bluuebunny/crossref_metadata_2025_split huggingface dataset:
Simplified nerve cell counting in the rat brainstem with the physical disector using a drawing‐microscope
Authors: GUNTINAS‐LICHIUS O. , MOCKENHAUPT J. , STENNERT E. , NEISS W. F. 
Published: August 2011

SummaryA simple modification of the physical disector is presented, which is used to count the number of neurons in the hypoglossal nucleus of the rat in a series of paraffin sections. One disector consists of two adjacent sections (6 μm thick) that have been Nissl‐stained with cresyl fast violet. In the first step of the procedure each of the two sections is investigated separately with a drawing‐microscope. The boundary of the hypoglossal nucleus and the position of neurons devoid of, or containing a part of, the cell nucleus in the plane of the section are marked on transparent paper. In the second step, these two drawings are placed one upon another, aligned and the number of cell profiles that show a cell nucleus in one but not in both drawings counted. This modification of the disector method for cell counting needs no specialized equipment, simply a light microscope with drawing apparatus, and can be combined with histochemical studies of other sections from the same tissue block.
Fractal characterization by frequency analysis. II. A new method
Authors: AGUILAR M. , ANGUIANO E. , PANCORBO M. 
Published: August 2011

SummaryA new frequency analysis method, fractal analysis by circular average (FACA), and an image replication procedure are proposed that together produce accurate measurements of the fractal dimension of surfaces and profiles, eliminating Fourier transform artefacts which arise from the lack of periodic continuity in real surfaces and profiles.
Evaluation of the precision of systematic sampling: Nugget effect and covariogram modelling
Authors: THIOULOUSE J. , ROYET J. P. , PLOYE H. , HOULLIER F. 
Published: August 2011

SummarySystematic sampling designs are widely used in stereology. When an estimator of the total amount, Q, of the sampled variable is evaluated by such a procedure, the coefficient of error can be predicted by applying Matheron's theory of regionalized variables. To evaluate the accuracy of the estimate of Q, it is necessary to study the behaviour of the regionalized variable and to model its covariogram. Histological data with a low short‐range variability and agronomic data with a pronounced nugget effect provided the biological material for extreme case studies. Results show that the short‐range variability, if present, cannot be detected when only small samples are available. An underestimation of the coefficient of error is then to be expected. We propose several models of the covariogram, which can be used to test for the presence of a nugget effect. If a nugget effect is present, these models will provide better estimates of the coefficient of error. If there is no nugget effect a simplified method can be used and will provide reliable estimates of the coefficient of error.
Edge detection in petrographic images
Authors: STARKEY J. , SAMANTARAY A. K. 
Published: August 2011

SummaryThe automatic detection of mineral grain boundaries in images obtained from a polarized‐light microscope requires special techniques. Observations in both plane‐ and cross‐polarized light may be necessary and the section must be rotated relative to the plane of polarization of the microscope to see all the grain boundaries. In computer‐based microscopy this can be accomplished by the sequential accumulation of individual images captured from one microscope field of view with different polarizer orientations.For real‐time implementation the sequential images are segmented individually by applying Canny's algorithm. A separable Gaussian mask is used for smoothing and a 3 times 3 convolution mask is used to generate 1‐pixel‐wide boundaries, which are located at the zero‐crossing of the second‐order derivative of the intensity gradient. The boundaries are extracted and accumulated in a composite image. The resulting composite image is a synoptic grain‐boundary image of the rock.
Development, quantitative performance and applications of a parallel electron energy‐loss spectrum imaging system
Authors: BOTTON G. , L'ESPÉRANCE G. 
Published: August 2011

