bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modelling of three‐dimensional fluorescence images of muscle fibres: an application of the three‐dimensional optical transfer function
Authors: SHEPPARD C. J. R. , GU M. 
Published: August 2011

SUMMARYStriated muscle fibres can be modelled by a simple geometry, which has allowed three‐dimensional (3‐D) images in conventional and confocal microscopes to be calculated. This model is useful for comparing different imaging methods and represents a simple example of an application of the 3‐D optical transfer function (OTF) for the system. The rejection of out‐of‐focus blur is demonstrated, and the effects of fibre thickness and confocal pinhole size on image contrast are investigated. The effects of using a simple filter for image enhancement are studied, elucidating the characteristics of the OTF.
Fluorescence photobleaching recovery in the confocal scanning light microscope
Authors: BLONK J. C. G. , DON A. , Van AALST H. , BIRMINGHAM J. J. 
Published: August 2011

SUMMARYMeasurement of mobilities of species in liquid systems is of great importance for understanding a number of dynamic phenomena. A well known method for measuring mobilities driven by diffusion is fluorescence photobleaching recovery (FPR), also known as fluorescence recovery after photobleaching (FRAP). New FPR recovery equations for three‐dimensional (3‐D) apertured scanning using a Gaussian approximation for the axial beam profile have been successfully developed and found to provide a solid basis for extraction of the lateral diffusion coefficient from confocal scanning light microscopy (CSLM)‐FPR experimental data. The 2‐D diffusion coefficients of fluorescent species can be successfully measured by FPR in the CSLM, which has the great advantage that bleaching can be targeted at a well‐defined volume element in bulk samples. Two‐dimensional diffusion coefficients of 45‐nm latex spheres, of FITC molecules and of a 2·45‐nm protein‐FITC complex in water‐glycerol mixtures, measured by FPR in the CSLM, are in close agreement with those calculated from the size of the diffusing species and viscosity of the medium. Diffusion coefficients as high as 2 times 10−6 cm2/s can be measured.
Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index
Authors: HELL S. , REINER G. , CREMER C. , STELZER E. H. K. 
Published: August 2011

SUMMARYThe effect of refractive‐index mismatch, as encountered in the observation of biological specimens, on the image acquisition process in confocal fluorescence microscopy is investigated theoretically. The analysis takes the vectorial properties of light into account and is valid for high numerical apertures. Quantitative predictions on the decrease of resolution, intensity drop and shift of focus are given for practical situations. When observing with a numerical aperture of 1·3 (oil immersion) and an excitation wavelength of 514 nm the centre of the focus shifts 1·7 μm per 10 μm of axial displacement in an aqueous medium, thus yielding an image that is scaled by a factor of 1·2 in the axial direction. Furthermore, it can be expected that for a fluorescent plane 20 μm deep inside an aqueous medium the peak intensity is 40% less than for a plane which is 10 μm deep. In addition, the axial resolution is decreased by a factor of 1·4. The theory was experimentally verified for test samples with different refractive indices.
Utility of the horizontal slice for stereological characterization of lineal features
Authors: GOKHALE A. M. 
Published: August 2011

SUMMARYThe efficient stereological procedure for estimating the length density Lv of lineal microstructural features is further improved: the length density of long or short (or those of any arbitrary geometry) lineal features can now be estimated from the measurements performed on the projected images of vertical and horizontal slices of unknown and variable thickness. It is not necessary to identify uniquely the intersections of the lineal features with the parallel faces of the slices in the projected images and distinguish them from the images of the feature end points contained in the slices. The new technique does not involve any assumptions, and it is applicable to the lineal features of any arbitrary geometry and anisotropy. As a ‘bonus’, the same input measurements also yield an assumption‐free estimate of the total number of true lineal‐feature end points per unit volume Ev, and the average slice thickness.
Stereological estimation based on isotropic slices through fixed points
Authors: KIEU K. , JENSEN E. B. VEDEL 
Published: August 2011

