogeneous bodies gave phosphorus signals which were even lower than those obtained from mitochondria, endoplasmic reticulum membranes or ribosomes.
EXELFS analysis of amorphous and crystalline silicon carbide
Authors: Martin J. M. , Mansot J. L. 
Published: August 2011

SUMMARYCore‐shell ionization edges from thin specimens of cubic silicon carbide (c‐SiC) are studied by means of electron energy‐loss spectroscopy (EELS) in a transmission electron microscope (TEM). Special attention is paid to the investigation of extended energy‐loss fine structures (EXELFS) to study local order effects around silicon atoms as a function of disorder in SiC.Two forms of SiC were used: c‐SiC for the crystalline form, and friction‐induced amorphous silicon carbide (a‐SiC) for the disordered form.EXELFS are generated by the elastic backscattering of outgoing electron waves by neighbouring atoms. Results show that both Si‐K and Si‐L edges contain structural information. EXELFS at the Si‐K edge can be used to calculate the radial distribution function (RDF) in the same way as extended X‐ray absorption fine structures (EXAFS). EXELFS at the Si‐L edge can be isolated by digital filtering. The high signal‐to‐noise ratio at the Si‐L edge permits the effect of larger interatomic distances to be observed, and thus the EXELFS technique is of promise for studying the effect of disorder in Si‐based ceramic materials with a high spatial resolution (nanometre scale).
Electron spectroscopic imaging and X‐ray microanalysis of acrylic fibres
Authors: Carboni V. , Lanzillotta F. , Carpanese G. F. , Mariani P. , Barbatelli G. , Sbarbati A. , Cinti S. 
Published: August 2011

SUMMARYAcrylic fibres are synthetic fibres produced by extruding viscous solutions of acrylonitrile co‐polymers. A spin finish is applied during the fibre‐forming process. In this work the structure of acrylic fibres has been correlated with spin finish distribution by a combined application of transmission electron microscopy (TEM), electron spectroscopic imaging (ESI) and X‐ray microanalysis (XRMA). Many irregularly shaped microcavities at the periphery of the fibres were detected by TEM. XRMA revealed that potassium and phosphorus are the elements most often found inside the spin finish material. ESI revealed that phosphorus is constantly present in all the microcavities. Therefore it seems likely that a preferential distribution of the spin finish is found inside the microcavities, just at the periphery of the fibre. This work is also an example of how both ESI and XRMA are powerful tools in morphological studies.
Stereology for pores in wheat bread: statistical analyses for the Boolean model by serial sections
Authors: Bindrich Ute , Stoyan Dietrich 
Published: August 2011

SUMMARYA simple approximative stereological method is proposed for obtaining the fundamental parameters of a three‐dimensional Boolean model. It uses serial sections for the determination of N+V, the specific convexity number. This procedure was applied to two different examinations of bread, which can be very well described by Boolean models.
A review of methods for the production and use of monoclonal antibodies to study zoosporic plant pathogens
Authors: Hardham A. R. , Gubler F. , Duniec J. , Elliott J. 
Published: August 2011

