bluuebunny/crossref_metadata_2025 huggingface dataset:
Subpixel position measurement using 1D, 2D and 3D centroid algorithms with emphasis on applications in confocal microscopy
Authors: Patwardhan A.
Published: November 2003

The accuracy with which centroid algorithms in 1D, 2D and 3D can estimate an object's position has been investigated. Three factors that can influence the method's accuracy have been investigated: systematic error of the algorithm, influence of photon noise and the influence of perturbations such as scanning nonlinearity. The variation of the accuracy with parameters that are relevant for confocal microscopy, such as object diameter and photon noise/pixel, has been considered. Theory and simulations presented show that the variation of the accuracy with respect to such parameters can differ drastically between the 1D, 2D and 3D cases. Experiments performed using microspheres show that the magnitudes of the three types of error can be approximately the same under normal operating conditions and that it is therefore necessary to take all three into account when assessing the total error.
Calcium in secretory vesicles of neurohypophysial nerve endings: quantitative comparison by X‐ray microanalysis of cryosectioned and freeze‐substituted specimens
Authors: THIRION S. , TROADEC J. D. , PAGNOTTA S. , ANDREWS S. B. , LEAPMAN R. D. , NICAISE G.
Published: November 2003

The calcium content of individual secretory vesicles in rat neurohypophysial nerve endings was measured by quantitative electron probe X‐ray microanalysis. Directly frozen control and potassium‐depolarized isolated endings were analysed using two presumably equivalent preparative techniques: (1) freeze‐substitution in presence of oxalic acid followed by sectioning of resin‐embedded pellets; or (2) direct cryosectioning of the frozen pellets followed by freeze‐drying in the column of the microscope. In the pellets of stimulated endings, both approaches revealed an increase in the calcium content of neurosecretory vesicles. This increase was statistically more significant in the specimens prepared by cryosectioning, probably because in this case the contribution of ‘dead’ nerve endings could be eliminated on the basis of excessive cytoplasmic sodium and chloride. The results demonstrate that an increase in cytosolic calcium can lead to an increase in intravesicular calcium, and that when this occurs, it occurs within a subpopulation of vesicles in a given nerve ending. In addition, measured intravesicular calcium was dispersed over a wide range of concentrations, as predicted by the hypothesis of intravesicular calcium priming. When the vesicular calcium content was averaged per nerve ending, a relatively wide distribution of concentrations was again observed, indicating that some nerve endings respond more strongly to the stimulation than others.
Imaging ‘intact’ myofibrils with a near‐field scanning optical microscope
Authors: SEIBEL E. J. , POLLACK G. H.
Published: November 2003

Fluorescently labelled myofibrils were imaged in physiological salt solution by near‐field scanning optical microscopy and shear‐force microscopy. These myofibrils were imaged in vitro, naturally adhering to glass while retaining their ability to contract. The Z‐line protein structure of the myofibrils was antibody labelled and easily identified in the near‐field fluorescence images. The distinctive protein banding structure of the myofibril was also seen clearly in the shear‐force images without any labelling requirement. With the microscope in the transmission mode, resolution of the fluorescence images was degraded significantly by excessive specimen thickness (>1 μm), whereas the shear‐force images were less affected by specimen thickness and more affected by poor adherence to the substrate. Although the exact mechanism generating contrast in the shear‐force images is still unknown, shear‐force imaging appears to be a promising new imaging modality.
Stereology of cubic particles: various estimators for the size distribution
Authors: OHSER J. , NIPPE M.
Published: November 2003

This paper deals with stereological problems for spatial systems of homothetic particles. In contrast to the traditional technique of estimating the size distribution of the particles from the sizes of section profiles we introduce a new method exploiting size and shape of the observed profiles. The shapes of the section profiles contain additional information about the sizes of the particles. This leads to improved stereological estimation of size distribution. As an example the case is studied when the particles form cubes with random edge length. The method is applied to a two‐phase WC–Co cermet structure with cubic particles.
Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen‐hydrated state by field‐emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies
Authors: Chandra S. , Morrison G. H.
Published: November 2003

We have employed field‐emission secondary electron microscopy (FESEM) for morphological evaluation of freeze‐fractured frozen‐hydrated renal epithelial LLC‐PK1 cells prepared with our simple cryogenic sandwich‐fracture method that does not require any high‐vacuum freeze‐fracture instrumentation (Chandra et al. (1986) J. Microsc.144, 15–37). The cells fractured on the substrate side of the sandwich were matched one‐to‐one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen‐hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane‐halves on the substrate; and (iii) cross‐fracture that passes randomly through the cells. The ectoplasmic face (E‐face) and protoplasmic face (P‐face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze‐dried cells fractured at the apical membrane revealed well‐preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K+, Na+ and Ca+). Structurally damaged cells revealed lower K+ and higher Na+ and Ca+ contents than in well‐preserved cells. Frozen‐freeze‐dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near‐field scanning optical microscopy.
Analysis of cardiac function by MRI and stereology
Authors: ROBERTS N. , CRUZ‐ORIVE L. M. , BOURNE M. , HERFKENS R. J. , KARWOSKI R. A. , WHITEHOUSE G. H.
Published: November 2003