SUMMARYStereological estimators of length and surface area based on measurements in an isotropic slice through a fixed point are described. Measurements of three‐dimensional angles are not needed. The estimators depend only on distance measurements. The variance of the length estimator is studied in some detail. Applications to particle analysis and second‐order stereology are discussed.
Contact distances and random free paths
Authors: SAXL I. 
Published: August 2011

SUMMARYThe first part of the present paper is aimed at a unified introduction of various random distances in a system of disjoint particles in ℝd or in its bounded subset. A general formula for the distribution function of distances is given and applied to several particular situations.In the second part, the detailed results for three selected types of random as well as regular structures are presented.
On the computation of the Minkowski dimension using morphological operations
Authors: KANMANI S. , RAO C. BABU , RAJ B. 
Published: August 2011

SUMMARYThe Minkowski dimension as a complexity measure, and its relation to the other dimensions in the case of self‐similar fractal objects, is discussed. An implementation of the Minkowski dimension, using morphological operations on a square grid, is presented. The effect of resolution, orientation and the symmetry of the structuring element used on the estimation of the Minkowski dimension is studied. The corrections required for free end points are presented.
Determination of microstructural parameters of random spatial surface fractals by measuring chord length distributions
Authors: HERMANN H. , OHSER J. 
Published: August 2011

SUMMARYAn experimental procedure for determining the micro‐structural parameters of random spatial surface fractals is proposed. The procedure is based on chord length distribution measurements of planar sections of the investigated material at different linear resolutions, and comparison of the experimental results with the corresponding parameters of a well‐defined structure model. The variability of the model allows microstructures with arbitrary volume fraction and fractal dimension to be considered. Furthermore, both discrete and continuous scaling behaviour have been studied.
Calibration of an electron back‐scattering pattern set‐up
Authors: LASSEN N. C. KRIEGER , BILDE‐SØRENSEN J. B. 
Published: August 2011

SummaryThe determination of lattice orientations from electron back‐scattering patterns (EBSPs) in a scanning electron microscope (SEM) requires accurate knowledge of the position of the pattern centre and the source point to screen distance. This paper outlines a new procedure that enables the determination of these parameters for any given set‐up of the EBSP/SEM system. The calibration procedure simply requires the positions and indices of at least four poles in a pattern obtained from an arbitrary specimen, and eliminates the need for standard specimens or special attachments to the EBSP/SEM system. The pattern centre is shown to be located with a precision of approximately 0·5° and the source point to screen distance can be determined with a relative precision of approximately 0·5%.
Observation of biological materials by X‐ray photoelectron‐conversion contact microscopy
Authors: WATANABE N. , MATSUMURA T. , INAGAKI Y. , KINOSHITA K. , SHIMANUKI Y. , FURUYA K. , TAGUCHI T. , TANIGUCHI M. , KIHARA H. 
Published: August 2011

SummaryDried biological specimens, such as fossil diatoms, collagen, nerve tissue and spicule of Trepang, were observed by X‐ray photoelectron‐conversion contact microscopy. A spatial resolution of 0·2 μm was attained. The fossil diatom image shows a clear difference below and above the carbon K‐absorption edge (4·46nm).
Observation of the loss of the hydroxyapatite sixfold symmetry in a human fetal tooth enamel crystal
Authors: BRES E. F. , STEUER P. , VOEGEL J.‐C. , FRANK R. M. , CUISINIER F. J. G. 
Published: August 2011