SUMMARYThe development of a parallel electron energy‐loss spectrum imaging system is presented. The analytical performance of the imaging technique was investigated and the system applied to materials science problems. The system, which allows acquisition and storage of a parallel electron energy‐loss spectrum at each pixel of an image, was developed by interfacing a multichannel analyser and a microscope to a computer workstation. In the experimental conditions used for imaging, detection limits and quantification errors were large and varied as a function of spatial resolution and the range of chemical elements of interest in the image. Applications of this imaging technique in materials science showed that quantitative chemical information is provided by the system and that the use of relative thickness maps and detailed statistical analysis of the spectrum‐image allowed an unbiased interpretation of the images. As energy‐loss spectra are available after processing, spectroscopic information about the analysed material can be used to provide supplementary information.
Semiautomated methods for cancellous bone histomorphometry using a general‐purpose video image analysis system
Authors: HUFFER W. E. , RUEGG P. , ZHU J.‐M. , LEPOFF R. B. 
Published: August 2011

SUMMARYSemiautomated methods are used to measure elongated, curved and complex branching profiles and isolated perimeter segments in monochrome video images with a general‐purpose analysis system. These methods are used to make the major primary measurements of bone histomorphometry. Accuracy and reproducibility of the image acquisition, processing and measurement system is documented by measuring a semicircular standard of known dimensions. Semiautomated applications of the Ar/Le method for measuring areas and perimeters, and calculating lengths and widths of osteoid seams, lengths of mineralization labels and mineral apposition rate, wall width, indirect measurements of eroded, osteoclastic and osteoblastic perimeters without tracing, and measurement of mineralized or total cancellous bone area and perimeter gave values comparable to measurements of the same parameters by tracing or grid counting techniques with equal or better reproducibility and much greater efficiency. Intraindividual variation in measuring multiple bone biopsies was comparable to that reported with current standard methods. Major sources of variability for semiautomated methods were image magnification and selection of profile edges by thresholding, and sources of variability for manual methods are image magnification, numbers of orthogonal intercepts, tracing speed and accuracy of the algorithm used to measure traced pixels. Semiautomated methods are accurate, reproducible and rapid methods suitable for bone histomorphometry.
Estimation of the directional distribution of spatial fibre processes using stereology and confocal scanning laser microscopy
Authors: MATTFELDT T. , CLARKE A. , ARCHENHOLD G. 
Published: August 2011

SUMMARYFibrous structures like polymers, glass fibres, muscle fibres and capillaries are important components of materials and tissues. A spatial fibre process is the union of smoothly curved or linear one‐dimensional features of finite length, arranged in an unbounded three‐dimensional reference space according to some random mechanism. Design‐based stereology was combined with confocal scanning laser microscopy to study samples of fibre‐reinforced composites, which were considered as realizations of not necessarily isotropic fibre processes. The methods enable the unbiased estimation of the intensity and of the directional distribution of spatial fibre processes from arbitrarily directed pairs of registered parallel optical sections a known distance apart. The directions of fibres sampled by a frame of observation on the reference plane are estimated from the coordinates of the intersection points of the fibres with both optical planes using confocal scanning laser microscopy. The true directional distribution of the fibre process is estimated by weighting each measured direction by the reciprocal of its chance of being sampled, which can be inferred from the data. The concept of complete directional randomness for uniformly and independently distributed spatial directions is introduced. The cumulative distribution function of the angular distances between different directions and other exact relations are derived for complete randomness of vectorial and axial directions. A Monte Carlo method is constructed to test spatial fibre processes, whose fibres have negligibly small curvature, for complete directional randomness. Confocal scanning laser microscopy was used to study the angular distribution of glass fibres in a polymer composite which was subjected to increasing hydrostatic extrusion. The hypothesis of complete directional randomness had to be rejected for all samples with 1% probability of error. The directional distribution was of the bipolar type, with the principal axis directed parallel to the axis of extrusion. Progressive stretching of the material increased the degree of anisotropy of the glass fibres. Although presented for an application in polymer physics, the methods are general and may also be applied in biological investigations.
Tip artefacts in scanning force microscopy
Authors: SCHWARZ U. D. , HAEFKE H. , REIMANN P. , GÜNTHERODT H.‐J. 
Published: August 2011

