bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrophoretic transfer of protein‐pigment complexes from non‐denaturing gels to electron microscopy grids
Authors: GRÄBER S. , LYON M. K.
Published: August 2011

Different fractions of a mixture of protein–pigment complexes have been separated from one another by non‐denaturing gel electrophoresis. These complexes were prepared for observation by inserting electron microscope grids directly into the focused bands of the pigment–protein complexes and resuming electrophoresis for a brief time, so that the complexes were deposited onto the grids. It was found that complexes deposited from each band exhibited distinctly different appearances. It was also found that the exact conditions of electrophoretic deposition onto the grids affected the appearance of the complexes. The protein–pigment complexes were characterized additionally by spectroscopy and denaturing gel electrophoresis.
Restoration of confocal images for quantitative image analysis
Authors: VAN DER VOORT H. T. M. , STRASTERS K. C.
Published: August 2011

Quantitative studies of three‐dimensional (3‐D) structure of microscopic objects have been made possible through the introduction of microscopic volume imaging techniques, most notably the confocal fluorescence microscope (CFM). Although the CFM is a true volume imager, its specific imaging properties give rise to distortions in the images and hamper subsequent quantitative analysis. Therefore, it is a prerequisite that confocal images are restored prior to analysis. The distortions can be divided into several categories: attenuation of areas in the image due to self‐absorption, bleaching effects, geometrical effects and distortions due to diffraction effects. Of these, absorption and diffraction effects are the most important. This paper describes a method aimed at the correction of diffraction‐induced distortions. All the steps necessary in restoring confocal images are discussed, including a novel method to measure instrumental properties on a routine basis. To test the restoration procedure an image of a fluorescent planar object was restored. The results show a considerable improvement in the z‐resolution and no ringing artefacts. The relevance of the method for image analysis is demonstrated by a comparison of results of applying 3‐D texture analysis to restored and unrestored images of a synthetic object. Furthermore, the method can be successfully applied to noisy fluorescence images of biological objects, such as interphase cell nucei.
Holography of holes, with electrons and photons
Authors: KREUZER H. J. , FINK H.‐W. , SCHMID H. , BONEV S.
Published: August 2011

Submicrometre holes in a gold film are illuminated with coherent low‐energy electrons in the low‐energy electron point‐source microscope. The images are interpreted as holograms and simulated using the Fresnel‐Kirchhoff formula. A reconstruction scheme yields the wave front at the object, in good agreement with transmission electron microscope images of the same hole structures. Laser experiments of similarly shaped, but scaled appropriately, hole structures reveal the same features.
Correction for the effects of elastic scattering in core‐loss quantification
Authors: WONG K. , EGERTON R. F.
Published: August 2011

In electron energy‐loss spectroscopy (EELS) elemental quantification, the measured intensity ratio has been observed to change with thickness (t) when a collector aperture is used. This change has been shown semi‐quantitatively to be a result of elastic scattering. In this paper, a method based on the elastic Lenz model is used to correct for the effects of elastic scattering in amorphous materials. The validity of the Lenz model is confirmed by studying the zero‐loss component of EEL spectra recorded from several amorphous materials as a function of thickness and collection angle. The experimental data are found to be consistent with calculations based on the Lenz model and Poisson statistics. Based on these calculations, a simple scheme is proposed to obtain the important scattering parameters t/Λe, where Λe is the elastic mean free path, and θ0, the characteristic angle of elastic scattering, with adequate accuracy from the low‐loss data. These values are then used to correct for the effect of elastic scattering in core‐edge quantification. The procedure is applied to Al2O3 and good agreement with experimental data up to t = 2Λe is found; beyond 2Λe the method can serve as a partial correction.
Use of light and scanning electron microscope techniques to monitor microstructural changes in aluminium‐based metal matrix composites
Authors: WHITEHOUSE A. F. , SHAHANI R. A. , CLYNE T. W.
Published: August 2011

