bluuebunny/crossref_metadata_2025 huggingface dataset:
Using the Hough transform for HOLZ line identification in convergent beam electron diffraction
Authors: KRÄMER  , MAYER
Published: March 2003

The Hough transform as a means for automatic line detection is applied for higher order Laue zone (HOLZ) line identification in convergent beam electron diffraction. The HOLZ line positions are commonly used to measure the strain state in the investigated material. Besides the automation, a very important aspect is the accuracy of the detected line positions. The limits are determined by the amount of noise in the pattern relative to the contrast of the HOLZ lines and the line width. Our investigations show that sub‐pixel resolution can be achieved routinely. We have also developed a new strain analysis procedure in which the dynamical shift of each individual line can be taken into account using a model based on several Ewald spheres with different radii. In comparison to the effective high voltage method, finer details of the dispersion surface can be considered, which increases the accuracy of the strain analysis. A measurement of the thermal expansion of aluminium is presented as a model experiment. The lattice constants were determined with an accuracy of about 10−4.
Using GaAsP photodiodes to characterize ultrashort pulses under high numerical aperture focusing in microscopy
Authors: MILLARD A. C. , FITTINGHOFF D. N. , SQUIER J. A. , MÜLLER M. , GAETA A. L.
Published: March 2003

We demonstrate for the first time that the two‐photon response of GaAsP photodiodes can be used to conveniently characterize the spatial and temporal profiles of ultrashort pulses at the focus of a high numerical aperture system.
The effect of large solid angles of collection on quantitative X‐ray microanalysis in the AEM
Authors: Watanabe  , Ackland  , Williams
Published: March 2003

Increasing the solid angle of X‐ray collection is a major factor in improving the analytical sensitivity of X‐ray energy‐dispersive spectrometry (XEDS) in the analytical electron microscope (AEM). A new scanning transmission electron microscope, the VG HB 603, is equipped with two XEDS detectors with the largest collection angles (0.30 and 0.17 sr) available in commercial AEMs. However, large collection angles result in a large range of take‐off angles, from ~ 4° to 36°, and the low angles can cause strong X‐ray absorption. In order to investigate possible detrimental effects of the low (and of the range of) take‐off angles on quantitative microanalysis of specimens exhibiting significant absorption, a stoichiometric Ni3Al thin‐film, in which the Al Kα line is significantly absorbed, was analysed. Furthermore, the effect of different values of the collection angle on X‐ray intensities was theoretically evaluated by numerical calculations and spectral simulation. These theoretical approaches permitted correlation of changes in the X‐ray take‐off angle (and hence X‐ray absorption) with changes in the collection angle. It is demonstrated that ~ 0.30 sr detectors, with minimum take‐off angles as small as 4°, only result in maximum errors of 4% in the quantification of Al in Ni3Al and, therefore, further increases in collection angle can be pursued while maintaining current levels of accuracy of quantification.
Interferometric measurement of femtosecond optical pulses emitted from a fibre probe
Authors: Kawashima H. , Furuki M. , Tani T.
Published: March 2003

An interferometric technique is applied to a temporal analysis of the far‐field radiation from a near‐field optical fibre probe. A balanced homodyne scheme combined with lock‐in detection at dual harmonics allows one to evaluate the temporal form of weak signal pulses, even in the presence of some environmental vibrations that disturb the optical interference. The transmission properties of a small aperture are formulated theoretically. It is shown that the sub‐wavelength aperture should not distort the femtosecond optical pulses temporally, which is consistent with the experimental observation.
Optical‐fibre scanning near‐field optical microscope for cryogenic operation
Authors: Tokizaki T. , Sugiyama K. , Onuki T. , Tani T.
Published: March 2003

We have developed a new type of scanning near‐field optical microscope (SNOM) utilizing optical fibres. The probe tip is controlled by shear force feedback with a fibre interferometer and signal light is collected directly by a multimode fibre. These features make the SNOM head more compact and less sensitive to vibration. Further advantages of this new type of SNOM are that it obviates the need for optical windows in the cryostat and offers easy optical alignment.
Coaxial probes for scanning near‐field microscopy
Authors: Leinhos T. , Rudow O. , Stopka M. , Vollkopf A. , Oesterschulze E.
Published: March 2003

This paper deals with the development of coaxial aperture tips integrated in a cantilever probe for combined scanning near‐field infrared microscopy and scanning force microscopy. A fabrication process is introduced that allows the batch fabrication of hollow metal aperture tips integrated on a silicon cantilever. To achieve the coaxial tip arrangement a metal rod is deposited inside the hollow tip using the focused ion beam technique. Theoretical calculations with a finite integration code were performed to study the transmission characteristics of coaxial tips in comparison with conventional aperture probes. In addition, the influence of the geometrical design parameters of the coaxial probe on its optical behaviour is investigated.
Fabrication and observation of a standard sample for near‐field optical microscopy
Authors: Kato M. , Kiguchi M. , Ishibashi M. , Heike S.
Published: March 2003

We fabricated a standard sample for a near‐field optical microscope using scanning probe lithography. The sample contains a wedged pattern, which allows the measurement of various sizes within one image. The optical resolution of our near‐field optical microscope has been evaluated as 40 nm, which was obtained by measuring the narrowest separable gap width of the wedged pattern. Thus a standard sample containing the wedged pattern enables clear evaluation of the resolution.
Crystal structure retrieval by maximum entropy analysis of atomic resolution incoherent images
Authors: Mcgibbon  , Pennycook  , Jesson
Published: March 2003