Design‐based stereology and phase contrast magnetic resonance imaging (MRI) were combined to monitor changes in the volume of the four chambers of the human heart during the cardiac cycle. The data set consisted of 18 adjacent slices (or ‘scanning levels’) of 0.5 cm thickness, perpendicular to the long axis of the body, and encompassing the whole heart of a healthy volunteer. At each scanning level, a cardiac gated MR image was obtained at each of 16 equally spaced time frames within the cardiac cycle. Given stationarity with respect to time, absence of image artefacts and appropriate definition of chamber boundaries, for each time frame unbiased estimates of total blood volume in the relevant heart chambers were efficiently obtained using the Cavalieri method and point counting. Combined with a proper MRI acquisition, modern stereological methods constitute an efficient and reliable tool to quantify cardiac function noninvasively.
The optical rotator
Authors: Tandrup T. , Gundersen H. J. G. , Vedel Jensen E. B.
Published: November 2003

The optical rotator is an unbiased, local stereological principle for estimation of cell volume and cell surface area in thick, transparent slabs. The underlying principle was first described in 1993 by Kiêu &38; Jensen ( J. Microsc170, 45–51) who also derived an estimator of length. In this study we further discuss the methods derived from this principle and present two new local volume estimators.

The optical rotator benefits from information obtained in all three dimensions in thick sections but avoids over‐/underprojection problems at the extremes of the cell. Using computer‐assisted microscopes the extra measurements demand minimal extra effort and make this estimator even more efficient when it comes to estimation of individual cell size than many of the previous local estimators. We demonstrate the principle of the optical rotator in an example (the cells in the dorsal root ganglion of the rat), evaluate its efficiency and compare it with other unbiased, local stereological principles available for estimation of cell volume and surface area.
Direct‐view microscopy: experimental investigation of the dependence of the optical sectioning characteristics on pinhole‐array configuration
Authors: FEWER D. T. , HEWLETT S. J. , McCABE E. M. , HEGARTY J.
Published: November 2003

We present a comparison between theoretical and experimental results for the axial response to a plane mirror specimen of the direct‐view microscope employing multiple‐pinhole arrays. The effects of pinhole size, pinhole spacing and array geometry are investigated in detail with a view to (i) achieving good optical sectioning characteristics and (ii) maximizing the amount of light available for imaging. The implications of our results for practical systems as regards pinhole‐array design and fabrication are also discussed.
Three‐dimensional reconstruction of senile plaques in Alzheimer's disease
Authors: RICKERT C. H. , FILLER T. J. , GULLOTTA F.
Published: November 2003

So far, the three‐dimensional approach to senile plaques, one of the principal histopathological hallmarks in Alzheimer's disease besides neurofibrillary tangles, has been scarce. Two main problems in three‐dimensional reconstruction of histological specimens are the horizontal distortion during the preparation of serial thin tissue‐slides and the need for consecutive vertical readjustment. This is greatly facilitated by the reflection contrast microscope (Leica, Germany) which is a light microscopical instrument causing interference patterns and reflections along interfaces by means of circularly polarized epi‐illumination. Using this technique, one can obtain distinct optical sections of a depth of 1.5 μm within specimens up to 30 μm in thickness, thus preserving the integrity of the observed object and rendering a manual alignment superfluous. We applied the reflection contrast microscope (RCM) on thick tissue‐slides of the cerebral cortex of a patient suffering from Alzheimer's disease which had been dyed according to Campbell. This is a silver‐based staining method detecting βA4‐amyloid, the main component of senile plaques. Under the RCM, these silver‐stained extracellular amyloid deposits cause reflections which allow the assessment of their three‐dimensional distribution by focusing through the specimen. The optical sections obtained in this way were digitized, and the identified senile plaques reconstructed by the grey‐scale image analysis system VIDAS 2.5 (Zeiss/Kontron, Germany).
X‐ray contact microscopy using an excimer laser plasma source with different target materials and laser pulse durations
Authors: ALBERTANO P. , REALE L. , PALLADINO L. , REALE A. , COTTON R. , BOLLANTI S. , DI LAZZARO P. , FLORA F. , LISI N. , NOTTOLA A. , VIGLI PAPADAKI K. , LETARDI T. , BANAI D. , CONTI A. , MORET M. , GRILLI A.
Published: November 2003

Soft X‐ray contact microscopy (SXCM), using a pulsed X‐ray source, offers the possibility of imaging the ultrastructure of living biological systems at sub‐100 nm resolution. We have developed a table‐top pulsed plasma X‐ray source for this application, generated by a large‐volume XeCl laser, achieving a good conversion efficiency to ‘water‐window’ X‐rays (hν≈280–530 eV).