SummaryA deviation from the hydroxyapatite hexagonal symmetry of a human tooth enamel crystal observed by high‐resolution electron microscopy is reported. This symmetry deviation is characterized by: (1) ‘preferential’ planes that can be indexed as (100) with an intensity that differs from the (300) and the other {100} hexagonal equivalent planes; and (2) streaking of higher order reflections in the optical diffractogram of the image of the crystal. Computer simulations show that similar ‘preferential’ planes can also be observed at specific crystal tilt angles (and/or beam tilt and/or objective aperture misalignment) and at crystal thickness/microscope defocus values in images of hydroxyapatite crystals observed along the [0001] or [] zone axes. The streaking of higher order reflections in the optical diffractogram is related to a deformation of the crystal itself and does indeed show a symmetry deviation of the crystal under observation.
The adsorption staining technique applied to isolated premessenger ribonucleoprotein particles: A comparison with conventional techniques using electron microscope tomography
Authors: LÖNNROTH A. , SKOGLUND U. , TOKUYASU K. T. , DANEHOLT B. 
Published: August 2011

SummaryA specific type of premessenger RNP particle, Balbiani ring granules from the dipteran Chironomus tentans, was biochemically isolated and visualized in three dimensions with electron microscope tomography. The particles were prepared for electron microscopy in three different ways: positively stained, negatively stained and adsorption‐stained (embedded in polyvinyl alcohol, PVA, and concomitantly stained). The results were compared with those obtained for RNP particles studied in situ in ultrathin sections of plastic‐embedded cells. The positively stained particles were compacted and heavily deformed with little or no internal structure. The negatively stained and the adsorption‐stained particles were well preserved; the outer contours and the central cavities of the particles were outlined. The internal structure, i.e. the folded 7‐nm elementary fibre, could not be recognized in the negatively stained particles. In the adsorption‐stained particles, however, the fibre was discernable, although not quite as distinctly demarcated as in the plastic‐embedded samples. We conclude that embedding in PVA with concomitant staining with uranyl acetate is a rapid method to obtain both good preservation and staining of isolated RNP particles. The PVA‐embedded particles were also found to be sufficiently resistant to irradiation to permit a comprehensive tilt‐series to be taken for electron microscope tomography.ribonucleoprotein particles.
Methods for large data volumes from confocal scanning laser microscopy of lung
Authors: OLDMIXON E. H. , CARLSSON K. 
Published: August 2011

SummaryConfocal scanning laser microscopy makes it possible to obtain series of optical sections in precise registration. Certain studies of lung parenchyma, however, require both the fine resolution obtainable with high‐numerical‐aperture (NA) objectives and the extensive fields of view that usually would be achieved only with low‐NA objectives. This article presents a technique that resolves this conflict by using a sequence of operations: (i) to correct intensity variations on individual sections due to non‐uniform illumination/detection characteristics of the microscope; (ii) to correct intensity variations between successive sections in a series due to, for example, depth‐related absorption or step changes in detector sensitivity; (iii) to adjust adjacent, overlapping stacks of sections to a common intensity level; and (iv) to fuse a group of such overlapping stacks into a single series of larger sections. This resulting stack may contain, for example, a complete cross‐section of an alveolar ductal unit about 500 μm or more in diameter at about 1‐μm pixel resolution.
Orthogonal‐plane fluorescence optical sectioning: Three‐dimensional imaging of macroscopic biological specimens
Authors: VOIE A. H. , BURNS D. H. , SPELMAN F. A. 
Published: August 2011

SummaryAn imaging technique called orthogonal‐plane fluorescence optical sectioning (OPFOS) was developed to image the internal architecture of the cochlea. Expressions for the three‐dimensional point spread function and the axial and lateral resolution are derived. Methodologies for tissue preparation and for construction, alignment, calibration and characterization of an OPFOS apparatus are presented. The instrument described produced focused, high‐resolution images of optical sections of an intact, excised guineapig cochlea. The lateral and axial resolutions of the images were 10 and 26 μm, respectively, within a 1·5‐mm field of view.
Applications of medium‐voltage STEM for the 3‐D study of organelles within very thick sections
Authors: BEORCHIA A. , HELIOT L. , MENAGER M. , KAPLAN H. , PLOTON D. 
Published: August 2011