SummarySince its invention in 1986, scanning force microscopy (SFM) has experienced great success as a characterization method for topography on small scales. In spite of the enormous potential of the method, it is limited by the quality of the tip used for probing the surface topography. Convolutions of non‐ideal tip shapes with the real topography and tip bending, flexing and jumping effects produce artefacts in the resulting images.A brief description of the preparation and characteristics of the most commonly used SFM tips is given. A variety of different artefacts originating from tip properties is presented and illustrated with selected scanning force micrographs. Methods to minimize tip artefacts in SFM images are described.
Contribution of energy‐filtering TEM to the detection of calcium: Application to mast cells
Authors: HOROYAN M. , SOLER M. , MARTIN J. M. , BENOLIEL A.‐M. , FRATERNO M. , PASSEREL M. , KATCHBURIAN E. , BONGRAND P. , FOA C. 
Published: August 2011

SummaryThe ultrastructural distribution and quantification of calcium in mast cells prepared by anhydrous processing was investigated by energy‐filtering transmission electron microscopy (EFTEM) using a Zeiss 902 electron microscope. Optimal conditions for calcium detection were determined using inorganic (calcium phosphate) and organic (calcium‐loaded chelex beads) standards with known amounts of calcium. Electron energy‐loss spectroscopy (EELS) revealed calcium at the L2,3 edge and also at the M2,3 edge for all specimens examined. Comparison with X‐ray microanalysis confirmed the results obtained with EELS. Electron spectroscopic imaging (ESI) was applied for mapping calcium both in standards and in cells and we showed that mast cell granules were the main site of calcium localization. Although, results have shown that a combination of analytical techniques is required to obtain reliable results.
Morphometric assessment of surface conformation as an estimate of roughness
Authors: SILAGE D. A. , BARAN G. R. 
Published: August 2011

SummaryOne of the standard methods for assessing the roughness of a material subjected to wear uses the surface arithmetic means and root‐mean‐square deviation. However, these parameters often do not provide a qualitative assessment of the difference in materials worn under the same conditions of load and elapsed time. The profile and surface roughness parameters are frequently inconsistent. Such measurements are also required to determine the wear characteristics of various materials under different conditions. A morphometric assessment of wear characteristics, based on the surface area fraction of localized deviations in the surface texture and stress fractures, is provided, and clearly indicates the observed difference.
Method for the study of the three‐dimensional orientation of the nuclei of myocardial cells in fetal human heart by means of confocal scanning laser microscopy
Authors: USSON Y. , PARAZZA F. , JOUK P.‐S. , MICHALOWICZ G. 
Published: August 2011

SummaryA series of three‐dimensional image analysis tools are used to measure the three‐dimensional orientation of nuclei of myocardial cells. Confocal scanning laser microscopy makes it possible to acquire series of sections up to 100 μm inside thick tissue sections. A mean orientation vector of unit length is calculated for each segmented nucleus. The global orientation statistics are obtained by calculating the vectorial sum of the nuclear unit vectors. The final orientation is expressed by a mean azimuth angle, an elevation angle and a measure of the angular homogeneity. The method is illustrated for two different regions of the myocardium (interventricular septum and papillary muscle) of a normal human fetal heart. This quantitative method will be used to assess and calibrate the information provided by polarized light microscopy.
Severe shrinkage of Microfil during tissue clearing with the Spalteholz technique
Authors: MØLLER J. , ROBERTSEN K. , HANSEN E. S. 
Published: August 2011

SummaryThe effect of two commonly used tissue clearing methods on the morphology of vascular casts formed by Microfil, a silicone rubber injection compound, was investigated. Microfil undergoes extreme shrinkage when the casted tissue is cleared by the alchohol‐methyl salicylate clearing technique. No shrinkage is observed when the alternative glycerin clearing method is used. The alcohol‐methyl salicylate clearing technique should be avoided in studies employing Microfil.
Influence of zero‐loss filtering on electron optical phase contrast
Authors: HIRSCH P. , REIMER L. 
Published: August 2011

