bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved quantitative electron probe X‐ray microanalysis of biological cryosections using an EDS germanium detector and a super‐atmosphere thin window (SuperATW)
Authors: Schultz  , Wendt‐Gallitelli
Published: March 2003

A new Link energy‐dispersive GEM detector with SuperATW window was tested for quantitative electron probe microanalysis of low calcium and sodium concentrations ([Ca], [Na]) in intracellular compartments of cardiac myocytes. We compare Ca profiles with high count statistics and similar peak area collected under the same conditions with either a Be‐windowed Si and a Ge SuperATW detector. The height of the Ca peak was increased by 7%, the full width at half‐maximum height was reduced by 9% with the Ge detector. The counts statistics of the Ca Kα peak improved by 9% and the partial overlap with the K Kβ peak was better deconvoluted. We calculate [Ca] and errors of the single measurement in mitochondria of guinea‐pig cardiac myocytes from spectra acquired with a Si or Ge detector. For identical analysis conditions, the [Ca] were identical; however, with the Ge SuperATW detector, the calculated error of the single measurement was only 1/2.7 of that calculated from measurements with the Si detector. We compare the peak area of identical [Na] in spectra collected with the Be‐windowed Si detector and Ge SuperATW detector. The peak area was significantly higher with the SuperATW Ge than with the Si detector and Be window, whereas the continuum in the range 4–10 keV was comparable, demonstrating the improved sensitivity for low atomic elements such as Na of the Ge SuperATW detector. [Na] and errors of the single measurement in mitochondria of quiescent guinea‐pig cardiac myocytes were calculated from spectra acquired with the Si or the Ge detector. The use of the Ge SuperATW detector improved the detectability limit for sodium by more than 80% and reduced the error of the single measurement by a factor of 7–8.
The effect of three‐fold astigmatism on measurements of grain boundary volume expansion by high‐resolution transmission electron microscopy
Authors: Merkle K. L. , Csencsits R. , Rynes K. L. , Withrow J. P. , Stadelmann P. A.
Published: March 2003

In the absence of high‐order aberrations, the lattice fringe technique should allow measurement of grain boundary rigid‐body displacements to accuracies about an order of magnitude better than the point‐to‐point resolution of the transmission electron microscope. The three‐fold astigmatism, however, introduces shifts of the lattice fringe pattern that depend on the orientation of the lattice relative to the direction of the three‐fold astigmatism and thus produces an apparent shift between the two grains bordering the grain boundary. By image simulation of grain boundary model structures, the present paper explores the effect of these extraneous shifts on grain boundary volume expansion measurements. It is found that the shifts depend, among others, on zone axis direction and the magnitude of the lattice parameter. For many grain boundaries of interest, three‐fold astigmatism correction to better than 100 nm appears necessary to achieve the desired accuracies.
Why don't high‐resolution simulations and images match?
Authors: Boothroyd C. B.
Published: March 2003

Computer simulations have been used for many years to understand experimental high‐resolution electron microscope images in a qualitative fashion, but the trend nowadays has been to attempt more quantitative image matching. This has led to the discovery that the contrast in experimental images is much less than in simulated images, typically by a factor of about three. There are many possible causes for this discrepancy, ranging from the mechanisms of scattering of electrons by the specimen through the calculations of the diffracted beam intensities and their focusing by the objective lens to the point spread function of the recording device. No single cause can explain all of the experimental contrast loss, although a combination of many factors could.
Retrieval of object information by inverse problems in electron diffraction
Authors: Scheerschmidt K.
Published: March 2003

The imaging of crystal defects by high‐resolution transmission electron microscopy or with the help of the electron diffraction contrast technique is well known and routinely used. However, a direct and phenomenological analysis of electron micrographs is mostly not possible, but requires the application of image simulation and matching techniques. The trial‐and‐error matching technique is the indirect solution to the direct scattering problem applied to analyse the nature of the object under investigation. Alternatively, inverse problems as direct solutions of electron scattering equations can be deduced using either an invertible linearized eigenvalue system or a discretized form of the diffraction equations. This analysis is based on the knowledge of the complex electron wave at the exit plane of an object reconstructed for the surrounding of single reflections by electron holography or other wave reconstruction techniques. In principle, it enables directly the retrieval of the local thickness and orientation of a sample as well as the refinement of potential coefficients or the determination of the atomic displacements, caused by a crystal lattice defect, relative to the atom positions of the perfect lattice. Considering especially the sample orientation as perturbation the solution is given by a generalized and regularized Moore–Penrose inverse, where the resulting numerical algorithms imply ill‐posed inverse problems.
Towards 1.0 Å resolution: taking advantage of dynamical scattering, and the benefits for structure retrieval
Authors: Barry J. C.
Published: March 2003

This paper is an exploration of the behaviour of high‐resolution transmission electron microscope (HRTEM) images at up to 1 Å resolution. The ultimate limits to HRTEM (structure) resolution and the manner in which strong scattering may lead to weak diffraction in heavy fcc metals are discussed. A resolution of 1.0 Å is somewhat better than the ultimate resolution presently achievable in a 400‐kV electron microscope. In heavy metals, such as platinum, it is found that the lattice fringe contrast is very low in the [011] projection, but that fringe contrast may be improved by imaging in the [111] projection. For atomic resolution imaging of the heavy metals in the [111] projection a resolution of 1.2 Å is required. For the study of oxygen position in high‐temperature superconducting (HTS) oxides a resolution of between 1.2 and 1.4 Å is required. At better than 1.2 Å resolution the thick crystal images in HTS oxides remain simple and are easily interpreted. At such resolution all atomic columns are separated for the HTS [010] projection and the dynamical diffraction effects improve the contrast of oxygen atoms relative to the metal atoms.
Secondary phase distribution analysis via finite body tessellation
Authors: Boselli  , Pitcher  , Gregson  , Sinclair
Published: March 2003