Microstructural developments in discontinuously reinforced aluminium composites have been monitored during a variety of thermo‐mechanical treatments. The material, made by powder blending or casting, followed by extrusion, contains particulate or short‐fibre alumina in a matrix of commercial purity aluminium. Attention is centred on the distribution of reinforcement, the fine alumina particles derived from the prior oxide layers on the aluminium powder, and the effects that these inclusions have on cavitation, recovery and recrystallization processes. Optical metallography, by viewing anodized sections in polarized light, is used to reveal grain and coarse subgrain structures. Scanning electron microscopy has also been employed to reveal fine oxide stringers, cavities and (in backscattered mode) fine subgrain structures. The emphasis in this paper is on the type of information that can be obtained for these materials from careful application of standard microscopy techniques.
Evaluation of triple‐band filters for quantitative epifluorescence microscopy
Authors: LOWY R. J.
Published: August 2011

The performance characteristics of two sets of triple‐band epifluorescence filters have been evaluated for use with digitally enhanced fluorescence microscopy. Use of such filters, at most, requires movement of the excitation filter, while the dichroic and emission filters remain fixed, allowing multi‐wavelength imaging to be performed on standard microscopes. The dyes appropriate for use with these particular filters include Texas Red (TR), Bodipy (BD), FITC and Cascade Blue (CB), four fluorophores now commonly conjugated to both immunochemical probes and other proteins and lipids of biological interest. Good spectral separation existed for most experimental conditions allowing accurate localization of the different fluorophores during multi‐wavelength imaging. Anomalous responses were observed during near‐UV excitation at high concentration for some dyes. Scanning spectrofluorometry demonstrated that concentration‐dependent spectral shifts occurred, resulting in large increases in near‐UV absorbance. Despite the complexity of concentration and dye‐interaction effects, quantitative measurements of dye concentration could be made, even in regions of multiple dye co‐localization. Therefore, multi‐band pass filters are an additional valuable approach for performing quantitative fluorescence microscopic imaging.
Analysis of the spatial distribution of AgNOR proteins in cell nuclei using simultaneous confocal scanning laser fluorescence and transmitted light microscopy
Authors: PARAZZA F. , BERTIN E. , WOZNIAK Z. , USSON Y.
Published: August 2011

The selective staining of nucleolar acidic proteins by silver dyes (AgNOR technique) is a promising tool to discriminate malignant cells from benign cells in tumour pathology. Until now, the existing methods to measure the distribution of the AgNOR proteins were performed in two‐dimensional space. Confocal scanning laser microscopy (CSLM) and progress in computer sciences make it possible to image biological specimens in three dimensions. We have used these techniques to develop a method to discriminate cells by comparing the three‐dimensional (3‐D) spatial distribution of the AgNOR proteins. The distribution of the AgNOR proteins was described by the following measurements: the volume fraction of the nucleus occupied by the AgNOR‐stained aggregates, the distances between the aggregates, their number per nucleus, the distance of each aggregate from the nuclear border and their anisotropy. CSLM was used to acquire simultaneously the 3‐D data set of the nucleus (confocal fluorescence) and the 3‐D data set of the location of the AgNOR proteins (brightfield transmission). Two biases, due to the different modes of microscopy and the silver labelling technique, had to be taken into account in the evaluation of the measurements. A preliminary study on three lines of lymphocytic cells shows that these parameters are discriminatory. Thus, this method makes it possible to combine two different modes of scanning laser microscopy for a 3‐D quantitative analysis and is potentially useful for assessing the spatial distribution of AgNOR proteins in tumour pathology.
A new confocal scanning beam laser MACROscope using a telecentric, f‐theta laser scan lens
Authors: DIXON A. E. , DAMASKINOS S. , RIBES A. , BEESLEY K. M.
Published: August 2011

A new confocal scanning beam system (MACROscope) that images very large‐area specimens is described. The MACROscope uses a telecentric, f‐theta laser scan lens as an objective lens to image specimens as large as 7·5 cm × 7·5 cm in 5 s. The lateral resolution of the MACROscope is 5 μm and the axial resolution is 200 μm. When combined with a confocal microscope, a new hybrid imaging system is produced that uses the advantages of small‐area, high‐speed, high‐resolution microscopy (0·2 μm lateral and 0·4 μm axial resolution) with the large‐area, high‐speed, good‐resolution imaging of the MACROscope. The advantages of the microscope/MACROscope are illustrated in applications which include reflected‐light confocal images of biological specimens, DNA sequencing gels, latent fingerprints and photoluminescence imaging of porous silicon.
The three‐dimensional point spread functions of a microscope objective in image and object space
Authors: TAO L. , NICHOLSON C.
Published: August 2011

