bluuebunny/crossref_metadata_2025_split huggingface dataset:
Freeze‐substitution without aldehyde or osmium fixatives: ultrastructure and implications for immunocytochemistry
Authors: Monaghan Paul , Robertson David 
Published: August 2011

SUMMARYCryo‐fixation followed by freeze‐substitution without aldehyde or osmium fixation has been investigated as a method for preparing biological specimens with a view to minimizing antigenic alteration. Samples of both solid tissues (mouse small intestine and human kidney) and a human tumour cell line grown in vitro were rapidly frozen by impact (slammed) onto a copper block cooled with liquid nitrogen. They were freeze‐substituted at −80°C in methanol, and embedded at low temperature in Lowicryl K4M or HM20. Resin blocks were polymerized by ultraviolet light. Well‐preserved ultrastructure was observed in the outer 10–15 μm of all samples. Positive immunocytochemical localization of fixation‐resistant and fixation‐labile antigens was obtained on sections of human kidney and the human breast tumour cell line ZR‐75‐1 at both light and electron microscope levels.
Primary considerations for image enhancement in high‐pressure scanning electron microscopy
Authors: Farley A. N. , Shah J. S. 
Published: August 2011

SUMMARYThe mechanisms of electron beam scattering are examined to evaluate its effect on contrast and resolution in high‐pressure scanning electron microscopy (SEM) techniques reported in the literature, such as moist‐environment ambient‐temperature SEM (MEATSEM) or environmental SEM (ESEM). The elastic and inelastic scattering cross‐sections for nitrogen are calculated in the energy range 5–25 keV. The results for nitrogen are verified by measuring the ionization efficiency, and measurements are also made for water vapour. The effect of the scattered beam on the image contrast was assessed and checked experimentally for a step contrast function at 20 kV beam voltage. A considerable degree of beam scattering can be tolerated in high‐pressure SEM operation without a significant degradation in resolution. The image formation and detection techniques in high‐pressure SEM are considered in detail in the accompanying paper.
TEM analysis of near‐coincidence grain boundaries in silicon
Authors: Bary A. , Nouet G. , Delavignette P. 
Published: August 2011

SUMMARYHigh‐precision characterization of grain boundaries with a deviation from the ideal coincidence is presented. The analysis is based on the shift of Kikuchi lines at grain boundaries. The accuracy was controlled using Frank's relationship applied to intrinsic secondary dislocations which accommodate the orientation deviation. An application is presented to coincidences with Σ = 25 and 3 observed in polycrystalline silicon.
Reflection contrast microscopy performed on epi‐illumination microscope stands: Comparison of reflection contrast‐ and epi‐polarization microscopy
Authors: Velde I. Cornelese‐ten , Bonnet J. , Tanke H. J. , Ploem J. S. 
Published: August 2011

SUMMARYIn reflection contrast microscopy (RCM) special optics such as a quarter‐wave plate and a central stop are used, the latter being unique for Leitz microscope stands and contributing much to contrast enhancement of the image. In this paper we demonstrate that a cap or prism, linked by oil immersion to the glass slide, can be used instead of this central stop. Thus, microscope stands with incident illumination may be used for RCM.We systematically investigated RCM and epi‐polarization microscopy (EPM), both incident light microscopical methods, for visualization of immunogold‐silver‐stained cells, autoradiographic stained cells and immunoperoxidase‐diaminobenzidine stained chromosomes. RCM was found to be superior (1) in terms of the intensity of reflected light from reflecting objects, (2) in the visualization of unstained structures and background and (3) with respect to the spatial resolution that is achieved, especially in the tiny stained structures in the image.
The influence of electron beam direction and atomic weight on the ionization cross‐section of metals by high‐energy electrons
Authors: Chen Z. , Bozdogan R. , Loretto M. H. , Smallman R. E. , Long S. J. 
Published: August 2011