In this paper, we discuss the application of the maximum entropy method to atomic resolution Z‐contrast images acquired in a scanning transmission electron microscope. Z‐contrast is an incoherent imaging technique, and can be described as a convolution between an object function (the real‐space map of the columnar scattering cross‐section to high angles) and a point spread function (the effective electron probe). As such, we show that the technique is ideally suited to maximum entropy analysis which can, given an electron probe distribution, retrieve the ‘most likely’Z‐contrast object function. Using both simulated and experimentally acquired data, we explore the capabilities of maximum entropy analysis when applied to atomic resolution Z‐contrast images, drawing conclusions on both the range of applicability of the technique and the nature of the retrieved crystal structures. Ultimately, we show the way in which the combination of Z‐contrast imaging with maximum entropy analysis can be used to yield important information on unexpected atomic structures.
Influence of grain orientations on the initiation of fatigue damage in an Al–Li alloy
Authors: TAYLOR C. J. , ZHAI T. , WILKINSON A. J. , MARTIN J. W.
Published: March 2003

The variation in microstructure and texture in a rectangular bar extruded from a billet of spray‐cast 8090 Al–Li alloy has been examined. The fine grain size of the as sprayed billet and the moderate extrusion ratio (≈ 25 : 1) were seen to cause geometric dynamic recrystallization (GDR) in regions of higher strain towards the edge of the bar. The grain morphology varied from the expected elongated grains at the centre of the bar to equiaxed grains where GDR occurred at the bar edges. A <111> + <100> double fibre texture, significantly distorted towards rolling components and varying through the bar thickness, was found using electron backscatter diffraction. Fatigue resulted in a high density of short secondary cracks, many of which had arrested at grain boundaries. The cracks preferentially nucleated in grains from the <100> fibre texture corresponding to high Schmid factors.
Electron backscatter diffraction: applications for nuclear materials
Authors: MEDEVIELLE A. , HUGON I. , DUGNE O.
Published: March 2003

The diffraction of electrons was first observed in 1928 by Kikuchi. The phenomenon results in the formation of characteristic diagrams of the crystalline lattice and the orientation of the phase. Backscattered electrons are diffracted by the different crystallographic planes (hkl) according to the Bragg angle θb. These describe, by symmetry, two cones of axes normal to the diffracting plane. Information is collected on a phosphor screen, leading to the acquisition of a diffraction pattern called a Kikuchi diagram. Several improvements now give a wide range of applications such as phase identification (carbides or complex compounds in a (U,Zr,O) structure), analysis of materials interfaces (ZrO2/UO2), as well as solidification studies and local texture determination (molybdenum sheets). In these applications, EBSD, as a type of quantitative metallography, is a powerful tool.
Three‐dimensional spectral precision distance microscopy of chromatin nanostructures after triple‐colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome
Authors: Esa A. , Edelmann P. , Kreth G. , Trakhtenbrot L. , Amariglio N. , Rechavi G. , Hausmann M. , Cremer C.
Published: March 2003

Topological analysis of the three‐dimensional (3D) chromatin nanostructure and its function in intact cell nuclei implies the use of high resolution far field light microscopy, e.g. confocal laser scanning microscopy (CLSM). However, experimental evidence indicates that, in practice, under biologically relevant conditions, the spatial resolution of CLSM is limited to about 300 nm in the lateral direction and about 700 nm in the axial direction. To overcome this shortcoming, the use of a recently developed light microscopical approach, spectral precision distance microscopy (SPDM) is established. This approach is based on the precise localization of small labelling sites of a given target in spectrally differential images. By means of quantitative image analysis, the bary centres (intensity weighted centroid analogous to the centre of mass) of these independently registered labelling sites can be used as point markers for distance and angle measurements after appropriate calibration of optical aberrations (here, polychromatic shifts). In combination with specific labelling of very small chromatin target sites with dyes of different spectral signatures by fluorescence in situ hybridization (FISH), SPDM presently allows us to analyse the nuclear topology in three‐dimensionally conserved nuclei with a ‘resolution equivalent’, many times smaller than the conventional optical resolution.

Chronic myelogeneous leukaemia (CML) is genetically characterized by the fusion of parts of the BCR and ABL genes on chromosomes 22 and 9, respectively. In most cases, the fusion leads to a translocation t(9; 22) producing the Philadelphia chromosome. SPDM was applied to analyse the 3D chromatin structure of the BCR region on the intact chromosome 22 and the BCR‐ABL fusion gene on the Philadelphia chromosome (Ph) by using a new triple‐colour FISH protocol: two different DNA probes were used to detect the BCR region and the third DNA probe was used to identify the location of the ABL gene. Consistent 3D distance measurements down to values considerably smaller than 100 nm were performed. The angle distributions between the three labelled sites on the Philadelphia chromosome territory were compared to two state‐of‐the‐art computer models of nuclear chromatin structure. Significant differences between measured and simulated angle distributions were obtained, indicating a complex and non‐random angle distribution.
Polymer composites in 2000: structure, performance, cost and compromise
Authors: Bader M. G.
Published: March 2003