SummaryScanning transmission electron microscopy at 300 kV enables the visualization of nucleolar silver‐stained structures within thick sections (3–8 μm) of Epon‐embedded cells at high tilt angles (–50°; + 50°). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6‐μm‐thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e—/Å2/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4‐μm‐thick sections with a spot‐size of 5·6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 μm. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.
Spurious contributions from scattered electrons in thin‐film X‐ray microanalysis
Authors: LI Y. H. , CHEN Z. , LORETTO M. H. 
Published: August 2011

SummaryCarbon films supported on half copper grids have been used in a JEOL 4000FX to investigate the source of Cu X‐rays generated when the electron probe is focused on the carbon film. Both horizontal and high take‐off angle detectors were used to collect the spectra with accelerating voltages at 200, 300 and 400 kV. Energy‐dispersive X‐ray results obtained with the electron beam focused either on the carbon film or to avoid the film have been compared. By tilting the copper grid along its cut edge, the contribution of X‐rays generated by electrons scattered by the sample has been distinguished from the contribution of specimen‐generated Bremsstrahlung. These observations, together with measurements made both with and without an objective aperture, show that the main spurious contribution is from the electrons, which interact with the lower objective pole‐piece, generating both backscattered electrons and high‐energy X‐rays which subsequently interact with the sample. This result is further confirmed by the observations on epitaxial CdTe and CdHgTe layers on a single‐crystal GaAs substrate.
Cerium methods for light and electron microscopical histochemistry
Authors: VAN NOORDEN C. J. F. , FREDERIKS W. M. 
Published: August 2011

SummaryMethods based on the use of cerium to detect the activity of oxidases and phosphatases are rapidly expanding. Both classes of enzymes can be demonstrated with cerium at the electron and light microscopical level. Thein situdetection of H2O2production with cerium is another application that has great potential, particularly in experimental pathological research. The fine precipitate of the cerium‐containing final reaction product, cerium perhydroxide or cerium phosphate, enables a very precise localization. With such techniques, important advances have been made in cell biology, such as the discovery of new organelles, functional subcompartmentization of peroxisomes, tubular lysosomes and the elucidation of the function of extracellular ATPases.At the light microscopical level, the activity of enzymes can be quantifiedin situbecause the production of final reaction product in the cerium methods is proportional to enzyme activity in tissue sections or cells. Cerium precipitates have strong reflectance properties and this enables their use in confocal scanning laser microscopy. In the present review, the principles of cerium methods are outlined and applications in cell biology and pathology are discussed.
Imaging modes for bilateral confocal scanning microscopy
Authors: BRAKENHOFF G. J. , VISSCHER K. 
Published: August 2011

SummaryThe bilateral scanning approach to confocal microscopy is characterized by the direct generation of the image on a two‐dimensional (2‐D) detector. This detector can be a photographic plate, a CCD detector or the human eye, the human eye permitting direct visualization of the confocal image. Unlike Nipkow‐type systems, laser light sources can be used for excitation. A design called a carousel has been developed, in which the bilateral confocal scan capability can be added to an existing microscope so that rapid exchange and comparison between confocal and non‐confocal imaging conditions is possible. The design permits independent adjustment of confocal sectioning properties with lateral resolutions better than, or, in the worst case equivalent to, those available in conventional microscopy. The carousel can be considered as a stationary optical path in which certain imaging conditions, such as confocality, are defined and operate on part of the imaging field. The action of the bilateral scan mirror then extends this image condition over the whole field. A number of optical arrangements for the carousel are presented which realize various forms of confocal fluorescence and reflection imaging, with point, multiple point or slit confocal detection arrangements. Through the addition of active elements to the carousel direct stereoscopic, ratio, time‐resolved and other types of imaging can be achieved, with direct image formation on a CCD, eye or other 2‐D detectors without the need to modify the host microscope. Depending on the photon flux available, these imaging modes can run in real‐time or can use a cooled CCD at (very) low light level for image integration over an extended period.
An efficient method for calculating the least‐squares background fit in electron energy‐loss spectroscopy
Authors: BECKERS A. L. D. , GELSEMA E. S. , DE BRUIJN W. C. 
Published: August 2011