SUMMARYThe intensities of X‐rays generated from samples of Ni3Al, Ni15 at. % A1 and (TiNb)3Al were examined over a range of accelerating voltages as a function of diffracting conditions. It was found that the intensities of the X‐rays from the heavier element in the alloys may be enhanced, with respect to the signal from the lighter element, when the sample is orientated at symmetry. This occurs for Ni and Ti with respect to the Al intensity in Ni3Al and (TiNb)3Al, even when the systematic row which is excited does not contain a superlattice reflection, and in Nil5 at. % A1 which is effectively a disordered alloy. The situation in (TiNb)3Al is more complex, since although the Ti signal is enhanced with respect to the Al signal at symmetry for all reflections, the NbK intensity is not increased relative to the Ti signal but is increased relative to the Al intensity; the magnitude of this increase is about twice as great for reflections which lie in rows which contain superlattice reflections than for rows which do not. In contrast, the NbL intensity is decreased with respect to the TiK intensity when the crystal is set at symmetry for all types of systematic rows. These observations are interpreted in terms of the influence of atomic number on the sizes of electron shells and hence on their ionization cross‐sections. The results are briefly discussed in terms of the technique of alchemi, which is used to determine site occupancy in alloys, and in terms of the quantification of energy dispersive X‐ray data from thin samples.
Contact microscopy with a soft X‐ray laser
Authors: Skinner C. H. , DiCicco D. S. , Kim D. , Rosser R. J. , Suckewer S. , Gupta A. P. , Hirschberg J.G. 
Published: August 2011

SUMMARYA soft X‐ray laser of output energy 1–3 mJ at 18–2 nm was used to record high resolution images of biological specimens. The contact images were recorded on photoresist which was later viewed in a scanning electron microscope. A composite optical X‐ray laser microscope ‘COXRALM’ of novel design is also described.
Efficient estimation of cell volume and number using the nucleator and the disector
Authors: Møller A. , Strange P. , Gundersen H. J. G. 
Published: August 2011

SUMMARYThe nucleator allows the unbiased estimation of absolute structural quantities of suitably sampled, arbitrarily shaped structures from observations made from arbitrary points using isotropic probes. A number of time‐saving modifications using the nucleator and the consequences of the modifications are studied in terms of their bias and efficiency. Using rat neocortex as an example, a description is given of how to estimate mean neuronal volume and total neuron number efficiently from only a few pairs of plastic sections with a thickness of about 3 μm.
Sources of error in the estimation of Leydig cell numbers in control and atrophied mammalian testes
Authors: Mendis‐Handagama S. M. L. C. , Ewing L. L. 
Published: August 2011

SUMMARYThe effects of assuming (i) that testicular tissue shrinks equally regardless of species or treatment at fixing and processing, (ii) that all Leydig cells in a given testis have spherical nuclei of identical size, and (iii) that testicular volume (i.e. the reference volume) is constant regardless of species or treatment, on the estimation of Leydig cell numbers in mammalian testes were investigated. This was accomplished by comparing the results of stereological analyses of Leydig cell numerical density and Leydig cell number in control testes of hamster, guinea‐pig, and rat and in atrophied testes of hamster, and rat, obtained via the disector method which is unbiased with respect to the particle shape under study, and the Floderus equation which assumes that the particles under study are identical spheres. In control hamster, and also in guinea‐pig, the effects of the three assumptions on the estimates of Leydig cell number per testis were negligible, because in these two treatment groups, the total shrinkage of testis tissue at fixing and processing (ST%) was low, Leydig cell nuclear profiles were circular in section, and the average volume of a testis was close to unity (i.e. 1 cm3). By contrast, in hamsters, and rats with atrophied testes, these assumptions produced incorrect estimates in Leydig cell number per testis, because the ST% was high, the majority of Leydig cell nuclear profiles were pleomorphic, and the average volume of a testis was lower than control.In summary, this study documents that the assumptions of equal shrinkage in testis tissue at fixing and processing, a constant testicular reference volume, and spheroidal shape of Leydig cell nuclei may contribute significant errors in estimates of Leydig cell number in mammalian testes. The magnitude of the errors introduced by these assumptions depends upon the species and the experimental treatment.
Efficiency of grain‐density autoradiographs with 125I for the light microscope
Authors: Semmler M. , Junger E. 
Published: August 2011

SUMMARYEfficiencies of grain‐density autoradiographs with 125I for the light microscope were determined using Ilford L4 emulsion and Microdol X development. Calibrated layers of 125I‐bovine serum albumin and polymer sections of autoradiographic [125I] micro‐scales were used as reference sources. Efficiency was determined as a function of the radiation dose, source thickness and thickness of the absorber foils (e.g. non‐radioactive material between the source and the emulsion), respectively. By describing the dependencies of these factors quantitatively this study provides a means for planning autoradiographic experiments.
A simple procedure for demonstrating the overall morphology of fibroblasts in routine histological preparations of adult tissues, using silver impregnation
Authors: Novotny G. E. K. , Gommert‐Novotny E. 
Published: August 2011