The concept of a Dirichlet tessellation has been extended to that of a ‘finite body’ tessellation to provide a more meaningful description of the spatial distribution of non‐spherical secondary phase bodies on two‐dimensional sections. A finite body tessellation consists of a network of cells constructed from the interfaces of each individual secondary phase body such that every point within a cell is closer to the corresponding body than to any other. Spatial distribution related cell characteristics derived from Dirichlet tessellations have been extended to finite body tessellations. Quantitative comparisons between the two methods indicate that finite body tessellation measurements are more physically representative as well as more sensitive to local distribution characteristics of secondary phases. To reflect the potential application of finite body tessellations, a methodology is described for analysing the effects of particle distribution and morphology on short crack behaviour in particulate reinforced metal matrix composites.
Diffraction of circularly polarized light from near‐field optical probes
Authors: Shin D. J. , Chavez‐Pirson A. , Lee Y. H.
Published: March 2003

Diffracted fields from 100‐nm aperture near‐field scanning optical microscopy (NSOM) probes and uncoated tapered fibres are measured and analysed. Using a solid angle scanner, the two‐dimensional intensity distribution and polarization state of the diffracted light are resolved experimentally. Polarization analyses show that circularly polarized input light does not maintain its polarization state for all diffraction angles, and is completely filtered into linearly polarized light at large polar diffraction angles. This drastic decomposition originates from the vector nature of light diffracted by the sub‐wavelength aperture. There is a fundamental difficulty in generating circularly polarized light near the aperture of NSOM probes owing to polarization‐dependent diffraction in the near‐field regime. This is illustrated by the Bethe‐Bouwkamp model using circularly polarized input light.
Single molecule mapping of the optical field distribution of probes for near‐field microscopy
Authors: Veerman J. A. , Garcia‐Parajo M. F. , Kuipers L. , Van Hulst N. F.
Published: March 2003

The most difficult task in near‐field scanning optical microscopy (NSOM) is to make a high quality subwavelength aperture probe. Recently, we have developed high definition NSOM probes by focused ion beam (FIB) milling. These probes have a higher brightness, better polarization characteristics, better aperture definition and a flatter end face than conventional NSOM probes. We have determined the quality of these probes in four independent ways: by FIB imaging and by shear‐force microscopy (both providing geometrical information), by far‐field optical measurements (yielding throughput and polarization characteristics), and ultimately by single molecule imaging in the near‐field. In this paper, we report on a new method using shear‐force microscopy to study the size of the aperture and the end face of the probe (with a roughness smaller than 1.5 nm). More importantly, we demonstrate the use of single molecules to measure the full three‐dimensional optical near‐field distribution of the probe with molecular spatial resolution. The single molecule images exhibit various intensity patterns, varying from circular and elliptical to double arc and ring structures, which depend on the orientation of the molecules with respect to the probe. The optical resolution in the measurements is not determined by the size of the aperture, but by the high optical field gradients at the rims of the aperture. With a 70 nm aperture probe, we obtain fluorescence field patterns with 45 nm FWHM. Clearly, this unprecedented near‐field optical resolution constitutes an order of magnitude improvement over far‐field methods like confocal microscopy.
Interactions between hydrophobic and hydrophilic silicon surfaces using a tapered probe in near‐field scanning optical microscopy
Authors: Wei P. K. , Fann W. S.
Published: March 2003

We examined the tip–sample interaction of a Si (111) surface processed with HF solution. The interaction length of the probe with its hydrophobic surface is ≈30, and the frequency responses show an interaction force of elastic type. By contrast, the damping curve on a hydrophilic surface, Si with thin oxide, operates on a longer length of 100. Here, the frequency responses indicate an interaction force of the viscous type.
3D simulations of the experimental signal measured in near‐field optical microscopy
Authors: Martin O. J. F.
Published: March 2003

We present three‐dimensional simulations of the image formation process in near‐field optical microscopy. Our calculations take into account the different components of a realistic experiment: an extended metal coated tip, a subwavelength sample and its substrate. We investigate all possible detection (transmitted, reflected and collected field) and scanning (constant height, constant gap) modes. Our results emphasize the strong influence of the tip motion on the experimental signal. They also show that it is possible, by controlling the polarization of both the illumination and the detected field, to strongly reduce these artefacts.
Crystallographic analysis of facets using electron backscatter diffraction
Authors: RANDLE V.
Published: March 2003

Applications of electron backscatter diffraction (EBSD), also known as backscatter Kikuchi diffraction in the scanning electron microscope (SEM) are first and foremost microtexture and grain boundary misorientation analysis on a single polished section in the specimen. A more subtle and revealing approach to analysis of these data is to use EBSD to probe the orientations of planar surfaces, i.e. facets, which bound crystals. These surfaces include: • grain or phase boundaries • fractures • cracks It is of great interest to know the crystallography of such facets since it provides a key to understanding the physical properties of them.