The three‐dimensional point spread function (3‐D PSF) of an optical system in image space is distinguished from the 3‐D PSF in object space and the relation between the two 3‐D PSFs is derived. By using this relation one 3‐D PSF can be easily obtained from the other. The 3‐D PSFs are given in a single integral expression, which can be computed numerically. The results of this study can be used in 3‐D image processing for microscopy and have been applied to the analysis of the diffusion of fluorescent molecules in a 3‐D porous medium.
Propagation function: constant time algorithms
Authors: SCHMITT M.
Published: August 2011

This paper deals with constant time algorithms computing the propagation function of X, assumed to be a simply connected planar set. This function, extensively used for object measurements and shape characterization, is defined as the geodesic distance to the furthest point in X, where the geodesic distance between two points in X is the lower bound of the length of paths entirely lying inside X and linking the two points. When the plane is equipped with a distance derived from a regular n‐sided polygon (distances on image lattices), efficient algorithms for the propagation function are based on the following remark: the furthest points to any point in X may have only a few possible locations Y. In this paper, it is shown that, as in the convex case, there exists a set Y with at most n elements, giving rise to constant time algorithms. Algorithms designed to compute this set Y and an implementation for the square lattice equipped with the four‐connectivity distance, by means of at most seven geodesic balls, whatever the shape of X, are described.
Improved specimen holders for the scanning electron microscopy of insects and other small objects
Authors: BEATON C. D. , TAYLOR R. W.
Published: August 2011

Two specimen holders for use in scanning electron microscopy (SEM) of insect and other specimens glued to triangular cardboard points are described. They have important advantages over standard metal stub mounts. Diverse, precisely orientated, viewing angles are possible using single specimens, which can afterwards be re‐pinned for return to the collection.
A new tool for the observation of embryos and other large specimens: confocal theta fluorescence microscopy
Authors: STELZER E. H. K. , LINDEK S. , ALBRECHT S. , PICK R. , RITTER G. , SALMON N. J. , STRICKER R.
Published: August 2011

A theta microscope for three objective lenses was designed and built at the European Molecular Biology Laboratory (EMBL). It was used to evaluate the principles of confocal theta fluorescence microscopy and to prove its ability to handle biological specimens. A resolution improvement over confocal fluorescence microscopy has been predicted due to a detection at an angle to the illumination axis. This is verified using fluorescent beads and a fluorophore‐coated capillary. The suitability of theta microscopy for the observation of biological specimens is verified by observing a FITC‐labelled mouse embryo. In addition, the design of a theta microscope and the handling of biological material are explained in detail.
Imaging of cytoskeletal elements by low‐temperature high‐resolution scanning electron microscopy
Authors: CHEN Y. , CENTONZE V. E. , VERKHOVSKY A. , BORISY G. G.
Published: August 2011

Actin filaments and microtubules, both in situ and in vitro, were imaged using high‐resolution scanning electron microscopy (HRSEM) at low temperature. For visualization of cytoskeletal elements in situ, fibroblasts were first extracted and fixed; for cytoskeletal elements in vitro, purified proteins were polymerized and fixed. Both types of specimen were then subjected to plunge freezing, controlled freeze‐drying, cryo‐sputter coating with a thin chromium layer, cryo‐ transferring and cryo‐observation in an FESEM. The three‐dimensional architecture of the cytoskeleton was well preserved, permitting examination of the structural relationships among cytoskeletal elements. Actin filaments and microtubules were identified by their characteristic helical features. Two periodicities of actin filaments, the short pitch of the left‐handed helix measured at 5·5 nm and the 37‐nm‐long pitch helix, were revealed. Individual protofilaments were seen in microtubules as well as the characteristic 4‐nm repeat of tubulin subunits along the protofilament. Clathrin cages were also observed. This technique provides a powerful approach for direct imaging of macromolecular structures with high contrast and high signal‐to‐noise ratio at a resolution of 2–3 nm.
A ‘mini‐microtome’ for preparing serial sections directly on a light microscope stage
Authors: BISERKOV V. , KOSTADINOVA A.
Published: August 2011