Polymer matrix composites are based on the combination of stiff, strong reinforcing fibres with either thermosetting or thermoplastic polymer matrices. Since their introduction in the early 1940s, the world market has increased to some 5 million tonnes per annum, and some composites may now be considered commodity materials. The spectrum of fibre‐reinforced plastics ranges from very high‐performance speciality materials costing more than $1000/kg to these commodity composites, with more modest properties, at less than $10/kg. The performance of composites is determined by the properties of the fibre, the fraction of fibre in the composite and the structure, or fibre architecture. Processing technologies have been developed which maximise fibre content and precisely control the fibre architecture allowing for the manufacture of components with mechanical properties tailored to service requirements. Many composites offer significant advantages in specific stiffness and/or specific strength over metals. This makes them attractive for applications where high mechanical performance and minimum weight are important. However, the wider acceptance of composites is based on their ability to offer a more cost‐effective alternative. In particular, composites also allow a dramatic reduction in the parts count in many applications, which leads to significant manufacturing advantages and greater economy.
Thermal imaging of composites
Authors: Almond D. P. , Peng W.
Published: March 2003

Active thermal imaging techniques and their applications to composite materials are reviewed. The techniques included are transient thermography, scanning thermal microscopy and scanning thermal probe microscopy. The factors that affect the images produced by both pulsed and periodic forms of active heating are considered. For pulsed heating, experimental and numerical modelling results for carbon fibre‐reinforced plastic are used to show how the resolution of subsurface features depends on their size and depth and on the anisotropy in thermal materials properties common in such composites. For periodic heating, thermal wave characteristics are introduced to show how the resolution of subsurface features also depends on modulation frequency and focal spot radius. Examples are given of the applications of scanning thermal microscopy and scanning thermal probe microscopy that illustrate the potential of these techniques for the imaging of composite materials.
Localization of strain in metal matrix composites studied by a scanning electron microscope‐based grating method
Authors: Crostack H.‐A. , Fischer G. , Soppa E. , Schmauder S. , Liu Y.‐L.
Published: March 2003

The deformation characteristics of the metal matrix composites Ag/Ni and Al/Al2O3 are studied at microstructural level by a scanning electron microscopebased grating method and finite element (FE) simulation. The measured strain was found to localize in narrow bands in the ductile matrix of both composites. In the case of the Al/Al2O3 composite, the bands are preferentially initiated in Al regions adjacent to the interface of large Al2O3 particles, leading to local strain maxima. The band positions found in the Ag/Ni composite are also affected by the less deformable Ni phase, but strain localization first occurs by sliding of single Ag grains sometimes located away from the Ni phase. Using a FE model of real phase geometry and measured border displacements as boundary conditions, the simulation agrees reasonably with the experiment. The differences in the case of the Al/Al2O3 composite are due to particle cracks and voids at the particle/matrix interface. This effect was found in the experiment but not considered in modelling. For the Ag/Ni composite the band positions agree fairly well. However, the level and gradient of strain is clearly different as the crystallographic orientation of the Ag grains was not accounted for in modelling.
Femtosecond near‐field scanning optical microscopy study of molecular thin films
Authors: Kawashima H. , Furuki M. , Tatsuura S. , Tian M. , Sato Y. , Pu L. S. , Tani T.
Published: March 2003

A near‐field scanning optical microscope has been combined with a two‐colour time‐resolved pump‐probe measurement system. It has a noise‐equivalent transmittance change of 5.0 × 10−5 for a probe pulse with an intensity of 30 nW. The system has been used for evaluating molecular thin films that have a domain structure, particularly for observing a gate action of the single domains. The results include key features to understand an origin of the domains and suggest that the film composition is uniform over a distance of several micrometres.
Morphology, fluorescence properties, and their photothermal changes of poly(substituted thiophene) films revealed by near‐field fluorescence microspectroscopy
Authors: Kurokawa N. , Yoshikawa H. , Masuhara H. , Hirota N. , Hyodo K.
Published: March 2003

The morphology and fluorescence spectrum of poly{3‐[2‐(N‐dodecylcarbamoyloxy)ethyl]thiophene‐2,5‐diyl} film were examined with spatial resolution of 100 nm using near‐field fluorescence microspectroscopy. Fluorescence spectra observed at protruding domains were blue‐shifted compared with flat areas, and further blue‐shift was observed there more appreciably by long‐time irradiation via a near‐field scanning optical microscope probe. It is considered that the polymer chains at the protruding domains take disordered conformations, in which conjugated lengths are shorter and further disordering can be induced more easily by irradiation compared with those in the flat areas.
Near‐field optics on silicon–electrolyte junctions
Authors: Diesinger H. , Bsiesy A. , Hérino R. , Huant S.
Published: March 2003

A technique allowing near‐field photocurrent (PC) mapping of silicon surfaces in contact with an electrolyte is presented. The illumination source is an optical fibre tip with a 100‐nm aperture. A shear force detection system controls the tip–sample distance while scanning the tip across the silicon–electrolyte interface. Topographic and PC images on SiO2/Si mesas both show 300 nm resolution. It is shown that this PC contrast is induced by the tip–topography interaction and hence the PC resolution is limited by the resolution of the topography. Indeed, PC mapping on topography‐less patterned porous‐silicon/silicon samples shows that the lateral resolution is only limited by the aperture size which is of the order of 100 nm.
Moulded photoplastic probes for near‐field optical applications
Authors: Kim B. J. , Flamma J. W. , Ten Have E. S. , Garcia‐Parajo M. F. , Van Hulst N. F. , Brugger J.
Published: March 2003