As far as investigation methodology is concerned, surfaces or facets associated with polycrystals are of two types: exposed or unexposed. Exposed facets, such as a fracture surface, can be viewed directly in the SEM, whereas unexposed facets, such as a grain boundary, are usually revealed as an etched trace on a polished surface. Photogrammetric methods can be used to obtain the positional orientation of an exposed facet, and the crystallographic orientation is obtained either directly from the surface or by indirect sectioning. Calibrated sectioning is required to obtain the equivalent parameters for an internal surface. The present paper compares the methods for obtaining and interpreting the crystallography of facets, with illustrations from several materials.
The effect of a weak W/SiC interface on the strength of Sigma silicon carbide monofilament
Authors: Dyos  , Shatwell
Published: March 2003

Fractography studies have shown that the strength‐determining flaws in silicon carbide monofilaments are generally at the core/silicon carbide interface or in the vicinity of the outside, carbon‐based coating. In tungsten‐cored monofilaments like DERA Sigma, the W/SiC flaws primarily determine the strength. Fracture is accompanied by brittle failure of the tungsten. The crack propagates simultaneously outwards through the silicon carbide, inwards through the tungsten and also around the W/SiC interface before being deflected into the tungsten or out through the silicon carbide. Experiments depositing boundary layers between the tungsten and silicon carbide have resulted in significantly different fracture behaviour. The tungsten fails in a ductile manner and the strength‐determining flaws are located predominantly at the outside surface of the silicon carbide. This behaviour is discussed in terms of models proposed by E. Martin and W. Curtin. It is thought that the work will ultimately lead to a significantly stronger, tungsten‐based monofilament.
CARS microscopy with folded BoxCARS phasematching
Authors: Müller  , Squier  , De Lange  , Brakenhoff
Published: March 2003

Three‐dimensional microscopy based on coherent anti‐Stokes Raman scattering (CARS) is a powerful new imaging technique, in which the contrast arises from molecular vibrations. Based on a simple numerical model, it is shown how the CARS interaction volume depends on the focusing parameters and the type of phasematching used. Collinear phasematching yields an ellipsoidal interaction volume, with lateral dimensions that readily cause vignetting of the CARS signal emission at the collection microscope objective. A folded BoxCARS phasematching geometry, on the other hand, results in an almost cylindrical interaction volume — at the cost of a reduced resolution, for which the possible vignetting of the CARS emission is much reduced. In addition, this type of phasematching provides spatial separation of the signal from the input laser beams, permitting simple signal detection of low frequency vibrational modes. Calculations show that when CARS is performed in a microscopic volume, the phasematching restraint on tuning over the vibrational band is strongly relaxed. A first example of CARS imaging using a folded BoxCARS imaging geometry is shown.
Identification of weak interfaces in composites using transmission electron microscopy
Authors: Perez‐Rigueiro  , Herrero  , Llorca
Published: March 2003

A new experimental technique was developed to identify crack paths with a resolution of nanometres in fibre‐reinforced composites. Cracks were introduced through Vickers indentations on one side of the sample prior to starting the thinning process. Indentations were placed close to the fibres in order to get enough cracks at the fibre/matrix interface in the electron‐transparent region of the thinned sample. The technique was used in a Nicalon‐fibre Al2O3matrix composite prior to and after a heat treatment at 1200 °C for 1 h. The analysis of the crack paths allowed the identification of the weakest interface in each condition.
Methodological principles for fractal analysis of trabecular bone
Authors: Parkinson  , Fazzalari
Published: March 2003

A standardized methodology for the fractal analysis of histological sections of trabecular bone has been established.

A modified box counting method has been developed for use on a PC‐based image analyser. The effect of image analyser settings, magnification, image orientation and threshold levels was determined. Also, the range of scale over which trabecular bone is effectively fractal was determined and a method formulated to calculate objectively more than one fractal dimension from the modified Richardson plot.

The results show that magnification, image orientation and threshold settings have little effect on the estimate of fractal dimension. Trabecular bone has a lower limit below which it is not fractal (λ < 25 μm) and the upper limit is 4250 μm. There are three distinct fractal dimensions for trabecular bone (sectional fractals), with magnitudes greater than 1.0 and less than 2.0.

It has been shown that trabecular bone is effectively fractal over a defined range of scale. Also, within this range, there is more than one fractal dimension, describing spatial structural entities. Fractal analysis is a model‐independent method for describing a complex multifaceted structure, which can be adapted for the study of other biological systems. This may be at the cell, tissue or organ level and complements conventional histomorphometric and stereological techniques.
Secondary ion mass spectrometry imaging of the fixation of 15N‐labelled NO in pollen grains
Authors: Lhuissier  , Lefebvre  , Gibouin  , Demarty  , Thellier  , Ripoll
Published: March 2003