SUMMARYA silver impregnation procedure for the total visualization of fibroblasts, with all their processes, is described. The method is applicable to routine formalin‐fixed paraffin sections, or to formalin‐fixed or native cryostat sections. The results are illustrated with examples from loose and dense connective tissue. Fibroblasts are visualized as cells with long, branching dendritic processes. The fibroblasts maintain contact with each other via these processes, thus forming a reticulum of cells, even in adult connective tissue.
Cytochemical observations on the centriolar adjunct of grasshopper spermatids in relation to its silver stainability
Authors: de la Torre J. , Gosálvez J. , Stockert J. C. 
Published: August 2011

SUMMARYThe silver stainability of the centriolar adjunct from grasshopper spermatids was analysed after blocking ‐SH groups with heavy cations (lead, mercury), or following the conversion of ‐SH and ‐S‐S groups into sulphonates (permanganate oxidation). These treatments produced the loss of silver affinity in the centriolar adjunct. Likewise, the selective fluorescence of this structure by mercuridibromfluorescein was abolished after these treatments. Our results indicate that ‐SH groups are involved in staining reactions of the centriolar adjunct by silver and mercuridibromfluorescein.
Fluorescence quenching; a practical problem in flow cytometry
Authors: Chapple M. R. , Johnson G. D. , Davidson R. S. 
Published: August 2011

SUMMARYDual fluorescence analysis with a single‐laser fluorescence‐activated cell sorter is dependent on the use of two fluorochromes with similar excitation wavelengths but different emission wavelengths. The dye pair fluorescein and R‐phycoerythrin (RPE) have been widely employed for this purpose and interaction between the two dyes has not been observed. Here evidence is presented to show that at high concentrations RPE can completely quench the fluorescein signal in dual fluorescence analysis of human tonsil lymphocytes labelled with pairs of monoclonal antibodies. Reduction in the fluorescein signal correlated with the intensity of red (RPE) staining. This phenomenon can seriously compromise interpretation of dual immunofluorescence carried out on a single laser instrument and can best be avoided by careful analysis of single colour controls.
Flow cytometric evaluation of DNA digestion with micrococcal nuclease on isolated HeLa nuclei
Authors: Prosperi E. , Giangaré M. C. , Supino R. , Bottiroli G. 
Published: August 2011

SUMMARYFlow cytometric assessment of DNA digestion with micrococcal nuclease has been performed on isolated HeLa nuclei by determining the relative reduction in stainability with the DNA‐specific fluorochrome, propidium iodide. At the nuclease concentrations used, DNA histograms of digested nuclei showed the typical bimodal pattern, when the enzymatic reaction was performed in a medium maintaining chromatin in its native (i.e. condensed) or partially decondensed form. In contrast, when nuclei were digested in a buffer lacking both the mono‐ and divalent cations K+ and Mg2+, an extensive decrease in fluorescence intensity, with loss of the histogram shape, was observed. In nuclei with native chromatin, DNA stainability decreased as a function of time and enzyme concentration, to reach a lower limit of about 46%, as compared with undigested control samples. Removal of the histone H1 induced a significant increase (approximately by a factor of 2) in the extent of digestion, although only in nuclei with partially decondensed chromatin. These results suggest that the sensitivity of DNA to digestion with micrococcal nuclease can be quantitatively monitored with flow cytometry when appropriate reaction conditions are chosen.
Flow cytometric analysis of the functional ability of neutrophils from patients with autoimmune neutropenia
Authors: Macey M. G. , Sangster J. , Veys P. A. , Newland A. C. 
Published: August 2011