SUMMARYFor many species of Oomycetes, the infection of host plants is initiated by motile biflagellate zoospores. In recent years, studies of these zoospores have utilized monoclonal antibodies directed towards a variety of zoospore components. Although only two species of fungi, Phytophthora cinnamomi and Pythium aphanidermatum, have been used for immunization and initial screening, the reactions of these antibodies with over thirty species of fungi in the Peronosporales or Saprolegniales have now been determined. The present paper reviews the methods employed to produce the monoclonal antibodies and to use them to study the biology of the zoospores and the infection process.The lack of a cell wall means that fixation protocols for zoospores for immunization and screening must be chosen carefully so that cell‐surface or intracellular sites will be accessible to the antibodies. The inclusion of glutaraldehyde in the fixative helps keep the zoospore plasma membrane as intact as possible, and screening with cells fixed in the presence of glutaraldehyde selects for antibodies that bind to the surface of the zoospores. Five different patterns of labelling to the zoospore surface have been found. Other antibodies bind with three distinct patterns to the surface of zoospores and/or cysts.The use of formaldehyde alone in the fixative solution allows fragmentation of the plasma membrane and the exposure of intracellular components. In P. cinnamomi attempts to obtain antibodies directed against intracellular antigens were hampered by the presence of an immunologically dominant component that is stored in small vesicles in the zoospore cortex and secreted onto the surface of cysts. This problem was resolved by immunotolerizing mice neonatally before proceeding with immunization 2 months later. Antibodies directed towards a number of novel sites were obtained in this way.Monoclonal antibodies generated by these methods have been used to identify taxonomically specific spore components, to locate surface molecules that might be responsible for the induction of zoospore encystment and to characterize molecules involved in spore adhesion to potential hosts.
Freeze‐substitution of scleractinian coral for confocal scanning laser microscopy and X‐ray microanalysis
Authors: Marshall A. T. , Wright O. P. 
Published: August 2011

SUMMARYFreeze‐substitution in combination with confocal scanning laser microscopy provides a unique means by which the microstructure of undecalcified, and chemically untreated, polyps of scleractinian corals can be examined free of preparation artefacts. Mucocytes and intercellular spaces are particularly well preserved and the relationships of cell layers to each other and to the skeleton are undisturbed. Freeze‐substitution also permits X‐ray microanalysis of bulk samples and thin sections, a procedure which has hitherto been impossible to carry out on corals except on fixed tissue samples. Analyses indicated a high retention of Na+ and Cl− in spaces thought to be filled with fluid similar in composition to sea water. This increases confidence in freeze‐substitution as a means of retaining diffusible ions. Zooxanthellae contained metal/Ca ratios within the range of those previously reported for extracted zooxanthellae. It is shown that the mucocytes contain very high concentrations of S, K, Ca and Sr which are specifically localized in mucous granules. The concentrations differ between, and are characteristic of, different epithelial cell layers. Remarkably good correspondence was obtained between the two entirely different X‐ray analytical methods. This is the first time such a comparision between methods has been made. It is suggested that the uptake of transepithelially transported Ca2+ and Sr2+ by mucocytes may be a means of regulating the deposition of these ions in the skeleton.
Immunoelectron microscopic localization of elastic tissue components in archival tissue samples
Authors: Fanning Joseph C. , White Jacinta F. , Polewski Roman , Cleary Edward G. 
Published: August 2011

SUMMARYTissue samples that have been stored for many years, in different media and under a variety of conditions, have been examined by modern techniques of immunoelectron microscopy, using antibodies against elastic tissue components. A range of post‐embedding restorative procedures has been identified, which will allow reliable immunolocalization of antibodies against the elastic tissue component of such specimens. These methods have been applied successfully to autopsy‐derived material, fixed in buffered formaldehyde, to archival material stored frozen at −70 or −20·C, to specimens fixed for electron microscopy and stored for many years in buffer, and even to archival material from formaldehyde‐fixed, paraffin‐embedded blocks, reprocessed for electron microscopic examination.The successful restorative methods included pre‐treatment of the sections with 6***M guanidine hydrochloride, or 1M Tris/saline, each containing 100 ***mM dithiothreitol (a reducing agent) followed by alkylation with 220 mM iodoacetamide. The application of these techniques allowed reliable study of elastic tissue antibody distributions in archival tissues that could not be obtained again, as well as comparative studies with tissues processed many years previously.
Preliminary characterization of an intrinsic germanium detector on a 400‐keV microscope
Authors: White T. J. , Cousens D. R. , Auchterlonie G. J. 
Published: August 2011

SUMMARYAn evaluation was made of an intrinsic germanium X‐ray detector fitted to a 400‐keV electron microscope. Its characteristics as a function of accelerating voltage, pulse processor settings and count rate were investigated. Comparisons are drawn between intrinsic Ge and Si(Li) detectors at both an experimental and theoretical level. The software requirements for the successful collection of spectra are discussed.