The inexpensive fabrication of high‐quality probes for near‐field optical applications is still unsolved although several methods for integrated fabrication have been proposed in the past. A further drawback is the intensity loss of the transmitted light in the ‘cut‐off’ region near the aperture in tapered optical fibres typically used as near‐field probes. As a remedy for these limitations we suggest here a new wafer‐scale semibatch microfabrication process for transparent photoplastic probes. The process starts with the fabrication of a pyramidal mould in silicon by using the anisotropic etchant potassium hydroxide. This results in an inverted pyramid limited by < 111 > silicon crystal planes having an angle of ∼ 54°. The surface including the mould is covered by a ∼ 1.5 nm thick organic monolayer of dodecyltrichlorosilane (DTS) and a 100‐nm thick evaporated aluminium film. Two layers of photoplastic material are then spin‐coated (thereby conformal filling the mould) and structured by lithography to form a cup for the optical fibre microassembly. The photoplastic probes are finally lifted off mechanically from the mould with the aluminium coating. Focused ion beam milling has been used to subsequently form apertures with diameters in the order of 80 nm. The advantage of our method is that the light to the aperture area can be directly coupled into the probe by using existing fibre‐based NSOM set‐ups, without the need for far‐field alignment, which is typically necessary for cantilevered probes. We have evidence that the aluminium layer is considerably smoother compared to the ‘grainy’ layers typically evaporated on free‐standing probes. The optical throughput efficiency was measured to be about 10−4. This new NSOM probe was directly bonded to a tuning fork sensor for the shear force control and the topography of a polymer sample was successfully obtained.
Local phase measurements of light in a one‐dimensional photonic crystal
Authors: Flück E. , Otter A. M. , Korterik J. P. , Balistreri M. L. M. , Kuipers L. , Van Hulst N. F.
Published: March 2003

For the first time the local optical phase evolution in and around a small, one‐dimensional photonic crystal has been visualized with a heterodyne interferometric photon scanning tunnelling microscope. The measurements show an exponential decay of the optical intensity inside the crystal, which consists of a periodic array of subwavelength air rods fabricated in a conventional ridge waveguide. In addition it is found that the introduction of the air rods has a counterintuitive effect on the phase development inside the structure. The heterodyne detection scheme allows the detection of low‐intensity scattered waves. In the vicinity of the scattering air rods phase singularities are found with a topological charge of plus or minus one.
Diamond colour centres as a nanoscopic light source for scanning near‐field optical microscopy
Authors: Kühn S. , Hettich C. , Schmitt C. , Poizat J‐PH. , Sandoghdar V.
Published: March 2003

Recently it was shown that a single molecule at cryogenic temperatures could be used as a local light source for illumination of a sample in the near field. Conventional light‐emitting systems such as dye molecules and semiconductor quantum dots could also be used for this purpose, but they suffer from lack of photostability. However, colour centres in diamond have been found to be remarkably stable against bleaching and blinking effects. Here we present the first SNOM images taken with nanoscopic diamond crystals as a light source.
In situ observation of oxygen deficiency occurring during electron irradiation in high Tc superconductor YBa2Cu3Oy
Authors: Akase Z. , Tomokiyo Y. , Watanabe M.
Published: March 2003

The decreasing process of oxygen in YBa2Cu3Oy is investigated through high resolution electron microscopy (HREM) and convergent beam electron diffraction (CBED). Measurements of the axial length in HREM images show that oxygen content y decreases faster near a twin boundary than at the inner part of a twin lamella. The transformation from an orthorhombic to a tetragonal phase starts at a twin boundary and the transformed region propagates to an inner region of lamella. Lattice strains are observed near boundaries between transformed and non‐transformed regions. The transformation is almost complete within 30 s during observation of HREM images at 400 kV and at room temperature. A value of y was quantitatively measured by analysing observed intensities of energy‐filtered CBED patterns with the dynamical theory. The value of y decreases from 6.9 to 6.5 when 200 kV electrons are irradiated for 160 s in a microscope at 108 K. More precise analysis of the intensities provides information on charge distribution along the c‐axis as well as local oxygen content at a spatial resolution of several nanometres.
Centrifuge polarizing microscope. II. Sample biological applications
Authors: Inoué S. , Goda M. , Knudson R. A.
Published: March 2003

The rationale, design and general performance of the CPM (centrifuge polarizing microscope) were described in Part I of this study (Inouéet al. J. Microsc. 201 (2001) 341–356. In this second part, we describe observations on several biological samples that we have explored over the past two years using the CPM.