SummaryComplex scattering amplitudes are used to calculate the phase contrast of colloidal gold particles. Comparison of measurements of the phase contrast intensity at the centre of the gold particle as a function of defocus for unfiltered and zero‐loss filtered images demonstrates the increase in phase contrast achieved by zero‐loss filtering even for a thick carbon substrate film. The granulation of amorphous germanium films is measured by the spatial rms (root mean square) values of image intensity in a defocus series.
Image‐EELS: Simultaneous recording of multiple electron energy‐loss spectra from series of electron spectroscopic images
Authors: KÖRTJE K.‐H. 
Published: August 2011

SummaryA new approach for element microanalysis with energy‐filtering transmission electron microscopy (EFTEM) is presented which was accomplished with the CEM 902 electron microscope (Zeiss, Germany). This method is called Image‐EELS, because it is a synthesis of electron energy‐loss spectroscopy (EELS) and electron spectroscopic imaging (ESI). Series of energy‐filtered images at increasing energy losses are recorded from one area with a TV camera. In a second step the intensity of selected regions in the image stack is measured with an image analysis system and plotted as a function of the energy loss. Thus many spectra from different objects can be calculated from one image series and compared with each other. The spatial resolution of EELS is considerably enhanced, the noise is decreased because many pixels from irregular objects are integrated, and the information from ESI can be analysed as a function of the energy loss.
Kinetics of intracellular Ca2+ concentration changes and cell contraction of electrically stimulated cardiomyocytes as analysed by automated digital‐imaging microscopy
Authors: SCHNEIDER H. , FALLERT M. , WACHSMUTH E. D. 
Published: August 2011

SummaryEnzymatically disaggregated, electrically stimulated cardiomyocytes from adult rats were examined by television‐mediated vital microscopy for intracellular Ca2+ concentration and contractile activity. Using an inverted microscope in the epifluorescence mode, the Ca2+ signal was imaged with a low‐light‐level CCD camera and traced by means of the intracellular concentration of the fluorescent complex of Ca2+ with its indicator Fluo‐3. Using the transmitted‐light mode, cardiomyocytes that were not loaded were imaged with a conventional CCD camera with automatic gain control and traced by length measurements. Optical images of at least 40 cardiomyocytes per batch of cells from one heart were recorded in up to 20 microscopic fields of observation on videotape within 20 min. They were consecutively analysed by a personal computer installed with an image analysis card at a time‐resolution of 20 ms, employing a discrete convolution operation, filtering and threshold setting for fluorescence measurements, and contour description and vectorial analysis for length measurements. Frames of fluorescent images were corrected for the halo effect caused by the increase in the Ca2+‐dependent fluorescence signal after electrical stimulation. The cell contraction had to be measured in the transmission mode without Fluo‐3 due to the inhibition caused by the intracellular Fluo‐3. The following coefficients of variation (V) were determined: Vfluorescence < 0·033 and Vtransmission < 0·003 for the precision of measurement, and Vfluorescence < 0·05 and Vtransmission < 0·04 for the reproducibility. The system was validated with isoprenaline and ouabain as agents to modify the Ca2+‐signal and the contraction. The response of cardiomyocytes of various rats to electrical stimulation, with respect to amplitude and its time point, had a V < 0·08 for both the Ca2+‐signal and the contraction.
Particle‐surface interaction in thin vitrified films for cryo‐electron microscopy
Authors: CYRKLAFF M. , ROOS N. , GROSS H. , DUBOCHET J. 
Published: August 2011