SUMMARYFlow cytometry has been used to evaluate the functional ability of neutrophils and the expression of IgG Fc receptors (FcRII and FcRIII) in autoimmune neutropenia. Quantification of the neutrophil oxidative burst was made by assaying the production of 2′7′‐dichlorofluorescein (DCF) from non‐fluorescent 2′7′‐dichlorofluorescin trapped within the cell, by flow cytometric analysis of cellular fluorescence. In the present study the DCF assay was used to examine the response of neutrophils to stimulation by opsonized and non‐opsonized Staphylococcus aureus. In addition, the rate of uptake of S. aureus labelled with the red nuclear dye propidium iodide was determined.The presence of surface‐bound immunoglobulin, which may affect the phagocytic capacity of the neutrophil, was also measured. No correlation between the neutrophil count and level of membrane‐bound IgG or the rate of bacterial uptake was found. The studies were performed on twenty patients with autoimmune neutropenia, twelve with other autoimmune disorders and fourteen normal controls. The rate of uptake of bacteria was considered in relation to the expression of FcRII and FcRIII. Good correlation was found with the level of expression of FcRII, the major receptor for neutrophil activation, and the rate of uptake of bacteria (r = 0.64).
x‐y‐Recording in transmission electron microscopy. A versatile and inexpensive interface to personal computers with application to stereology
Authors: Rickmann M. , Siklós L. , Joó F. , Wolff J. R. 
Published: August 2011

SUMMARYAn interface for IBM XT/AT‐compatible computers is described which has been designed to read the actual specimen stage position of electron microscopes. The complete system consists of (i) optical incremental encoders attached to the x‐ and y‐stage drivers of the microscope, (ii) two keypads for operator input, (iii) an interface card fitted to the bus of the personal computer, (iv) a standard configuration IBM XT (or compatible) personal computer optionally equipped with a (v) HP Graphic Language controllable colour plotter.The small size of the encoders and their connection to the stage drivers by simple ribbed belts allows an easy adaptation of the system to most electron microscopes. Operation of the interface card itself is supported by any high‐level language available for personal computers. By the modular concept of these languages, the system can be customized to various applications, and no computer expertise is needed for actual operation.The present configuration offers an inexpensive attachment, which covers a wide range of applications from a simple notebook to high‐resolution (200‐nm) mapping of tissue. Since section coordinates can be processed in real‐time, stereological estimations can be derived directly “on microscope”. This is exemplified by an application in which particle numbers were determined by the disector method.
Estimation of surface area and length with the orientator
Authors: Mattfeldt Torsten , Mall Gerhard , Gharehbaghi Hassan , Möller Peter 
Published: August 2011

SUMMARYThe orientator is a new technique for the estimation of length and surface density and other stereological parameters using isotropic sections. It is an unbiased, design‐based approach to the quantitative study of anisotropic structures such as muscle, myocardium, bone and cartilage. A simple method for the practical generation of such isotropic planes in biological specimens is described. No special technical equipment is necessary. Knowledge of an axis of anisotropy can be exploited to optimize the efficiency. To randomize directions in space, points are selected with uniform probability in a square using various combinations of simple random, stratified random, and systematic random sampling. The point patterns thus produced are mapped onto the surface of a hemisphere. The mapped points define directions of sectional planes in space. The mapping algorithm ensures that these planes arc isotropic, hence unbiased estimates of surface and length density can be obtained via the classical stereological formulae. Various implementations of the orientator are outlined: the prototype version, the orientator‐gencrated ortrip, two systematic versions, and the smooth version. Orientator sections can be generated without difficulty in large specimens; we investigated human skeletal muscle, myocardium, placenta, and gut tissue. Slight practical modifications extend the applicability of the method to smaller organs like rat hearts. At the ultrastructural level, a correction procedure for the loss of anisotropic mitochondrial membranes due to oblique orientation relative to the electron beam is suggested. Other potential applications of the orientator in anisotropic structures include the estimation of individual particle surface area with isotropic nucleators, the determination of the connectivity of branching networks with isotropic disectors, and generation of isotropic sections for second‐order stereology (three‐dimensional pattern analysis).
A direct method for fast three‐dimensional serial reconstruction
Authors: Odgaard Anders , Andersen Kurt , Melsen Flemming , Gundersen Hans Jørgen G. 
Published: August 2011

SUMMARYA method for accurate three‐dimensional reconstruction of openly connected porous structures is described. The method is based on embedding of a specimen in a contrast coloured epoxy resin and serial sectioning in a standard hard tissue microtome. A PC‐based image processing system is used for direct digitization of the cut surface, and by thresholding two‐phase images are obtained. The process is fully automated, and about 170 sections can be produced, digitized, dichotomized, and stored per hour. As an example of its applications, the method is used on trabecular bone, which is an anisotropic porous structure.
Compton‐scattering X‐ray artefacts observed in an STEM with high take‐off angle EDS detector
Authors: Wilson A. R. , Lambrianidis L. T. 
Published: August 2011