As described in the first part of the study, although the CPM was basically designed as a high‐extinction centrifuge polarizing microscope, it also allows observations of the specimen exposed to high centrifugal fields up to 10 500 × g (earth's gravitational acceleration) in fluorescence (532‐nm excitation) and in DIC (differential interference or Nomarski contrast).
On the coupling efficiency of metal‐coated near‐field probes
Authors: Alvarez L. , Xiao M.
Published: March 2003

A theory for calculating the optical transmission of nanometric circular apertures in a thick and perfectly conducting screen coated upon an optical fibre has been developed. The theory is intended for the study of near‐field probes and differs from other well known theories of radiation transmission through subwavelength apertures in the fact that it includes an optical fibre, making possible to distinguish which part of energy passing through the aperture is effectively coupled in guided modes. In a scanning near‐field optical microscope tip, only the guided modes will reach the photodetector, and will contribute to the final read‐out. A numerical calculation for different fibre parameters, to show the dependence of the guided transmission coefficient for the guided modes of the fibre, is presented. The agreement of the theory with earlier calculations where the optical fibre is not included is emphasized.
Fluorescence scanning near‐field optical microscopy of polyfluorene composites
Authors: Stevenson R. , Milner R. G. , Richards D. , Arias A. C. , Mackenzie J. D. , Halls J. J. M. , Friend R. H. , Kang D.‐J. , Blamire M.
Published: March 2003

Fluorescence scanning near‐field optical microscopy (SNOM) is used to investigate binary polyfluorene‐based composites of varying composition. The samples investigated contain blends of the polymer poly(9,9′‐dioctylfluorene‐cobenzothiadiazole), F8BT, with similar polyfluorenes of wider band gap. Images acquired from a film containing 50% by weight F8BT exhibit a high degree of correlation between the topography and fluorescence, with an F8BT‐rich phase which protrudes from the surface of the film forming isolated regions with sizes from hundreds of nanometres to several micrometres. A film containing 10% by weight F8BT also has micrometre‐size F8BT‐rich regions, but also present are small and locally varying proportions of F8BT in the other polyfluorene component phase, indicating a hierarchy of phases within this sample. The fluorescence and topographic images of a third sample studied, containing 90% by weight F8BT, display no correlation, demonstrating that it is not always appropriate to use topographic information to determine the phase structure within polymer blends. The fluorescence SNOM images acquired from these samples are able to assist our understanding of the photovoltaic efficiency of devices fabricated from these films, which are governed by the extent of the interfacial area between these two constituent polymers.
Near‐field optical microscopy based on microfabricated probes
Authors: Eckert R. , Freyland J. M. , Gersen H. , Heinzelmann H. , Schürmann G. , Noell W. , Staufer U. , De Rooij N. F.
Published: March 2003

We demonstrate high resolution imaging with microfabricated, cantilevered probes, consisting of solid quartz tips on silicon levers. The tips are covered by a 60‐nm thick layer of aluminium, which appears to be closed at the apex when investigated by transmission electron microscopy. An instrument specifically built for cantilever probes was used to record images of latex bead projection patterns in transmission as well as single molecule fluorescence. All images were recorded in constant height mode and show optical resolutions down to 32 nm.
An improved method for preparing thick sections for immuno/histochemistry and confocal microscopy and its use to identify rare events
Authors: Monaghan P. , Watson P. R. , Cook H. , Scott L. , Wallis T. S. , Robertson D.
Published: March 2003

Detection of rare events within solid tissues by immunocytochemistry is aided by imaging thick sections. Sections of 40–100 µm thickness of paraformaldehyde‐fixed solid tissue can be prepared by use of a vibrating microtome and when immunolabelled these sections can be imaged in a confocal microscope. This approach provides excellent preservation of the structure of the sample and imposes minimal antigenic damage. In studies of the invasion of the bovine intestinal epithelium by Salmonella, this method has allowed detection of individual invading bacteria within large samples. The thick vibrating microtome sections were also used for the detection of rare apoptotic cell nuclei identified by TUNEL staining.
Microscopy and computational modelling to elucidate the enhancement factor for field electron emitters
Authors: Edgcombe C. J. , Valdrè U.
Published: March 2003

We report on the computation of the electric field at the surface of single‐tip field emitters for a variety of geometries and wide range of geometrical parameters. In conjunction with experimental work, this has allowed the determination of quantities useful for characterizing and comparing the performance of field emitters. The ratio of the field at the tip surface to field at a tip supporting base (enhancement factor) has been calculated for hemispherical tips with parallel or conical shanks, for ratios of tip length to tip radius from 1 to 3000. Enhancement factors greater than 1000 are achievable with suitable tip geometry. The threshold voltage dependence on the tip–anode separation for cylindrical tips facing a flat anode has also been calculated and reported.
Electron energy‐loss near‐edge structure – a tool for the investigation of electronic structure on the nanometre scale
Authors: Keast V. J. , Scott A. J. , Brydson R. , Williams D. B. , Bruley J.
Published: March 2003

Electron energy‐loss near‐edge structure (ELNES) is a technique that can be used to measure the electronic structure (i.e. bonding) in materials with subnanometre spatial resolution. This review covers the theoretical principles behind the technique, the experimental procedures necessary to acquire good ELNES spectra, including potential artefacts, and gives examples relevant to materials science.
Is plastic flow always controlled by dislocation mobility? An answer from in situ transmission electron microscopy straining tests
Authors: Louchet F. , Doisneau‐Cottignies B. , Calonne O. , Fraczkiewicz A. , Janecek M. , Guelton N.
Published: March 2003

In situ transmission electron microscopy (TEM) straining experiments are used to illustrate in two extreme cases the possible role of dislocation nucleation and exhaustion as a controlling factor in plastic flow. In the first example (FeAl intermetallic compounds), a thermally activated dislocation exhaustion is responsible for an anomalous stress–temperature dependence and an associated small strain rate sensitivity, the latter being evidenced during in situ experiments through unstable localized slip. The second example (heavily drawn pearlite) shows specific dislocation loop nucleation processes that may account for the Hall–Petch law breakdown characteristic of fine scale nanostructures.
Nano‐optical reversible switching of organic photochromes*
Authors: Rath S. , Mager O. , Klingler T. , Port H.
Published: March 2003