SummaryThe concentration of particles in thin vitrified films of suspensions is described as a function of various parameters such as the type of particles observed, the time the sample is left on the grid and the effect of different washing procedures. The thin films are prepared for cryo‐electron microscopy by the classical, single‐side blotting method or by blotting both sides of the grid simultaneously. The two‐side blotting method gives the most faithful representation of the bulk solution. The single‐side blotting method results in particles preferentially adsorbing to the non‐blotted surface. This has the advantage that the concentration of particles in the thin vitrified film is higher than in the original suspension. The energy involved in adhesion of particles to the surface seems to be generally small. In most cases, it does not cause significant deformation of the particles or of the surface of the film. However, there are cases, as for example with lipid vesicles, where the particles are broken as a result of adsorption.Since particles remain adsorbed to the air‐liquid interface, it is possible to wash or dialyse the solution directly on the grid with negligible loss of particles. This represents a very rapid and handy method for micro‐dialysis. A thin film is then formed by blotting the specimen and vitrified by rapid cooling.
Preparation of cultured airway smooth muscle for study of intracellular element concentrations by X‐ray microanalysis: Comparison of whole cells with cryosections
Authors: WARLEY A. , CRACKNELL K. P. B. , CAMMISH H. B. , TWORT C. H. C. , WARD J. P. T. , HIRST S. J. 
Published: August 2011

SummaryMethods for growing and preparing smooth muscle cells, isolated from rabbit trachealis, for X‐ray microanalysis studies are presented. The cells are grown on Pioloformcovered gold grids supported on Thermanox coverslips. This provides a growth‐compatible substrate which is easy to handle and is easily incorporated into routine cell culture studies. The cells are analysed as whole mounts after removal of growth medium by washing, followed by cryofixation and freeze drying. The effects of different washing media (0·3msucrose, 0·15mammonium acetate and distilled water) on cytoplasmic elemental content are discussed. A method for growing the cells as monolayers and mounting the cryofixed monolayers for cryosectioning is also given. Comparison of elemental concentrations in the cytoplasm of distilled‐water washed cells with those of the cytoplasm of cryosectioned cells obtained from the same animal showed good agreement between values obtained from the two preparative procedures. These methods are therefore easily applied to the study of changes in intracellular element concentrations which may be important in understanding the mechanisms of proliferation which lead to increased airway smooth muscle mass in persistent severe asthma.
The measurement of intracellular antigens and DNA by multiparametric flow cytometry
Authors: CAMPLEJOHN R. S. 
Published: August 2011

SummaryThe aim of this paper is to provide a strategy for measuring intracellular antigens combined with DNA content in cells or nuclei. A series of protocols are included which enable the majority of such antigens to be labelled and further information is provided for cases in which the standard methods prove to be inadequate. The basic principles of cell permeabilization/fixation are described, thus explaining how methods can be divided into three basic categories: (a) alcohol fixation with or without detergent pretreatment; (b) paraformaldehyde fixation followed by permeabilization with alcohol or detergents; (c) permeabilization of unfixed cells. The preparation of nuclear suspensions from paraffinembedded material is described and the possibilities and problems of staining such suspensions for nuclear antigens are discussed. Examples of results obtained with the detailed protocols are given for staining with antibodies directed against proliferating cell nuclear antigen (PCNA), Ki‐67 antigen and Ki‐Sl antigen. Details of published studies of a variety of intracellular antigens are given in two tables. The power of multiparametric flow cytometry in the study of cell proliferation, differentiation and response of cells to damage is underlined.
Direct imaging in a water layer of human chromosome fibres composed of nucleosomes and their higher‐order structures by laser‐plasma X‐ray contact microscopy
Authors: KINJO Y. , SHINOHARA K. , ITO A. , NAKANO H. , WATANABE M. , HORIIKE Y. , KIKUCHI Y. , RICHARDSON M. C. , TANAKA K. A. 
Published: August 2011

SummaryX‐ray contact microscopy with a 300‐ps‐duration laser‐plasma X‐ray source has been used to image hydrated human chromosomes. Clearly imaged are individual nucleosomes and their higher‐order particles (superbeads), elementary chromatin fibrils c. 30 nm in diameter and their higher‐order fibres of various sizes up to c. 120 nm in diameter. The results demonstrate that X‐ray microscopy is now capable of opening a new path of investigation into the detailed structures of hydrated chromosome fibres in their natural state.