Optimum plasma conditions for SXCM are discussed, including the effect of pulse duration, target material and resist development time on image resolution. Soft X‐ray contact images of Chlamydomonas dysosmos (unicellular alga) and of the cyanobacterium Leptolyngbya are shown.
The use of single image random dot stereograms for presenting 3D microscopic confocal images
Authors: Hallett M. B. , Pettit E. J.
Published: November 2003

Presenting 3D images produced by confocal optical slicing techniques in a static and easily publishable form can be difficult. Here, we demonstrate the presentation of the data as a single image random dot stereogram (SIRD), which can be viewed as a 3D object by ‘defocusing’ the eyes. The production of the SIRD employs three steps: (i) acquisition of the optical slices using confocal techniques, (ii) allocation of a suitable grey level to the object in each slice to provide depth‐encoding information for the final image and (iii) calculation of the SIRD from the composite depth‐encoded image. The technique is demonstrated with a limited number of optical slices through an acridine‐orange‐stained neutrophil (diameter =10 μm), in order to show the relative positions of the nuclear lobes in the cell.
Freezing of aqueous specimens: an X‐ray diffraction study
Authors: Lepault J. , Bigot D. , Studer D. , Erk I.
Published: November 2003

The effects on water of two cooling methods, immersion in a liquid cryogen and high‐pressure freezing, were studied by X‐ray cryodiffraction on different sucrose solutions. The nature of the ice formed by each method depends on both the sucrose concentration and the specimen thickness. In order to compare the two methods, we mainly studied specimens having a thickness of 0.2 mm. Under these conditions, freezing by immersion gives rise to hexagonal (IH), cubic (IC) and amorphous (IV) ices when the sucrose concentration (weight/weight) has a value within the range 0–30%, 30–60%, 60% and higher, respectively. The temperature of the phase transitions IV–IC, IC–IH depends on the sucrose concentration. High‐pressure freezing gives rise to two specific forms of ice: an amorphous and a crystalline ice (ice III). Ice III is observed when pure water samples are high‐pressure frozen provided that the sample temperature does not rise above −150 °C. Above this temperature, ice III transforms into hexagonal ice. Amorphous ice is formed when the sucrose concentration is higher than 20%. The amorphous ice formed under high pressure has a similar, but not identical, X‐ray diffraction pattern to that of amorphous ice formed at atmospheric pressure. While the X‐ray diffraction pattern of amorphous ice formed at atmospheric pressure (IV) shows a broad ring at a position corresponding to 0.37 nm, that of high‐pressure amorphous ice (IVHP) shows a broader ring, located at 0.35 nm. IVHP presents a phase transition (IVHP–IV) at temperatures that depend on the sucrose concentration. We also observed that some precautions have to be taken in order to minimize the alcohol contamination of high‐pressure frozen samples. The ice‐phase diagram presented in this paper should be taken into account in all methods dedicated to the structural study of frozen biological specimens.
Estimation of the connectivity of a synthetic porous medium
Authors: Roberts N. , Reed M. , Nesbitt G.
Published: November 2003

Highly connected continuous phases in a porous medium can usefully be described in terms of their connectivity. Appropriate descriptions of connectivity are the connectivity number density or the related quantity Euler number density. Broadly speaking, Euler number density is equal to the number of discrete objects which comprise the phase plus the number of enclosed cavities within those objects less the number of tunnels through the objects, per unit volume. The conneulor is a convenient stereological method for estimating Euler number density. The relevant probes (i.e. disectors) from which the estimate is obtained are randomly positioned and arbitrary orientated pairs of closely spaced parallel planes.

We have applied the conneulor method to estimate the Euler number density of the pore space in a cell of sintered silica imaged using a magnetic resonance (MR) microscope. By flooding the cell simultaneously with a mixture of oil and water prior to imaging we were able to obtain a series of 16 contiguous perfectly registered serial MR sections depicting the pore space. The Euler number density of the porous medium estimated from four systematic pairs of consecutive MR images is −5.6 per mm3 with a coefficient of error (CE) of 10–15%. The Euler number density was lower near the walls of the silica cell than the centre, which is consistent with the method of formation of the cell.

In contrast to the majority of stereological methods, and in common with the physical disector, the conneulor method requires that deductions are made regarding the changes in the morphology of the structure of interest that occur between the two parallel image planes comprising the sampling probe. We therefore compared estimates of the Euler number density of the pore space obtained by two independent observers. We conclude that careful thought and discussion between observers is required before a definitive set of counts is made. Once mastered the conneulor counting rules are straightforward to apply.
Far‐field fluorescence microscopy with three‐dimensional resolution in the 100‐nm range
Authors: Hell S. W. , Schrader M. , Van Der Voort H. T. M.
Published: November 2003

We report three‐dimensional (3D) microscopy with nearly isotropic resolution in the λ/5 − λ/10 range. Our approach combines 4Pi‐confocal two‐photon fluorescence microscopy with image restoration. The 3D resolution is demonstrated with densely clustered beads as well as with F‐actin fibers in mouse fibroblast cells. A comparison with unrestored two‐photon confocal images reveals a total reduction of the uncertainty volume up to a factor of 15.
Reliability of phases retrieved from 400‐kV spot‐scan images of purple membranes acquired on a slow‐scan CCD camera
Authors: Sherman M. B. , Chiu W.
Published: November 2003