SummaryIn quantitative electron energy‐loss spectroscopy, an estimate has to be made of the unspecific spectral background to obtain the element‐specific contribution of an ionization edge. For energy losses above c. 100 eV, such a background estimate is commonly performed by calculating the least–squares fit of a power‐law model to a part of the pre‐edge region of the spectrum and by extrapolating this model to the region beyond the edge. Existing methods are cumbersome in terms of computation time and stability, or yield biased estimates. Computation efficiency becomes important when processing spectral‐image sequences. In this paper a new, computationally efficient numerical method is presented, which appears to be extremely robust in practice.
Endopolygalacturonase‐gold complex: Parameters influencing the labelling of pectic acid in the cell walls of flax hypocotyl
Authors: CABIN‐FLAMAN A. , JAUNEAU A. , MORVAN C. , DEMARTY M. 
Published: August 2011

SummaryAn enzyme/colloidal‐gold complex was prepared using a purified endopolygalacturonase (endoPGH, E.C. 3.2.1.15). Gold particles with a radius of 4·8 nm were prepared. To stabilize the gold particles, it was necessary to add a minimal amount of enzyme (approximately 0·5 μg) at a pH higher than its isoelectric pH. Under these conditions, the number of protein molecules adsorbed per gold particle was approximately one.The parameters influencing the diffusion of the endoPGH‐gold complex to its target molecules were screened and tested through quantitative approaches: (i) the labelling efficiency q was calculated according to the equation for the diffusion of gold markers from the bulk solutions to target molecules and (ii) the labelling density N/S was measured by counting gold particles on electron micrographs.The highest value of N/S was obtained by determining the optimal values for the parameters outlined in the equation for q. To obtain the highest value of N/S, it was not necessary to choose values for these parameters which gave high values for q.The specificity of the probe towards homogalacturonic acid blocks was established through controls. The localization of these blocks was illustrated throughout the walls of different kinds of flax seeding cells.
Second‐order stereology of benign and malignant alterations of the human mammary gland
Authors: MATTFELDT T. , FREY H. , ROSE C. 
Published: August 2011

SummaryThe purpose of the present study was a quantitative characterization of the three‐dimensional arrangement of the epithelial component of benign and malignant alterations of the female breast by combining stereology with stochastic geometry. Twenty cases of fibrous mastopathy and 20 cases of invasive ductal mammary cancer were studied at the light microscopic level. Segmentation of the epithelial tissue component was performed with an image analyser. From the resulting binary images, unbiased estimates of the covariance C(r) and the intensity Vv of the epithelial volume component were obtained automatically by computer. From these data, estimates of the correlation function k(r), of the pair correlation function g(r), of the radial distribution function RDF(r) and of the reduced second moment function K(r) of epithelial volume were determined. The estimates of C(r) and RDF(r) differed between groups, but these functions depend on spatial pattern and Vv. As carcinomas showed a significantly higher epithelial volume density Vv than mastopathies, estimation of C(r) and RDF(r) alone did not permit a safe distinction between possibly different types of spatial arrangement of epithelium in the benign and malignant lesions. Analysis of the estimates of k(r), g(r) and K(r), which are not influenced by Vv, showed definite interaction between epithelial volume elements, with clustering at short distances and repulsion at long distances. In both groups, the null hypothesis of purely random arrangement of epithelium had to be rejected. The clearest distinction between groups was obtained by estimation of g(r), which showed that short‐range, tubular pattern as well as long‐range, lobular architecture are better preserved in benign than in malignant lesions. The low interindividual scatter of k(r), g(r) and K(r) indicates a high biological significance of spatial pattern, which is presumably under strict genetic control. Potential uses of the method are: (i) the identification of biomathematical models which could contribute to a better understanding of the growth processes involved, (ii) conditional simulation of the underlying three‐dimensional structures by computer, and (iii) supporting the diagnosis of mammary lesions with borderline histopathological appearance.