Photochromic fulgides are UHV‐deposited in ultrathin films exhibiting intrinsic nanostructures. The two isomeric states (C and E) are detected in absorption via scanning near‐field optical microscopy (SNOM). The optical image contrast measured in SNOM corresponds to the nanostructure topography observed in atomic force microscopy. Nano‐optical reversible switching of individual nanostructures is demonstrated and possible applications to rewritable optical recording on the nanoscale are discussed.
A new surface characterization technique: RIMAPS (Rotated Image with Maximum Average Power Spectrum)
Authors: Fuentes N. O. , Favret E. A.
Published: March 2003

This work introduces a new imaging technique, Rotated Image with Maximum Average Power Spectrum (RIMAPS), for use in determining orientation and characteristics of surface topography. It consists of computing the maximum value of the averaged power spectrum, given by one step of the two‐dimensional Fourier transform, for each angle of rotation of a digitized image. The basic measurement science of this technique is described and different cases are studied. The characterization of simple geometrical figures explains the meaning of peaks and their angular positions given by RIMAPS analysis. A known surface pattern made on a sample of pure copper, mechanically ground, is used to study reproducibility, dependence on image quality and topography scale relative to pixel size and magnification. Samples of pure zinc, mechanically ground and chemically etched, were used to show the main features of RIMAPS analysis when characterizing a more complicated pattern on a real surface. All the studies performed under different conditions for observation and acquisition of images give strong evidence of the stability and robustness of RIMAPS as a technique for the characterization of topography.
Efficient embedding technique for preparing small specimens for stereological volume estimation: zebrafish larvae
Authors: Hill A. J. , Howard C. V. , Cossins A. R.
Published: March 2003

Stereological sampling regimes, in particular volume and number estimation, often require systematic uniformly random sections throughout a specimen. A method has been developed to increase the efficiency of preparing fish larvae for sectioning prior to histological or stereological analysis. Embedding a group of larvae in a resin block using this technique greatly reduces the quantity of sections produced and allows easy assessment of sample groups. Saving time in this way therefore makes stereology a more viable research tool.
Simultaneously identifying S‐phase labelled cells and immunogold‐labelling of vinculin in focal adhesions
Authors: Owen G. RH. , Meredith D. O. , Gwynn I. Ap , Richards R. G.
Published: March 2003

A new combination of autoradiography and immunolabelling techniques is presented that allows the simultaneous identification of both S‐phase cells and their focal adhesions using scanning electron microscopy. The technique allows both labels to be discerned visually by their unique shapes and location within and on the cell. S‐phase cells were radio‐labelled with a pulse of tritiated thymidine, selectively incorporated into synthesizing DNA. The cells were then immunogold‐labelled for the focal adhesion protein, vinculin, prepared for autoradiography, and embedded in resin. The resin was then polymerized before removing the substrate, to expose the embedded cell undersurface. Electron‐energy ‘sectioning’ of the sample by varying the accelerating voltage of the electron beam allowed separate S‐phase cell identification in one electron‐energy ‘section’ and visualization of immunogold label in another ‘section’, within the same cell. As a result of applying this technique it was possible to positively identify S‐phase cells and immunogold‐labelled focal adhesions on the same cell simultaneously, which could be used to quantify focal adhesion sites on different substrates.
On the orientational analysis of planar fibre systems from digital images
Authors: Kärkkäinen S. , Jensen E. B. V. , Jeulin D.
Published: March 2003

The orientational characteristics of fibres in digital images are studied. The fibres are modelled by a planar Boolean model whose typical grain is a thick (coloured) fibre. The aim is to make stereological inference on the rose of directions of the unobservable central fibres from observations made on a digital image of the thick fibres. For central fibres, the relation between the rose of directions and the point intensity, observed on a sampling line, is known. We derive, under regularity conditions, the relation between the unobservable point intensity and the scaled variogram observed on the line in a binary and a greyscale image. Using such a relation, it is possible to draw inference about the rose of directions from the scaled variogram, which is easy and quick to determine in a digital image.
Presence of Gonadotropin‐Releasing Hormone (GnRH) mRNA in Rathke’s Pouch and Effect of the GnRH‐Antagonist ORG 30276 on Lactotroph Development
Authors: Van Bael  , Seuntjens  , Proesmans  , Denef
Published: March 2003