Glucose‐embedded purple membranes were used as a test specimen to evaluate the reliability of phases retrieved from 400‐kV spot‐scan images acquired on a 1024 × 1024 slow‐scan CCD camera. This specimen was chosen because it represents a broad class of low‐contrast radiation‐sensitive biological objects and its structure is well established. The amplitudes of computed reflections from these images were strongly damped by the modulation transfer function of the camera. Nevertheless, their phases on average were < 12° different from the reference data of Henderson et al. (1986), Ultramicroscopy, 19, 147–178, up to 8.8 Å resolution, which corresponds to 0.8 of the Nyquist frequency of the camera.
Improving DIC microscopy with polarization modulation
Authors: Holzwarth G. , Webb S. C. , Kubinski D. J. , Allen N. S.
Published: November 2003

It is demonstrated experimentally, as well as analytically, that when the polarization of the light incident upon the first Nomarski–Wollaston prism in a differential interference contrast (DIC) light microscope is switched by 90°, image highlights are changed into shadows and vice versa. Using an inexpensive ferroelectric liquid‐crystal modulator, which is easily installed in the microscope, this switching can be done at 30 frames s−1, synchronized to the camera. Subtraction of alternate digitized frames generates a stream of images in which contrast is doubled, compared with conventional video‐enhanced DIC, while image defects and noise tend to cancel. Subtraction of alternate images is carried out efficiently by frame buffer operations and amounts to massively parallel synchronous detection. The new method eliminates the problems inherent in obtaining a separate background image, as required by current video‐enhanced DIC practice, without loss of resolution.
X‐ray microscopy and imaging of Escherichia coli, LPS and DNA
Authors: Rajyaguru J. M. , Kado M. , Torres D. , Richardson M. , Muszynski M. J.
Published: November 2003

Ultrastructural examination by transmission and scanning electron microscopy involves a series of specialized preparation steps which may introduce artefacts in the micrographs. X‐ray microscopy can take instant images of speci‐mens but is mostly restricted to a few synchrotron X‐ray sources. We have utilized a bench‐top nanosecond laser‐plasma to produce a single‐shot source of nanosecond X‐rays tuned for maximum contrast with carbon‐rich material. To examine the ultrastructure by absorption profiles, we utilized a laser‐produced plasma generated by a single‐shot laser (1.06 μm wavelength, 5 × 1012 W cm−2 intensity) focused on to a silicon target as an X‐ray source for high‐resolution X‐ray microscopy. This approach eliminates the specimen preparation steps. Whole hydrated cells of Escherichia coli and purified preparations of lipopolysaccharide (LPS) and chromosomal DNA (cDNA) were streaked onto poly(methyl methacrylate) (PMMA)‐coated grids (resist). This resist was exposed to X‐rays under vacuum at a distance of 2.5 cm from the target disc. The silicon plasma produced by a 10‐ns burst of laser energy (at 20 J) radiates strong emission lines in the region of 300 eV. The X‐rays penetrate the sample and their absorption profile is transferred on to the resist where PMMA acts as a negative to generate an image. By atomic force microscopy imaging of this photoresist we have visualized layers around cells of E. coli, darker areas inside the cell probably corresponding to cDNA, and preliminary images of LPS and DNA molecules. This technique has resolution at the 100 Å level, produces images similar to the space‐filling models of macromolecules and may be of great value in the study of the ultrastructure of hydrated live biological specimens.
Undergraduate TEM instruction by telepresence microscopy over the Internet
Authors: Voelkl E. , Allard L. F. , Bruley J. , Williams D. B.
Published: November 2003

The use of telepresence microsopy (TPM) over the Internet for teaching is demonstrated. A high‐resolution field‐emission gun transmission electron microscope (TEM) at Oak Ridge National Laboratory was operated by undergraduate students at Lehigh University, 800 miles away. The remote operation used commercial software and freeware. The advantage of computer‐controlled TPM and is that students can access dedicated equipment not available in their home laboratory and also parallel hands‐on access and instruction can be given to a much larger group of students than usually can be taught on a TEM. Drawbacks include the bandwidth of the Internet.
Axial resolution of confocal Raman microscopes: Gaussian beam theory and practice
Authors: DE GRAUW C. J. , SIJTSEMA N. M. , OTTO C. , GREVE J.
Published: November 2003

A straightforward and transparent model, based on Gaussian beam optics, for the axial r0 resolution of a confocal microscope is presented. A confocal Raman microscope was used to determine the axial confocality in practice. The axial response of a thin planar object was measured for three different objectives, two pinhole sizes and a slit. The results show that, in the case of a confocal configuration, the response calculated with the model provides a good prediction of the axial resolution of the confocal microscope.
Single‐lens theta microscopy — a new implementation of confocal theta microscopy
Authors: Lindek S. , Stefany T. , Stelzer E. H. K.
Published: November 2003

A new microscopical technique based on the principle of confocal theta microscopy (Stelzer, E.H.K., Lindek, S. & Pick, R. (1996) Konfokales Mikroskop. German Patent Office DE 43 26 473 (filed 6.8.1993, granted 6.12.1996)) is described. It uses a single objective lens in combination with a mirror unit to achieve the theta configuration that leads to axial and volume resolution improvements. In this paper we present technical details of possible microscopical set‐ups, and we discuss different versions of mirror units.
High‐resolution, energy‐dispersive microcalorimeter spectrometer for X‐ray microanalysis
Authors: WOLLMAN D. A. , IRWIN K. D. , HILTON G. C. , DULCIE L. L. , NEWBURY D. E. , MARTINIS J. M.
Published: November 2003