A simple ‘mini‐microtome’ is described which can be attached to a standard compound microscope. The device enables small biological specimens, such as parasitic helminths, to be sectioned with precise orientation.
Holographic measurement of the wave aberration of an electron microscope by means of the phases in the Fourier spectrum
Authors: FU Q. , LICHTE H.
Published: August 2011

The resolution limit achievable by holographic correction of the aberrations of an electron microscope depends critically on the information available about the microscope parameters when the hologram was taken. The measuring technique based on symmetry relations of the phases in the Fourier spectrum of the reconstructed electron wave is outlined and experimentally tested.
The advantages of using linear and non‐linear second‐order finite element methods in electron lens design
Authors: TAHIR K. , HODKINSON J.
Published: August 2011

Powerful personal computers (PCs) and advanced software now enable electron optical designers to set up a proposed design, revise it and finally obtain a rapid overview of its electron optical properties. The finite element method (FEM) in general is useful for the computer modelling of electron lenses as the mesh layout is well adapted to the shape of electrodes and polepieces. The second‐order FEM is especially relevant to curved boundaries, for example in spherical‐double and single‐pole lenses. In the case of high‐resolution magnetic objective lenses, where marked saturation of the polepieces is an essential feature of the design, special care is needed to reduce the computational errors. Since the difference in saturation magnetization of soft iron and a typical iron–cobalt is around 10%, this means that for a meaningful comparison between the two materials, the computation errors should not exceed 2%. This level of accuracy can be achieved easily with second‐order, non‐linear computations even when very low mesh density is used. In first‐order, non‐linear computations a very high mesh density is needed, and this requires more care and effort to reach even lower levels of accuracy.
Use of the quasi‐classical approximation for projected potential calculation in HREM image simulation
Authors: TANG D. , DORIGNAC D.
Published: August 2011

A new method for high‐resolution electron microscope image simulation using the multislice approach is proposed: the calculation of the two‐dimensional projected potential of a crystal is performed directly in real space, from tabulated atomic potentials based on the relativistic Hartree–Fock–Slater model and following the quantum mechanical quasi‐classical approximation. The validity of the approximation is carefully examined by estimating the electron wave phase shift due to the crystal potential. The results are compared with those given by the conventional method, for which the projected potential is obtained by Fourier transforming the first Born electron scattering amplitudes.
Femtosecond pulse width control in microscopy by two‐photon absorption autocorrelation
Authors: BRAKENHOFF G. J. , MÜLLER M. , SQUIER J.
Published: August 2011

A technique for both the measurement and the control of the pulse width at the focal point of a high‐NA lens, based on two‐photon absorption interferometric autocorrelation, is presented. The technique is applied to measuring the pulse broadening induced on a pulse propagating through a high‐NA lens system for several objectives. It is known that the pulse width may increase by up to ≅50% of its original value by propagation through an objective which has wide field compensation for spherical and chromatic aberrations. The two‐photon absorption autocorrelation technique allows adjustment of the actual pulse width in the focus of a high‐NA lens through pre‐chirp compensation. The pulse width is shown to be almost independent of penetration depth into the sample, while the amplitude of the autocorrelation signal shows a strong decrease with depth. The ability to both measure and control the actual pulse width under strong focusing conditions is of direct importance to, among others, two‐photon absorption imaging approaches.
A novel force‐sensing arrangement for combined scanning force/scanning tunnelling microscopy applied to biological objects
Authors: AMREIN M. , SCHENK M. , VON NAHMEN A. , SIEBER M. , REICHELT R.
Published: August 2011

The force and compliance upon scanning tunnelling microscopy (STM) of macromolecular specimens and the conductivity of the specimens upon scanning force microscopy (SFM) have been studied, using a hybrid scanning force/scanning tunnelling microscope. Unlike conventional force microscopy, the sample lies on top of the force sensor. This gives full freedom in the choice of the shape, size and material of the probing tip. Hence, the setup is well suited for those combined scanning probe microscopies, where the probe to sample distance must be kept constant while some property of the sample is measured locally in its near field.
An efficient semi‐automatic algorithm for cell contour extraction
Authors: WU H.‐S. , BARBA J.
Published: August 2011