We used secondary ion mass spectrometry to image cellular targets of nitrogen oxides (widespread air pollutants) in pollen grains of birch (Betula verrucosa Ehrh.) and cockfoot (Dactylis glomerata L.). The pollen samples were exposed to air supplemented with high doses of 15NO. The pollen grains were then fixed, dehydrated using a newly developed ‘vapour phase’ preparation method and embedded in LRW resin. Semithin sections were then analysed. Imaging was performed in scanning mode. As usual, the two isotopes 14N and 15N were imaged as 12C14N− and 12C15N−, respectively. The isotopic percentages of 15N were quantitatively determined either by image processing or by direct analysis. We show that the preferential areas of NO fixation in the pollen cell are the sporoderm and discrete intracytoplasmic structures that we tentatively describe as globoid‐like structures similar to those encountered in seeds.
New controlled environment vitrification system for cryo‐transmission electron microscopy: design and application to surfactant solutions
Authors: Egelhaaf S. U. , Schurtenberger P. , Müller M.
Published: March 2003

A newly designed controlled environment vitrification system for cryo‐transmission electron microscopy of vitrified samples (cryo TEM) is presented. It significantly reduces the lag‐time from blotting to vitrification by means of an optimized blotting procedure. Furthermore, a modified transfer system allows rapid transfer of the vitrified sample into the microscope and reduces the equilibration time. The practicable throughput of samples is thereby markedly increased. This new equipment is used to determine the overall size of individual polymer‐like mixed lecithin–bile salt micelles, which very critically depends on the composition of the sample. The quantitative agreement with the results from independent light scattering experiments on the same sample supports the validity and performance of the presented sample preparation and handling procedures. In addition, these experiments demonstrate that cryo TEM experiments not only provide direct structural information, but furthermore permit a quantitative determination of micellar properties.
Image analysis of insulation mineral fibres
Authors: Talbot H. , Lee T. , Jeulin D. , Hanton D. , Hobbs L. W.
Published: March 2003

We present two methods for measuring the diameter and length of man‐made vitreous fibres based on the automated image analysis of scanning electron microscopy images. The fibres we want to measure are used in materials such as glass wool, which in turn are used for thermal and acoustic insulation. The measurement of the diameters and lengths of these fibres is used by the glass wool industry for quality control purposes. To obtain reliable quality estimators, the measurement of several hundred images is necessary.

These measurements are usually obtained manually by operators. Manual measurements, although reliable when performed by skilled operators, are slow due to the need for the operators to rest often to retain their ability to spot faint fibres on noisy backgrounds. Moreover, the task of measuring thousands of fibres every day, even with the help of semi‐automated image analysis systems, is dull and repetitive. The need for an automated procedure which could replace manual measurements is quite real.

For each of the two methods that we propose to accomplish this task, we present the sample preparation, the microscope setting and the image analysis algorithms used for the segmentation of the fibres and for their measurement. We also show how a statistical analysis of the results can alleviate most measurement biases, and how we can estimate the true distribution of fibre lengths by diameter class by measuring only the lengths of the fibres visible in the field of view.
Simulation and quantitative assessment of homogeneous and inhomogeneous particle distributions in particulate metal matrix composites
Authors: Yang N. , Boselli J. , Sinclair I.
Published: March 2003

Reinforcement distributions play an important role in various aspects of the processing and final mechanical behaviour of particulate metal matrix composites (PMMCs). Methods for quantifying spatial distribution in such materials are, however, poorly developed, particularly in relation to the range of particle size, shape and orientation that may be present in any one system. The present work investigates via computer simulations the influences of particle morphology, homogeneity and inhomogeneity on spatial distribution measurements obtained by finite‐body tessellation. Distribution inhomogeneity was simulated both by the segregation of particles away from specified regions within a microstructure and by generating point density peaks at random locations within a microstructure. Both isotropic and anisotropic inhomogeneous distributions were considered to simulate distribution patterns in PMMCs before and after mechanical working. It was found that the coefficient of variation of the mean near‐neighbour distance (COV(dmean)), derived from particle interfaces using finite‐body tessellation, was essentially independent of particle shape, size distribution, orientation and area fraction in homogeneous (random) distributions, but showed great sensitivity to inhomogeneity. Increased values of COV(dmean) were seen for both forms of inhomogeneous distributions considered here, with little influence of particle morphology. The COV(dmean) was also seen to be sensitive to anisotropic clustering, the presence of which was identified via nearest‐neighbour angles and cell orientations. Although generally formulated for PMMCs, the present results may be generalized to other systems containing low aspect ratio finite bodies of low to moderate area fraction.
Subsurface deformation of machined Al2O3and Al2O3/5vol%SiC nanocomposite
Authors: Wu H. , Inkson B. J. , Roberts S. G.
Published: March 2003

Under machine grinding, material removal in monolithic Al2O3is by intergranular fracture and grain pull‐out. In comparison, under the same grinding conditions, an Al2O3/5%SiC nanocomposite undergoes significant surface grooving and intragranular fracture. The subsurface deformation mechanisms were investigated by cross‐sectional transmission electron microscopy. For Al2O3, the residual deformation zone was localized very close to the surface in the first layer of grains, with dislocations occurring only within 1.5 µm of the top surface and a high density of basal twins penetrating to a depth of one single grain. Cracks were present along grain boundaries or basal twin interfaces. For Al2O3/SiC nanocomposites, the main residual plastic deformation is observed to be dislocations activated to a depth of about 10 µm (approx. 3–4 grains), with twinning rarely observed. Possible mechanisms by which the SiC particles influence the subsurface deformation and material removal modes are discussed.
Imaging of three‐dimensional objects in emission electron microscopy
Authors: Nepijko S. A. , Sedov N. N. , Schmidt O. , Schönhense G. , Bao X. , Huang W.
Published: March 2003