We have developed a prototype X‐ray microcalorimeter spectrometer with high energy resolution for use in X‐ray microanalysis. The microcalorimeter spectrometer system consists of a superconducting transition‐edge sensor X‐ray microcalorimeter cooled to an operating temperature near 100 mK by a compact adiabatic demagnetization refrigerator, a superconducting quantum interference device current amplifier followed by pulse‐shaping amplifiers and pileup rejection circuitry, and a multichannel analyser with computer interface for the real‐time acquisition of X‐ray spectra. With the spectrometer mounted on a scanning electron microscope, we have achieved an instrument response energy resolution of better than 10 eV full width at half‐maximum (FWHM) over a broad energy range at real‐time output count rates up to 150 s−1. Careful analysis of digitized X‐ray pulses yields an instrument‐response energy resolution of 7.2 ± 0.4 eV FWHM at 5.89 keV for Mn Kα1,2 X‐rays from a radioactive 55Fe source, the best reported energy resolution for any energy‐dispersive detector.
Acceleration of the ICTM image restoration algorithm
Authors: Verveer P. J. , Jovin T. M.
Published: November 2003

We have implemented a simple modification of the ICTM image restoration algorithm that reduces the computational requirements considerably. The modified algorithm converges to the same solution as alternative approaches for computing non‐negatively constrained Tikhonov solutions. It compares favourably with these algorithms in terms of speed.
Structure of electrodeposited graded composite coatings of Ni–Al–Al2O3
Authors: Barmak K. , Banovic S. W. , Petronis C. M. , Susan D. F. , Marder A. R.
Published: November 2003

The structure of electrodeposited composite coatings of Ni–Al–Al2O3, with Ni as the matrix and Al and Al2O3 as second‐phase particles, was investigated using light microscopy and scanning electron microscopy. Ni coatings with no particles, which were used as reference samples, had progressively coarser structures with increasing current density. Co‐deposition with Al resulted in refinement of the Ni matrix structure at high (>10 A dm−2) current densities. For single‐particle baths, the co‐deposition of Al2O3 was more strongly affected by current density and bath particle content than was the co‐deposition of Al. However, for baths containing both Al and Al2O3 the amount of incorporated Al2O3 no longer depended on current density. With the choice of appropriate conditions, coatings of Ni with up to 39 vol.% Al2O3 were made. Similar experiments with Al yielded a maximum of 17.5 vol.% only. Uniform and graded mixed‐particle coatings were also produced. When coatings containing Al were annealed, the reaction of the two elements resulted in the formation of either single‐phase γ or two‐phase γ–γ′ alloys, in agreement with the equilibrium phase diagram.
Influence of pitch air‐blowing and thermal treatment on the microstructure and mechanical properties of carbon/carbon composites
Authors: MENÉNDEZ R. , GRANDA M. , FERNÁNDEZ J. J. , FIGUEIRAS A. , BERMEJO J. , BONHOMME J. , BELZUNCE J.
Published: November 2003

This paper discusses the optimization of pitch properties as a matrix precursor of C/C composites by subjecting the pitches to different pretreatments to promote polymerization of the molecular components of pitch, which could improve carbon yield and density of the resultant carbon materials. Two series of pitches were prepared by air‐blowing of impregnating coal‐tar pitches at temperatures between 523 and 573 K, and thermal treatment at temperatures between 673 and 703 K, in an inert atmosphere. Effects of pitch treatments on resultant coke texture and porosity were monitored by polarized light microscopy. Unidirectional laminar composites were prepared using the original and treated pitches and commercial PAN‐based carbon fibres. Effects of pitch treatments on the main microstructural features of composites are determined by scanning electron microscopy and polarized light microscopy, and related to the mechanical properties of composites. Results show an improvement of the mechanical properties of composites in both series, particularly for pitch treatments at 523 K/air in the air‐blown series, and at 698 K–5 h in the thermally treated one.
Electron microscopy of crack/particle interactions in Al2O3/SiC nanocomposites
Authors: JIAO S. , JENKINS M. L. , DAVIDGE R. W.
Published: November 2003

Crack/particle interactions in alumina/silicon carbide nanocomposites have been investigated by scanning electron microscopy and transmission electron microscopy, with cracks induced by Vickers microindentation. Intergranular cracks are frequently deflected into grains by SiC particles on grain boundaries inclined to the average direction of crack propagation. This mechanism is proposed to explain the change in the fracture mode from intergranular fracture for monolithic alumina to predominantly transgranular fracture for the nanocomposites. Neither stress‐induced microcracking around SiC particles nor significant crack deflection by intragranular particles was found to occur in the nanocomposites. It is argued that an addition of nanoparticles may not be a promising approach for increasing the toughness of alumina.
Fibre/resin interaction in fragmentation tests: coaxial vs. simple specimens comparison
Authors: Favre J.‐P. , Auvray M.‐H. , Mavel A.
Published: November 2003