An interactive semi‐automatic procedure for extraction of cell contours from light microscope images is presented. The user is required to specify four contour points and the algorithm determines the rest of the contour automatically. The algorithm exploits the fact that cell contours have lower grey level than their immediate surrounding and are usually very similar in shape to some piece‐wise ellipses. A cost function is defined to detect the cell contours incorporating both the elliptical shape and local image intensities. The procedure is fast, reliable and well suited for routine interactive applications.
Signal standardization of the secondary ion mass spectrometry (SIMS) microscope for quantification of halogens and calcium in biological applications
Authors: JEUSSET J. , STELLY N. , BRIANÇON C. , HALPERN S. , ROSHANI M. , FRAGU P.
Published: August 2011

The secondary ion mass spectrometry (SIMS) microscope is able to map chemical elements in tissue sections. Although absolute quantification of an element remains difficult, a relative quantitative approach is possible for soft tissue by using carbon (12C) as an internal reference present at large homogeneous and constant concentration in specimen and embedding resin. In this study, this approach is used to standardize the signal of an SIMS microscope for the quantification of halogens (19F—, 35Cl— and 79Br—) and calcium (40Ca+). Standard preparation was determined based on homogeneity and stability criteria by molecular incorporation (halogens) or mixing (calcium) in methacrylate resin. Standard measurements were performed by depth analysis on areas of 8 μm (halogens) and 150 μm (calcium) in diameter for 10–30 min, under Cs+ (halogens) or O2+ (calcium) bombardment. Results obtained from 100–120 measurements for each standard dilution show that the relationship between the signal intensity measured and the elemental concentration (μg/mg of wet tissue or mm) is linear in the range of biological concentrations. This quantitative approach was applied firstly to bromine of the 5‐bromo‐2′‐deoxyuridine (BrdU) used as nuclear marker of rat hepatocytes in proliferation. The second model concerns depletion of calcium concentration in cortical compartment in Paramecium tetraurelia during exocytosis. Then signal standardization in SIMS microscopy allows us to correlate quantitative results with those obtained from other methods.
Easily prepared holey films for use in cryo‐electron microscopy
Authors: JAHN W.
Published: August 2011

Holey films or nets may be produced from an emulsion of glycerol/water in a solution of a mixture of Formvar® and Triafol® in dichloromethane, spread on mica. After being floated off the mica support the films may be treated with ethyl acetate to transform ‘pseudoholes' into holes or nets.
Observation by TEM of spontaneous alloying in nanometre‐sized atom clusters in the Au‐Al system
Authors: MORI H. , YASUDA H.
Published: August 2011

Cluster‐size dependence of alloying behaviour in nanometre‐sized atom clusters has been studied by transmission electron microscopy, using clusters in the Au‐Al system, which forms several intermetallic compounds in the phase diagram. In gold clusters of approximately 6–12 nm mean size, rapid dissolution of aluminium atoms took place and Au2Al or AuAl2 clusters were formed. When the size of gold clusters was increased to approximately 40 nm, no such rapid spontaneous alloying occurred. These observations suggest that there exists a critical size of initial gold clusters below which aluminide clusters are successfully formed by spontaneous alloying.
Quantification of the modulated structures in TiPdCr alloys
Authors: SCHWARTZ A. J. , PACIORNIK S. , KILAAS R. , TANNER L. E.
Published: August 2011