Under investigation by emission electron microscopy, the shape and size of three‐dimensional objects are distorted because of the appearance of a characteristic potential relief and a possible contact potential difference between the particles and the substrate. An estimation of these effects for spherical particles is made. It is shown that the apparent size of particles observed in an emission electron microscope (EEM) could be increased as well as decreased depending on the relation between the work functions of the particle and the substrate. The corresponding formulae are given and several possibilities are shown which permit us to determine from the EEM image the real size of particles and their work function relative to the substrate.
GELFISH – Graphical environment for labelling fluorescencein‐situhybridization images
Authors: Lerner B. , Dhanjal S. , Hultén M. A.
Published: March 2003

Signal (dot) counting in fluorescencein‐situhybridization (FISH) images that relies on an automatic focusing method for obtaining clearly defined images is a time‐consuming procedure prone to errors. Our recently developed system has dispensed with automatic focusing, and instead relies on a neural network classifying focused and unfocused signals into valid and artefact data, respectively, and thereby discriminating between in‐ and out‐of‐focus images. However, to train the classifier accurate labelling of the image signals is required. GELFISH is a Graphical Environment for Labelling FISH images that enables the rejection of unanalysable nuclei and labelling of FISH signals simply and rapidly. GELFISH is flexible and can be modified easily for additional FISH applications. Also, implemented using popular software, the environment can be employed on any computer by any user. Finally, GELFISH is proposed in controlling a classifier‐based dot counting system.
Multi‐field 3D scanning light microscopy of early embryogenesis
Authors: Czirók A. , Rupp P. A. , Rongish B. J. , Little C. D.
Published: March 2003

A computer‐controlled microscopy system was devised to allow the observation of avian embryo development over an extended time period. Parallel experiments, as well as extended specimen volumes, can be recorded at cellular resolution using a three‐dimensional scanning procedure. The resulting large set of data is processed automatically into registered, focal‐ and positional‐drift corrected mosaic images, assembled as montages of adjacent microscopic fields. The configuration of the incubator and a sterile embryo chamber prevents condensation of the humidified culturing atmosphere in the optical path and is compatible with both differential interference contrast and epifluorescence optics. As a demonstration, recordings are presented showing the large‐scale remodelling of the embryonic primordial vascular structure.
Energy dispersive spectroscopy analysis of aluminium segregation in silicon carbide grain boundaries
Authors: Zhang X. F. , Yang Q. , De Jonghe L. C. , Zhang Z.
Published: March 2003

The aluminium distribution in polycrystalline SiC hot‐pressed with aluminium, boron and carbon additives was studied using X‐ray energy‐dispersive spectroscopy (EDS) and transmission electron microscopy (TEM). The Al excess in homophase SiC grain boundary films was determined, taking into account dissolved Al in the SiC lattice. In the spot‐EDS analysis, an electron beam probe with a calibrated diameter was formed, and the total beam–specimen interaction volume was defined, taking the beam spreading through crystalline TEM foil into consideration. EDS spectra were collected from regions containing intergranular films and adjacent matrix grains, respectively. A theoretical treatment was presented and experimental errors were estimated, with a further discussion about the effects of foil thickness. Experimental examples are given, followed by statistical EDS analyses for grain boundary films in SiC samples hot‐pressed with increased amounts of Al additions. The results demonstrated a substantial Al segregation in the nanometer‐wide intergranular films in all samples. Al additions higher than 3 wt% saturated the Al concentrations in SiC grains and in grain boundary films. The effect of foil thickness, and the parameters for determining the optimum incident beam diameter in the EDS analysis are discussed.
Characterization of the cutting edge of glass and diamond knives for ultramicrotomy by scanning force microscopy using cantilevers with a defined tip geometry. Part II
Authors: Matzelle T. R. , Gnaegi H. , Ricker A. , Reichelt R.
Published: March 2003

The cutting edge of glass as well as diamond knives was studied at high resolution using a scanning force microscope (SFM). The local shape of the cutting edge was estimated from single line profiles of the SFM topographs taking into account the exact shape of the probing tip estimated by a high‐resolution field emission scanning electron microscope (FESEM). The glass knives were prepared by ‘balanced breaking’. The radius of the investigated cutting edges was found to be 3.2–4.4 nm and 4.3–6.0 nm for the 35° and 45° diamond knife, respectively, and 3.4–4.3 nm for the glass knives.

Besides the opening angle and the cutting edge radius, the friction of a knife during sectioning represents a significant factor influencing the quality of sections. Thus, the roughness of both the diamond clearance angle side and the back side was characterized as well. Corresponding RMS values of the roughness were found to be smaller on the back side (≈ 0.14 nm) than on the clearance angle side (≈ 0.26 nm).
Effect of Oestradiol and Photoperiod on TH mRNA Concentrations in A15 and A12 Dopamine Cell Groups in the Ewe
Authors: Beccavin Caroline , Malpaux Benoît , Tillet Yves
Published: March 2003