SUMMARYA new spectral artefact has been identified in a high take‐off angle energy‐dispersive X‐ray detector used in a JEOL 2000FX STEM. This artefact has been verified to be due to Compton scattering of Mo K X‐rays which are produced in the standard JEOL condenser lens Mo apertures. These Mo X‐rays are Compton scattered by a thick carbon insert in the upper objective lens pole‐piece of the microscope and are detected as two broad X‐ray peaks centred on 16.44 and 18.3 keV. The presence of this artefact severely limited attempts to quantify small concentrations of Nb in a nickel alloy. Possible ways to avoid or minimize the artefact include the use of large apertures, apertures constructed from different materials or thick apertures.
Submicrometre resolution measurement of the composition profile through E‐glass fibres subjected to stress‐corrosion
Authors: Caddock B. D. , Devenish R. W. , Evans K. E. 
Published: August 2011

SUMMARYA replication method has been developed to obtain high‐resolution composition profiles through E‐glass fibres subjected to corrosion by aqueous acids. STEM/EDX analysis of fragments of glass fibres has provided compositional information at a spatial resolution better than 0.1 μm. It has been shown that the core‐sheath profile observed in long‐time exposures of glass fibres to aqueous acids is indeed an extremely abrupt change, with calcium and aluminium ions fully leached from the sheath region. A surface region less than 0.2 μm thick of severe demineralization has also been detected in fibres undergoing a combination of aqueous acid corrosion and tensile load, which fail at short times and in which no core‐sheath morphology is detectable by SEM examination.
On the empirical variance of a fractionator estimate
Authors: Cruz‐Orive Luis M. 
Published: August 2011

SUMMARYThe fractionator consists of several sampling stages with systematic sampling at each stage; data are collected only at the last stage. Therefore, predicting the error variance of a fractionator estimator is a non‐trivial problem, because the observations are correlated in a complicated, unknown way. Gundersen proposed to split the material sampled at the first stage into two subsets, and to compute the variance of the pooled estimate empirically using the corresponding pair of observations made in these two subsets. The idea is very effective, but the estimator thus proposed needed some corrections. The purpose of this paper is to present an improved estimator of the coefficient of error of a fractionator estimator using Gundersen's design.
Characterization of low‐light‐level cameras for digitized video microscopy
Authors: Tsay Tsong‐Tseh , Inman Richard , Wray Barnaby , Herman Brian , Jacobson Ken 
Published: August 2011

SUMMARYThe use of low‐light‐level video cameras and solid‐state image detectors in conjunction with image digitizers for optical microscopy is increasing dramatically as more people learn about such systems, and as they become more powerful, less expensive, and easier to use. However, there is currently little information available allowing comparison between cameras, or for determining camera suitability for a given experiment. In this paper, we describe a series of tests designed to characterize the performance of low‐light‐level cameras. The results of these tests should assist in the selection of appropriate cameras for given video microscopy applications.
Experimental ionization cross‐sections of phosphorus and calcium by electron spectroscopic imaging
Authors: Heng Y. M. , Simon G. T. , Boublik M. , Ottensmeyer F. P. 
Published: August 2011

SUMMARYThe absolute partial electron scattering cross‐section for the phosphorus L2,3‐shell ionization was measured by electron spectroscopic imaging using poliovirus as a primary standard. The equivalent calcium cross‐section was obtained in relation to phosphorus using the stoichiometric ratio for these two elements in hydroxyapatite, Ca10(PO4)6(OH)2. At 80kcV, the partial cross‐section of phosphorus was 2.26 times 10−20 and 2.68 × 10−20cm2/atom for poliovirus and hydroxyapatite, respectively, at 150eV loss for a 15‐cV energy window and an acceptance angle of 15 mrad. Under the same conditions the calcium cross‐section was 0.49 × 10−20 cm2/atom at 360 eV loss. The experimental values are slightly higher than the theoretical cross‐sections calculated either by hydrogenic or Hartree—Slater approaches.