Reverse transcription‐polymerase chain reaction (RT‐PCR) with specific GnRH cDNA primers performed on RNA from Rathke’s pouches removed from pregnant rats at day 12 of gestation (e12) generated an amplified DNA fragment of the expected length (357 bp). The fragment hybridized with a labeled GnRH cDNA probe in Southern blotting. DNA sequencing demonstrated identity with the known nucleotide sequence of the corresponding segment of rat GnRH cDNA. To determine whether GnRH mRNA was located in the Rathke’s pouch cells or in remnants of surrounding tissue not completely removed during preparation, the pouches were treated with collagenase. Based on light and electron microscopic examination, this treatment disconnected virtually all contaminating tissue, allowing the ‘pure’ Rathke’s pouches to be picked‐up separately. Again, RT‐PCR generated a DNA fragment of the expected length, the fragment hybridized with the GnRH cDNA probe and showed the nucleotide sequence of the corresponding region of rat GnRH cDNA. In Rathke’s pouches established in explant culture on e12, lactotrophs were well developed when examined 9 days later by immunostaining of prolactin in paraffin‐embedded sections of the tissue. Computerized image analysis showed prolactin immunoreactivity in 8.0±1.1% of the section area. Addition of the potent and long‐acting GnRH antagonist ORG 30276 to the crude preparation of Rathke’s pouches caused a significant decrease in the relative area staining for prolactin. The latter effect was abolished by concomitant addition of GnRH. In preparations of pure Rathke’s pouches (collagenase‐treated), ORG 30276 failed to affect the relative area of prolactin immunoreactivity. GnRH mRNA remained expressed in explants of both crude and pure Rathke’s pouches until the end of the culture period. It is concluded that the GnRH gene is expressed in Rathke’s pouch as early as e12 and that GnRH may be a physiological paracrine/autocrine peptide stimulating the development of lactotrophs. Mesenchymal and/or neural factors may be essential for the latter system to function.
Interaction Between Glucocorticoids and Corticotropin Releasing Hormone (CRH) in the Regulation of the Pituitary CRH Receptor in vivo
Authors: Ochedalski Tomasz , Rabadan‐Diehl Cristina , Aguilera Greti
Published: March 2003

Acute stress causes biphasic changes in corticotropin releasing hormone (CRH) receptor mRNA expression with an early decrease followed by an increase. However, in the absence of glucocorticoids in adrenalectomized rats, stress results in prolonged CRH receptor (CRH‐R) mRNA loss, suggesting that interactions between glucocorticoids and hypothalamic factors are critical for regulation of CRH receptor mRNA. To address this question, CRH binding, type‐1 CRH‐R mRNA, POMC mRNA and POMC hnRNA expression were measured by binding autoradiography and in situ hybridization in pituitaries from intact and adrenalectomized rats. CRH‐R mRNA decreased by 59% 5 h after injection of corticosterone (10 mg sc) and returned to basal levels by 18 h, a time when plasma corticosterone concentrations were still elevated, and CRH binding and POMC hnRNA were significantly reduced. Elevations in plasma corticosterone in the range of acute stress by injection of 2 mg sc caused CRH‐R mRNA expression to return to near basal values by 6 h, after a 52% and 39% decrease at 2 h and 4 h. More transient changes were seen after a single injection of CRH (1 μg), with a 44% decrease in CRH‐R mRNA and a 175% increase in POMC hnRNA by 2 h, returning to basal values by 4 h. The transient effect of CRH was not due to clearance of CRH from the circulation or receptor desensitization since CRH receptor mRNA expression also recovered after injection of a higher dose (10 μg) or repeated injections of CRH which caused sustained increases in plasma CRH and pituitary POMC hnRNA levels. CRH injection in adrenalectomized rats decreased CRH‐R mRNA for up to 6 h, suggesting that glucocorticoids are permissive for the recovery of CRH‐R mRNA. Supporting this hypothesis, simultaneous injection of corticosterone and CRH restored CRH‐R mRNA expression by 4 h, and increased CRH binding 4 h and 6 h after injection. The data show that interaction between CRH and glucocorticoids counteracts individual inhibitory effects of these regulators alone, and that such effects are likely to contribute to the regulatory pattern of pituitary CRH receptors during acute stress.
Neuropeptide FF in the Rat Adrenal Gland: Presence, Distribution and Pharmacological Effects
Authors: Labrouche Sylvie , Laulin Jean‐Paul , Le Moal Michel , Tramu Gérard , Simonnet Guy
Published: March 2003

Neuropeptide FF (NPFF, FLFQPQRFamide) is an FMRFamide‐like octapeptide exhibiting antiopiate activity. The presence of both NPFF‐immunoreactivity (NPFF‐IR) and NPFF‐specific receptors has been described in the mammalian central nervous system (CNS). The peripheral effects of NPFF indicate that NPFF‐IR material is present outside the CNS. Biochemical and immunohistochemical methods enabled us to determine the presence and distribution of NPFF‐IR in the rat adrenal gland. The amount of NPFF‐IR material in whole gland was estimated by radioimmunoassay to be 19.00±4.00 fmol/gland. High performance liquid chromatography analysis of adrenal extracts revealed a single molecular form which coeluted with authentic NPFF. Demedullation decreased adrenal NPFF‐IR content, indicating that NPFF‐IR was present in both cortex and medulla. Light microscopy revealed NPFF‐IR in beaded fibers confined in the outer part of the cortex and in medullary cells. Double‐labeling with antityrosine‐hydroxylase and anti‐NPFF antibodies showed NPFF‐IR in cortical catecholaminergic postganglionic fibers restricted to the subcapsular and glomerulosa zonae. NPFF‐IR was also located in medullary chromaffin cells and in rays and islets of chromaffin cells dispersed throughout the cortex. Insulin‐induced hypoglycemia did not alter NPFF‐IR content. Denervation lowered adrenal NPFF‐IR content. These data indicate that this peptide is present in nerve fibers of extrinsic origin. In vitro approaches using adrenal slices have shown that NPFF inhibited aldosterone release in a dose‐dependent manner. Taken together, these data suggest that NPFF may participate in the control of aldosterone production and adrenal blood supply.
Photoperiod Affects the Ability of Testosterone to Alter Proopiomelanocortin mRNA, but not Luteinizing Hormone‐Releasing Hormone mRNA, Levels in Male Sheep
Authors: Hileman Stanley M. , Kuehl David E. , Jackson Gary L.
Published: March 2003