In the sheep, photoperiod, through melatonin, and oestradiol negative‐feedback are two major regulators of seasonal changes in luteinizing hormone (LH) and prolactin secretion. Melatonin and oestradiol act on dopamine neurons of the hypothalamus to modify the enzymatic activity of tyrosine hydroxylase (TH). To further understand how melatonin and oestradiol regulate TH activity, we have studied the level of TH mRNA by in situ hybridization with an homologous cDNA probe, in A12 and A15 dopamine neurons of four groups of ovariectomized ewes: long‐day exposed ewes with or without subcutaneous oestradiol implants and short‐day exposed ewes with or without oestradiol. Results were analysed in relation to the concentration of LH and prolactin in the peripheral circulation. In the A15 cell group, TH mRNA levels were elevated in the short‐day, oestradiol‐treated ewes compared to all other groups. In this group, the level of TH mRNA was elevated simultaneously with LH concentration. The low level of TH mRNA found in the long‐day, oestradiol‐treated ewes may indicate that the increase of TH enzymatic activity previously reported by this treatment is not caused by an increase of the level of enzyme. In the A12 cell group, the level of TH mRNA in both long‐day and short‐day oestradiol‐treated ewes was significantly higher than in ewes without oestradiol replacement. Prolactin concentrations were not correlated with TH mRNA variations in the A12 cell group.
Anterior Pituitary Cell Population Control: Basal Cell Turnover and the Effects of Adrenalectomy and Dexamethasone Treatment
Authors: Nolan Lesley A. , Kavanagh Emma , Lightman Stafford L , Levy Andrew
Published: March 2003

We have used an extremely accurate, dedicated, real time computerized image analysis system to facilitate the manual quantification of changes in the prevalence of mitotic figures and apoptotic bodies in male rat pituitary following surgical adrenalectomy and, 14 days later, dexamethasone treatment. Under basal conditions, the prevalence of mitotic figures and apoptotic bodies was 0.066±0.016% and 0.030±0.012% (mean±SE) respectively. Dexamethasone treatment reduced the prevalence of mitotic figures and, in adrenalectomized animals, produced a highly significant and reproducible burst of apoptotic activity that peaked 48 h after the beginning of treatment (0.261±0.022%) before falling sharply to control levels within a further 8 h. Two weeks after the start of dexamethasone treatment, total pituitary cell numbers continued to decline. The rate of accumulation of mitotic figures in vivo after colchicine treatment indicates that mitosis is histologically overt in 2 μm thick hematoxylin and eosin stained sections under the light microscope for around 80 min; that apoptosis — identified as classical apoptotic bodies — is overt for 44 min and that, on average, a young, adult, male rat anterior pituitary cell either dies or divides as frequently as once every 60–70 days. These data show that transient and apparently trivial fluctuations in the prevalence of apoptotic and mitotic events have a profound effect on pituitary cell population dynamics, and demonstrate that dexamethasone treatment of adrenalectomized rats produces a decline in total anterior pituitary cell numbers that continues for at least 2 weeks after the start of glucocorticoid treatment.
Enhanced Response of Growth Hormone to Growth Hormone‐Releasing Hormone and a Decreased Content of Hypothalamic Somatostatin in a Stress‐Induced Rat Model of Depression
Authors: Hamanaka Kenji , Soya Hideaki , Yoshizato Hideo , Nakase Shinji , Ono Junko , Inui Koji , Zhang Kai , Okuyama Ryuji , Ishikawa Yoko , Kitayama Isao , Nomura Junichi
Published: March 2003

This study was designed to evaluate changes in the hypothalamic somatostatin‐growth hormone axis (SRIF–GH axis) in a stress‐induced rat model of depression. We exposed male Wistar rats to intermittent walking stress for two weeks, and then measured their spontaneous running activities for 12 days. We divided the rats into the depression‐model group and the partial recovery group according to their spontaneous running activities after the termination of exposure to stress. We examined the secretion of GH from the anterior pituitary by injecting human GH‐releasing hormone (hGHRH) with intracardiac cannulae or by applying hGHRH or SRIF to isolated anterior pituitaries using a perifusion system. We also determined SRIF content in the stalk‐median eminence (SME) and the plasma concentration of GH. In the depression‐model group, intracardiac administration of hGHRH caused the enhanced release of GH into plasma, while application of hGHRH or SRIF to the anterior pituitary in vitro had similar effects on GH release in the control and partial recovery groups. Furthermore, the SRIF content was decreased in the SME and the GH concentration was increased in plasma. The partial recovery group gave similar values to the control group. The enhanced response of GH to hGHRH in the depression‐model group might have been caused by the reduced content of SRIF in the SME in view of the unchanged response of GH to the infusion of hGHRH or SRIF in the perifusion system.
Intracranial Androgenic Activation of Male‐Typical Behaviours in house Mice: Concurrent Stimulation of the Medial Preoptic Area and Medial Nucleus of the Amygdala
Authors: Sipos Maurice L. , Nyby John H.
Published: March 2003