Fragmentation tests of single carbon fibres embedded in small resin specimens are widely used to compare fibre surface treatments or sizes. A limitation of the test is that the matrix must have a high enough ultimate strain for the multiple fracture process to be fully achieved before specimen fractures. The so‐called coaxial geometry was introduced in order to extend the method to low strain‐to‐failure resin systems. In agreement with the Cox theory of the elastic stress transfer, interposition of a ‘high’‐modulus material between the fibre and the resin (the ‘low’‐modulus material) results in a shorter fragment length, i.e. a better stress transfer.
Analysis of composites using Raman and fluorescence microscopy — a review
Authors: Young R. J.
Published: November 2003

A review is presented of the application of analytical optical microscopy to the analysis of composite micromechanics in a number of different composites systems. A description is given of the use of Raman spectroscopy to follow fibre deformation in a variety of different aramid/epoxy composite test‐pieces. Various examples are presented including the single‐fibre pull‐out test, crack bridging with an array of fibres, stress concentrations around a hole in a unidirectional composite and a high‐volume fraction woven composite. In each case it is shown that the Raman technique gives a unique opportunity to measure local fibre strain leading to the possibility of analysing fully the deformation micromechanics. In addition it is shown how, for alumina‐based fibres such as PRD‐166, well‐defined fluorescence spectra can be obtained and that the stress‐induced fluorescence band shifts allow deformation micromechanics to be studied for PRD‐166 fibres in a glass matrix. In particular it is shown how it is possible to determine the level of thermal residual strain and follow the effect of externally imposed deformation upon the level of residual strain.
Interface considerations in high‐temperature titanium metal matrix composites
Authors: VASSEL A.
Published: November 2003

Near‐α titanium alloys are of interest for continuous fibre reinforced titanium‐based composites owing to their better creep resistance than other titanium alloys. The main advantage of these matrices is that they can improve transverse properties of the composite at high temperature. However, cracking at the fibre/matrix interface in the as‐consolidated condition has been reported with some near‐α matrices. Two composites were compared in the present work: SCS‐6/Ti‐6‐2‐4‐2 and SCS‐6/IMI 834. Very few defects were detected in the SCS‐6/Ti‐6‐2‐4‐2 composite whereas an extensive cracking of interfibre spacings was observed in the SCS‐6/IMI 834 composite manufactured in the same conditions. Our investigations revealed that the fibre/matrix interaction zone is very similar in both composites, so this region cannot explain the brittle behaviour of the IMI 834 matrix. It has been established that cracking of the SCS‐6/IMI 834 composite results from a combined effect of thermal residual stresses and matrix microstructure.
A quantitative comparison of image restoration methods for confocal microscopy
Authors: VAN KEMPEN G. M. P. , VAN VLIET L. J. , VERVEER P. J. , VAN DER VOORT H. T. M.
Published: November 2003

In this paper, we compare the performance of three iterative methods for image restoration: the Richardson–Lucy algorithm, the iterative constrained Tikhonov–Miller algorithm (ICTM) and the Carrington algorithm. Both the ICTM and the Carrington algorithm are based on an additive Gaussian noise model, but differ in the way they incorporate the non‐negativity constraint. Under low light‐level conditions, this additive (Gaussian) noise model is a poor description of the actual photon‐limited image recording, compared with that of a Poisson process. The Richardson–Lucy algorithm is a maximum likelihood estimator for the intensity of a Poisson process. We have studied various methods for determining the regularization parameter of the ICTM and the Carrington algorithm and propose a (Gaussian) prefiltering to reduce the noise sensitivity of the Richardson–Lucy algorithm. The results of these algorithms are compared on spheres convolved with a point spread function and distorted by Poisson noise. Our simulations show that the Richardson–Lucy algorithm, with Gaussian prefiltering, produces the best result in most of the tests. Finally, we show an example of how restoration methods can improve quantitative analysis: the total amount of fluorescence inside a closed object is measured in the vicinity of another object before and after restoration.
Structure‐orientated fluorescence photobleaching analysis: a method for double fluorescent labelling studies
Authors: GHAUHARALI R. I. , VAN DRIEL R. , BRAKENHOFF G. J.
Published: November 2003

Differences in the degree of photodegradation can be used for fluorophore identification in double fluorescently labelled specimens. Based on the use of morphological information, a noise‐insensitive method is presented for discriminating between the fluorophores, assuming spatially uniform photodegradation. Separate images of the labelled structures can be obtained. Alternatively, with spatially nonuniform photodegradation, the photodynamics of one fluorophore — i.e. photodegradation, concentration associated quenching, etc. — in relation to its microenvironment can be investigated.
Reproductively‐Relevant Stimuli Induce Fos‐Immunoreactivity within Progestin Receptor‐Containing Neurons in Localized Regions of Female Rat Forebrain
Authors: Auger Anthony P. , Moffatt Christopher A. , Blaustein Jeffrey D.
Published: November 2003