This study tested the hypothesis that photoperiod affects the ability of testosterone to reduce proopiomelanocortin (POMC) mRNA levels in the arcuate nucleus and luteinizing hormone‐releasing hormone (LHRH) mRNA levels in both the preoptic area (POA) or medial basal hypothalamus (MBH). Twenty castrated male sheep were assigned to one of four treatment groups (i): short days (SD;

n=5) (ii), short days with testosterone (SD+T; n=5) (iii), long days (LD; n=5), or (iii) long days with testosterone (LD+T; n=5). Blood samples were collected twice weekly for the last 3 weeks of photoperiod treatment and assessed for LH to validate the response to photoperiod. After evaluating LH levels, one animal each from the LD+T and SD+T groups was excluded from the analyses. Mean concentrations of LH were lower (P<0.01) in the LD+T group than in the other treatment groups, which did not differ (P>0.10) from each other. Neither POA nor MBH LHRH mRNA levels were affected (P>0.10) by treatment. Conversely, POMC mRNA levels were suppressed (P<0.01) in the LD+T males compared with the other treatment groups which did not differ (P>0.10) from each other. These observations suggest that photoperiod specific, testosterone‐induced alterations in LHRH mRNA levels are not a mechanism whereby testosterone suppresses LHRH release, and that increased beta‐endorphin synthesis and release do not mediate testosterone‐induced seasonal suppression of LHRH release.
Glucose Availability Modulates the Timing of the Luteinizing Hormone Surge in the Ewe
Authors: Medina Christopher L. , Nagatani Shoji , Darling Tiffany A. , Bucholtz§ David C. , Tsukamura Hiroko , Maeda Kei‐ichiro , Foster Douglas L.
Published: March 2003

To determine if glucose availability modulates the timing of the positive feedback action of oestrogen on gonadotropin secretion, we monitored the estradiol‐induced luteinizing hormone (LH) surge in sheep (n=5/group) made transiently hypoglycemic by insulin. Experiment 1 determined an effective insulin treatment, one which would depress tonic LH secretion. Two injections of insulin (5 IU/kg iv) 4 h apart were found to induce extended hypoglycemia (10–13 h) and to decrease the LH pulse frequency for 8 h (5.0±0.32 pulses/4 h before versus 2.5±0.34 pulses/4 h after insulin; P<0.05; mean±SEM). Using this same paradigm, experiment 2 determined the influence of the transient hypoglycemia on the LH surge mechanism. In control sheep, estradiol (subcutaneous implants at hour 0) evoked an LH surge with a latency period of 12.4±0.5 h. When insulin was administered either before (hours −4 and 0) or after the estradiol stimulus (hours 4 and 8, or 12 and 16), the onset of the LH surge was delayed to 29.0±2.4 h (average of all three time groups, P<0.05). Infusion of glucose from hours 12–30, along with insulin, prevented hypoglycemia and restored the normal timing of the oestrogen‐induced LH surgeto that of controls (15.4±0.93 h, P>0.05). These findings suggest that not only is the tonic mode of LH secretion sensitive to metabolic fuel availability, but the surge mode of LH secretion is as well.
Supraoptic Oxytocin and Vasopressin Neurones Show Differential Sensitivity to the Neurosteroid Pregnenolone Sulphate
Authors: Richardson  , Wakerley
Published: March 2003

The neurosteroid pregnenolone sulphate (PS) interacts allosterically with ionotropic glutamate receptors and thereby could be an important modulator of activity within the hypothalamic magnocellular nuclei. The present in‐vitro study therefore examined the effect of perifusion of PS (100 μM) on activity of supraoptic oxytocin (OT) and vasopressin (VP) neurones, in which firing was stimulated by local application of glutamate, NMDA or AMPA. In the presence of locally applied glutamate, PS significantly potentiated firing in putative VP neurones, but had little effect on putative OT neurones. In both cell types, PS increased firing in the presence of NMDA and depressed firing in the presence of AMPA. The action of PS on glutamate‐ and NMDA‐stimulated firing was unaffected by addition of the GABAA receptor antagonist, picrotoxin (50 μM). The suppressive action of PS on AMPA‐stimulated firing was, however, reversed by picrotoxin and therefore probably requires intact GABAergic transmission for its expression. When putative VP neurones were stimulated by local application of K+, in the presence of picrotoxin, PS evoked a small increase in the ongoing activity, although this did not reach significance. When the glutamate receptor antagonists, NBQX (20 μM) and AP5 (40 μM), were included in the medium, no change in K+–stimulated firing was observed. Hence PS has no effect on activity of putative VP neurones in the absence of exogenous and endogenous glutamate excitation. In conclusion, PS selectively potentiates glutamate‐stimulated activity in putative VP neurones, probably via NMDA receptors, thus providing a mechanism whereby this neurosteroid might exert rapid non‐genomic effects on VP secretion. The lack of effect of PS in putative OT neurones probably relates to the relatively small involvement of NMDA receptors in mediating glutamate excitation in this cell type.