This experiment examined whether testosterone proprionate (T) action in the medial preoptic area (MPO) would synergize with T action in the medial nucleus of the amygdala (AME) for the expression of androgen‐dependent behaviors in house mice. Cannulae containing T were bilaterally implanted into the MPO, the AME, or both areas concurrently (MPO/AME) of castrated males. In addition, other castrates were implanted subcutaneously with empty Silastic capsules (BSIL) or Silastic capsules containing T (TSIL). All subjects were examined for the following androgen‐dependent, male‐typical behaviors: mounting, urinary scent marking, preference for female urine over male urine, preference for female over male conspecifics and ultrasonic mating vocalizations. MPO implants restored ultrasonic vocalizations and preference for females, but had little or no effect upon urine marking, mounting or preference for female urine. In contrast, AME implants were ineffective at restoring any of these male‐typical behaviors. The combined MPO/AME implants were not more effective in restoring male‐typical behaviors than MPO implants alone, thus providing no evidence for synergy in hormone action between these two brain areas. In general, castration (BSIL) resulted in low levels of all behaviors whereas systemic T replacement (TSIL) resulted in high levels of behavior, verifying the androgen‐dependence of these behaviors. Group differences in male‐typical behavior could not be accounted for by differences in general activity levels. Moreover, none of the brain‐implanted groups had larger seminal vesicles than those of the BSIL. Thus, when the brain implants affected behavior, they most probably did so through their effects within the brain. Although the AME is a target for steroid hormones and is an important area for the expression of male‐typical behaviors, intracranial T implants into the AME did not demonstrate a role for androgen in the AME in restoring male‐typical behaviors in castrated mice.
Adrenal Responsiveness and the Timing of Parturition in Hypothalamo‐Pituitary Disconnected Ovine Foetuses With and Without Constant Adrenocorticotrophin Infusion
Authors: Poore  , Canny  , Young
Published: March 2003

Ovine parturition results from an increase in foetal cortisol secretion in late gestation which is dependant on an intact hypothalamo‐pituitary connection. The cortisol surge and parturition fails in hypothalamo‐pituitary disconnected (HPD) foetuses but, paradoxically, immunoreactive (ir)‐ACTH concentrations and secretory dynamics appear normal. This study compares the occurrence and timing of labour, basal ir‐ACTH and cortisol concentrations and adrenal responsiveness in HPD foetuses (HPD/ACTH) receiving constant ACTH(1–24) infusion (43 ng/h/kg) from surgery (114±1 days gestational age (GA)) with those of saline‐infused HPD or intact foetuses (HPD/SAL and INT/SAL). HPD/ACTH foetuses initiated labour at 147±2 days GA, which was not significantly different from INT/SAL foetuses (149±1 day GA). HPD/SAL foetuses were killed electively at 146±3 days GA with no signs of labour. Foetal ir‐ACTH concentrations in all groups were indistinguishable, but only HPD/ACTH and INT/SAL foetuses had a significant cortisol surge. Adrenal responsiveness to ACTH(1–24)(1 μg/kg) was greater in HPD/ACTH foetuses than in HPD/SAL or INT/SAL foetuses at all GAs studied. Adrenal responsiveness in HPD/SAL foetuses exceeded that in INT/SAL foetuses at 120 and 130 days GA but did not change with GA. In summary, the basal cortisol and parturition defect in HPD foetuses was reversed by low‐dose ACTH(1–24) infusion. Basal cortisol concentrations were unrelated to adrenal responsiveness. HPD/SAL foetuses had hyper‐responsive adrenals compared to those of INT/SAL foetuses until 130 days GA, suggesting that the foetal hypothalamus exerts a negative influence on adrenal cortisol responses before 130 days GA, after which time stimulatory influences predominate.
Progestins’ Rapid Facilitation of Lordosis When Applied to the Ventral Tegmentum Corresponds to Efficacy at Enhancing GABAAReceptor Activity
Authors: Frye  , Vongher
Published: March 2003

Progestins may have actions in the midbrain though γ‐aminobutyric acid (GABA)A/benzodiazepine receptor complexes (GBRs) that are relevant for sexual receptivity. The efficacy and time course of various progestins to enhance lordosis when applied to the ventral tegmental area (VTA), following progesterone to the ventral medial hypothalamus (VMH) was investigated. Ovariectomized, oestrogen‐primed rats and hamsters with contralateral VMH/VTA cannulae were tested for lordosis before and after implants of P to the VMH and progestins to the VTA. The progestins were P, 5α‐pregnan‐3,20‐dione (DHP), 5α‐pregnan‐3α‐ol‐20‐one(3α,5α‐THP), 5α‐pregnan‐3α,21‐diol‐20‐one (THDOC), 5β‐pregnan‐3α‐ol‐20‐one(3α,5β‐THP), 17α‐ol‐6‐methyl‐4,6‐pregnadiene‐3,20‐dione‐17‐acetate (megestrol acetate, MA), and 6‐chloro‐17‐ol‐4,6‐pregnadiene‐3,20‐dione‐17‐acetate (chlormadinone acetate, CA). Progestins’ effects on GABA‐mediated chloride influx and SR 95531 binding in cortical and midbrain tissue, respectively, were examined in rats and hamsters. 3α,5α‐THP and THDOC implants to the VTA were the most effective at immediately facilitating lordosis of rats and hamsters. Two hours later all other progestins, except MA and CA, increased lordosis in rats; only P, 3α,5α‐THP, and THDOC were effective in hamsters. The progestins’ effectiveness at facilitating lordosis were similar to their effects on GABA‐stimulated chloride influx and SR 95531 receptor binding (3α,5α‐THP and THDOC>P>DHP>3α, 5β‐THP>MA and CA). These findings suggest that progesterone lordosis enhancing effects in the rodent VTA may be via GBRs.
Mating Stimuli Influence Endogenous Variations in the Neurosteroids 3α,5α‐THP and 3α‐Diol
Authors: Frye  , Bayon
Published: March 2003