An experiment was conducted to determine if neurons that respond to stimuli associated with mating in female brain also contain progestin receptors. We found that a portion of the neurons that respond to stimuli associated with mating also contains progestin receptors. While the appropriate hormonal conditions are important for sexual receptivity, somatosensory information provided by the male also influences sexual behavior. One important stimulus provided by the male during copulation is vaginal‐cervical stimulation (VCS). VCS has been shown to elicit many different behavioral and endocrine changes in female rats, such as increases in lordosis, pseudopregnancy, and termination of sexual receptivity. VCS also increases the expression of the immediate early gene product, Fos, in areas associated with reproduction. A portion of the neurons responding to VCS with increased Fos‐immunoreactivity (Fos‐IR) in female rat forebrain also contains estrogen receptors, illustrating that hormonal and mating‐stimuli converge in a population of cells. As progesterone also plays an important role in female sex behavior, it is important to determine if some of the neurons also integrate information concerning serum progesterone levels and social interactions. Thus, we used a dual immunofluorescent technique to label both Fos‐IR and progestin receptor‐immunoreactivity (PR‐IR) in the brains of estradiol‐primed, ovariectomized female rats following VCS manually applied by the experimenter. Many of the neurons that respond to VCS with increased Fos‐IR within the medial preoptic area, the arcuate nucleus, and the progestin receptor‐rich areas of the rostral and caudal ventromedial nucleus of the hypothalamus also contain PR‐IR.
Inhibition of Steroid‐Induced Galanin mRNA Expression in GnRH Neurons by Specific NMDA‐Receptor Blockade
Authors: Rossmanith Winfried G. , Marks Daniel L. , Steiner Robert A. , Clifton Donald K.
Published: November 2003

Galanin mRNA levels in GnRH neurons increase in association with a steroid‐induced LH surge in female rats. Both the steroid‐induced LH surge and the concomitant increase of galanin mRNA in GnRH neurons are blocked by non‐specific inhibition of central nervous system activity imposed by pentobarbital and specific central alpha‐adrenergic receptor blockade. Based on these observations, we hypothesized that galanin gene expression in GnRH neurons is induced whenever GnRH neurons become activated to generate an LH surge. If this were the case, then any neurotransmitter receptor blocking agent that inhibits the LH surge by central mechanisms would likewise block the associated increase in galanin mRNA in GnRH neurons. We tested this hypothesis by examining the effects of an N‐methyl‐D‐aspartate (NMDA) receptor antagonist on the steroid‐induced LH surge and on levels of galanin mRNA in GnRH neurons. Three groups of ovariectomized rats were used: Group 1‐treated with estradiol and progesterone (E/P) and sacrificed at the peak of the LH surge; Group 2‐treated the same as Group 1 except that dizocilpine (MK801, an NMDA receptor antagonist) was used to block the LH surge; and Group 3‐treated the same as Group 1 except they received vehicle instead of E/P. Double‐and single‐label in situ hybridization followed by computerized image analysis were used to measure levels of galanin mRNA and GnRH mRNA in GnRH neurons [as grains/cell (g/c)]. E/P treatment induced a 3‐fold increase in LH levels and a 5‐fold increase in the galanin mRNA signal content of GnRH neurons. Treatment with MK801 completely prevented the LH surge in all animals and also blocked the steroid‐induced increase in galanin mRNA in GnRH neurons. As assessed by 2 independent GnRH single‐labeled assays, neither GnRH message content nor the number of identifiable GnRH neurons differed among the experimental groups. We conclude that the increase in galanin mRNA levels in GnRH neurons is tightly coupled to the occurrence of a steroid‐evoked LH surge, and we infer that induction of galanin gene expression in GnRH neurons is induced as a consequence of synaptic activation of GnRH neurons.
Nitric Oxide Induces Morphological Changes in Cultured Neurohypophysial Astrocytes
Authors: Ramsell Katrina D. , Cobbett Peter
Published: November 2003

Cultured pituicytes, derived from the neurohypophysis of adult rats, have previously been reported to change from a non‐stellate form to a stellate form when incubated in medium containing a β‐adrenoreceptor agonist. This study was designed to determine whether the same morphological change could be induced by direct activation of adenylate cyclase or of soluble guanylate cyclase. The fraction of stellate cells was normally low (<0.25) when the pituicytes were incubated (90 min) in a HEPES buffered salt solution (HBSS); most pituicytes had an amorphous protoplasmic appearance. The fraction of stellate cells was significantly increased when pituicytes were incubated in HBSS supplemented with isoproterenol (10 μM) or forskolin (5 μM) or with either of the nitric oxide donors nitroprusside (10–25 μM) and 3‐morpholinosydnonimine (SIN‐1; 10 μM). The effect of forskolin was mimicked by 8‐bromo cyclic AMP, a membrane permeable analog of cyclic AMP, but not by the inactive forskolin analog 1, 9 dideoxyforskolin. The effect of nitroprusside was blocked by methylene blue, an inhibitor of soluble guanylate cyclase, and was mimicked by 8‐bromo cyclic GMP, a membrane permeable analog of cyclic GMP. These results demonstrate that activation of adenylate cyclase and also of soluble guanylate cyclase can induce pituicytes to undergo morphological changes in vitro. The data suggest that the activity of both enzymes may be important in control of the plastic relationship that exists between neuronal and glial elements in the neurohypophysis in vivo.
Regulation of Activin Type‐II Receptor mRNA Levels in Rat Hypothalamus by Estradiol in vivo
Authors: Trudeau V. L. , Pope L. , de Winter J. P. , Haché R. J. G. , Renaud L. P.
Published: November 2003