Progesterone facilitates and 5α‐dihydrotestosterone (DHT) inhibits female sexual behaviour in rodents; their metabolites, 5α‐pregnan‐3α‐ol‐20‐one (3α,5α‐THP) and 5α‐androstane‐3α‐17β‐Diol (3α‐Diol), may influence the onset and termination of lordosis. Changes in these and related steroids in hormonal states associated with differences in receptivity were investigated. Rats were assigned to oestrus, metoestrus, dioestrus, pro‐oestrus, mated, gestational days 5–7, 12–14, 18–20, or post‐partum conditions; rats 9+ months of age were considered older. Pro‐oestrus rats were exposed to the mating arena, sight and smell of a male with no tactile contact, artificial vaginocervical stimulation, standard mating, or no mating. Progesterone, 5α‐pregnane‐3,20‐dione, 3α,5α‐THP, oestradiol, testosterone, DHT, 3α‐Diol, dehydroepiandrosterone, and corticosterone were measured in plasma and whole brain, midbrain, hypothalamus, cortex, amygdala, hippocampus. 3α,5α‐THP and 3α‐Diol changed with reproductive state and mating stimuli. Plasma and whole brain 3α,5α‐THP and 3α‐Diol were significantly increased in pro‐oestrus versus dioestrus rats; plasma 3α,5α‐THP was decreased and 3α‐Diol increased in mated versus pro‐oestrus rats. The midbrain and hypothalamus had the most evident changes in 3α,5α‐THP and 3α‐Diol between dioestrus versus pro‐oestrus and pro‐oestrus versus mated rats. 3α,5α‐THP and 3α‐Diol were altered differently in response to mating stimuli. 3α,5α‐THP was greater in the midbrain of mated versus pro‐oestrus rats; other mating‐relevant stimuli decreased 3α,5α‐THP. Midbrain 3α‐Diol was increased with exposure to a male <VCS< mating. 3α,5α‐THP was increased and 3α‐Diol was decreased in the hypothalamus of mated versus pro‐oestrus rats; exposure to the various mating stimuli decreased 3α,5α‐THP and 3α‐Diol. The neurosteroids, 3α,5α‐THP and 3α‐Diol, vary with mating in the hypothalamus and midbrain of rats.
Effects of Restraint Stress and Spontaneous Hypertension on Neuropeptide Y Neurones in the Brainstem and Arcuate Nucleus
Authors: Krukoff  , MacTavish  , Jhamandas
Published: March 2003

Neuropeptide Y (NPY) is found in autonomic neurones and participates in regulation of autonomic functions. To investigate the role of NPY in the stress response in normo‐ and hypertensive rats, activation of brainstem and arcuate nucleus (ARC) NPY neurones and levels of NPY mRNA in the ARC were measured in response to restraint stress in adult spontaneously hypertensive rats (SHRs) and two strains of normotensive rats. Controls from each strain were not restrained. Sections of the brain were prepared for Fos immunohistochemistry and NPY in‐situ hybridization to identify activated NPY neurones in the nucleus of the tractus solitarii (NTS), ventrolateral medulla (VLM), and ARC. NPY mRNA levels were quantified in the ARC. In the NTS and VLM of restrained rats, 33% and 75%, respectively, of NPY neurones were activated. No differences among strains were found. In the ARC, about 36% of neurones activated by restraint contained NPY mRNA with no differences found among strains. In unrestrained rats, NPY mRNA levels were significantly elevated in SHRs compared to the normotensive rats. Restraint led to significant decreases in mRNA levels in all strains and mRNA levels among strains were no longer different from one another. These data show that NPY likely participates as a neurotransmitter in the autonomic pathways utilized during stress and originating in the NTS, VLM, and ARC. On the other hand, the decreased gene expression of NPY in the ARC in response to restraint stress argues against a role for activation of autonomic pathways or the hypothalamo‐pituitary‐adrenal (HPA) axis by NPY from the ARC of stressed rats. The elevated NPY gene expression in resting SHRs compared to normotensive rats is abrogated after restraint, suggesting that this gene is differentially regulated in SHRs compared to normotensive rats.
Interactive Effects of Tamoxifen and Oestrogen Upon the Nigrostriatal Dopaminergic System: Long‐Term Treatments
Authors: McDermott  , Anderson  , Dluzen
Published: March 2003

In the present report adult female rats were ovariectomized (OVX) and assigned to one of four treatment conditions. Treatments consisted of administering pellets containing 17β‐oestradiol (E), tamoxifen (TMX), a combination of TMX and E or no further treatment (OVX). Animals received these treatments immediately following OVX and were maintained in these conditions for a 40‐day period. Subsequently, the corpus striatum (CS) was dissected from each animal and prepared for determinations of basal and amphetamine stimulated DA output using in‐vitro superfusion. No statistically significant differences among the four treatment groups were obtained for basal dopamine output. The highest levels of amphetamine‐stimulated dopamine responses were obtained from E treated rats. These values were significantly greater than that obtained from OVX rats and rats treated with a combination of TMX+E. The significance of these findings is that they indicate both a non‐traditional central nervous system site and mechanism of action through which tamoxifen–oestrogen interactions can function. Such data may have important implications for administration of tamoxifen to premenopausal women as this anti‐oestrogen may compromise nigrostriatal dopaminergic function under conditions where oestrogenic modulation is present.