A solution‐hybridization S1‐nuclease protection assay was used to evaluate the expression of messenger RNAs for the activin βA subunit and type II activin receptor in adult rat brain. Results indicate the presence of βA subunit mRNA in both hypothalamus and brainstem, with approximately two‐fold higher levels in brainstem. Levels of activin type II receptor mRNA were similar in the hypothalamus of young virgin and 15‐day lactating females, and in females in which pups were removed after a 5‐day lactation period. Male rats castrated prepubertally (30 days p.n.) had approximately 220% higher (P<0.05) hypothalamic activin type II receptor mRNA levels than postpubertal, 3‐month old age‐matched sham controls. Two month treatment of castrate rats with estradiol (200 ng/g, i.p. every 2 days) reduced hypothalamic activin type II receptor mRNA expression to control levels; the same dose of testosterone had no effect. The expression of the hypothalamic activin type II receptor gene may be estrogen‐regulated in vivo.
Adrenocorticotrophin and Luteinizing Hormone Responses to N‐Methyl‐D‐Aspartate During Fetal Development in Sheep
Authors: Brooks A. N. , Howe D. C.
Published: November 2003

The hypothalamic mechanisms which underlie the development of the fetal neuroendocrine system are unclear. However, in adult animals neuroactive amino‐acids, particularly those acting at the N‐methyl‐D‐aspartate (NMDA) receptor, have been shown to be important transmitters involved in the neuroendocrine regulation of the anterior pituitary gland. In this study we have investigated the potential role of NMDA in the neuroendocrine regulation of fetal pituitary function, by examining the ontogeny of LH and ACTH responses to NMDA during fetal development in sheep. Catheterized fetal sheep were injected with 3 intravenous doses of NMDA (1, 2 and 4 mg/kg; estimated fetal body weight) and saline vehicle on consecutive days between days 110–115, 120–125 and 135–140 gestation (term=145 days). At each gestational age fetuses also received a pituitary challenge test consisting of CRF (0.4 μg/kg), AVP (80 ng/kg) and GnRH (125 ng/kg). NMDA caused a significant dose‐related increase in ACTH at day 120–125 (P<0.02) and day 135–140 gestation (P<0.001). NMDA had no effect on ACTH secretion at day 110–115. The ACTH response to the highest does of NMDA (4 mg/kg) increased with advancing gestational age, in contrast to the ACTH response to CRF+AVP which was the same at all ages. NMDA caused a significant increase in LH secretion at all gestational ages with the greatest response observed at the latest gestational age studied (day 135–140). A similar increase in LH response to the GnRH challenge was observed late in gestation. Pretreatment of fetuses with the competitive NMDA antagonist CGP37849 (1 mg/kg) abolished the LH and ACTH responses to an intravenous injection of NMDA (4 mg/kg) given 5 min later. These data show that activation of NMDA receptors during fetal development elicits the secretion of ACTH and LH and demonstrate that NMDA receptors are functionally coupled to the neuroendocrine pathways regulating these two hormones during fetal life. The dramatic increase in ACTH repsonse to NMDA during the final days before birth occurs at a time of high adrenocortical activity and suggests an important functional role for these receptors at this time.
PACAP and VIP Stimulate Ca2+ Oscillations in Rat Gonadotrophs through the PACAP/VIP Type 1 Receptor (PVR1) Linked to a Pertussis Toxin‐Insensitive G‐Protein and the Activation of Phospholipase C‐β
Authors: Hezareh Marjan , Schlegel Werner , Rawlings Stephen R.
Published: November 2003

Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are hypothalamic factors that play roles in the regulation of anterior pituitary cell activity. PACAP exists in 2 forms physiologically, a 38 amino acid form (PACAP38) and a form possessing the N‐terminal 27 amino acids of PACAP38 (PACAP27). We have previously shown that PACAP38 stimulates an increase in [Ca2+]i in rat gonadotrophs. In an attempt to identify the PACAP receptor type underlying this effect, we compared the potency of PACAP38, PACAP27 and VIP to stimulate Ca2+ changes in identified single rat gonadotrophs. All 3 peptides at 100 nM were capable of stimulating high amplitude Ca2+ oscillations, which were also observed in the absence of extracellular Ca2+. The order of potency of these peptides was PACAP38>PACAP27>VIP, and a potent antagonist of the PACAP/VIP type II binding site ([4‐Cl‐D‐Phe6, Leu17]‐VIP) failed to block these responses, suggesting that these effects are mediated through a PACAP/VIP type 1 receptor (PVR1). The Ca2+ responses to PACAP38 and VIP were unaffected by overnight treatment of the cells with pertussis toxin (PTX; 250 ng/ml) indicating that these responses are mediated by a PTX‐insensitive G‐protein. Finally, the Ca2+ responses stimulated by PACAP38 and VIP were blocked by the phospholipase C‐β blocker U73122 (5 μM). In summary, PACAP stimulates Ca2+ oscillations in rat gonadotrophs through the activation of the PVR1 linked to a PTX‐insensitive G‐protein and the activation of phospholipase C‐β. VIP can stimulate the same pathway in rat gonadotrophs, although it is at least 100 fold less potent than PACAP38.