Ti50Pd(50‐x)Crxβ2 alloys (B2, CsCl‐type crystal structure) have been investigated as part of an overall study of the systematics of phase transformation behaviour and also for the commercial applicability of the shape memory effect at elevated temperatures. The phase transformations are unique in the sense that electron diffraction and high‐resolution transmission electron microscopy of Ti50Pd(50‐x)Crx alloys with x between 6 and 10 at.% reveal {110}B2 modulated structures with periodicities between 3 and 4·5 planes that vary linearly with composition. Satellites along certain <110>* lying at incommensurate positions are observed in electron diffraction patterns and are consistent with transverse lattice displacements. High‐resolution transmission electron microscopy (HRTEM) imaging usually reveals sinusoidal displacements; however, a few images appear ‘banded’ at three‐ and four‐plane intervals suggesting the possibility that three‐ and four‐plane commensurate packets combine to give the overall incommensurate satellite spacing. With the use of a newly developed technique for quantifying atomic displacements from HRTEM images, modulated phases in Ti50Pd43Cr7 and Ti50Pd42Cr8 alloys, where atoms are shifted from the cubic lattice sites by a transverse lattice displacement wave, have been studied. The real space displacements of the modulated structures were determined and compared to truly incommensurate and varying three‐ and four‐plane wave packet combinations by examination of the goodness of fit as assessed by the correlation coefficient for a least‐squares fit. In the cases of the Ti50Pd43Cr7 and Ti50Pd42Cr8 alloys, the incommensurate sine wave exhibited much better matching than the commensurate packets, indicating that these modulations are probably incommensurate.
New mechanism for field‐induced anisotropy of cobalt‐rich amorphous alloys
Authors: KIM C. K. , O'HANDLEY R. C.
Published: August 2011

It is generally assumed that amorphous magnetic alloys respond to field annealing by a process of local directional ordering which leaves the amorphous structure intact. We have made a comparative microstructural study of field‐annealed Co95‐xFe5(BSi)x amorphous alloys using transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) with thin sections parallel to the ribbon surface. Field annealing response was measured from anisotropy in low‐field hysteresis loops. These alloys show appreciable surface crystallization for annealing as much as 80 K below the bulk crystallization temperature. The surface crystallization proceeds by a known mechanism (selective oxidation) to which we have added a more detailed understanding. Three steps are involved: (1) formation of an amorphous borosilicate surface oxide layer during annealing; (2) depletion of glass stabilizing elements (boron and silicon) from the underlying amorphous metal substrate; (3) primary crystallization of the destabilized, metal‐enriched subsurface layer to an fee or hep cobalt‐rich phase. Striking differences in the microstructural morphology were revealed for different glass former ratios B/Si. For high B/Si ratios, the surface crystallites are predominantly fee Co and show a high density of oxygen faults. For low B/Si ratios, the surface crystallites are predominantly hep Co and almost free of faults. Response to field annealing is proportional to the B/Si ratio and correlates with the presence of oxygen faults in surface crystallites. Electron diffraction and microprobe analysis indicate that the surface oxide in silicon‐rich alloys is a dense silica glass which appears to be an effective diffusion barrier to oxygen. The surface oxide in boron‐rich alloys is a more porous oxide richer in B2O3. These observations appear to be related to those from perminvar alloys where oxygen was found to be necessary for field annealing to be effective.
Stimulated emission on microscopic scale: Light quenching of Pyridine 2 using a Ti:sapphire laser
Authors: HELL S. W. , SCHRADER M. , BAHLMANN K. , MEINECKE F. , LAKOWICZ J. R. , GRYCZYNSKI I.
Published: August 2011

We report the realization of stimulated emission on a microscopic scale. An experiment is presented describing significant depopulation of the excited state of the fluorophore Pyridine 2 with a mode‐locked Ti: Sapphire laser. Stimulated emission is performed at 750 nm and excitation at the frequency doubled wavelength of 375 nm. The pulses are synchronized so that the stimulating pulse follows the excitation pulse after 5 ps. The set‐up is a modified 4Pi‐confocal microscope employing one of the objective lenses for excitation and the opposing one for stimulated emission.
X‐ray microanalysis of ion distribution in frozen salt/dextran droplets after freeze‐substitution and embedding in anhydrous conditions
Authors: ORLOVICH D. A. , ASHFORD A. E.
Published: August 2011

A model system using dextran droplets of different salt solutions either frozen together, or sandwiched together after freezing and then freeze‐substituted, embedded and dry sectioned has been investigated by X‐ray microanalysis. X‐ray maps and spot measur ements taken in transects through the interface of both droplets have shown that the P, K and Ca remain well localized in their original droplet. This validates freeze‐substitution as a method for localization of these elements in biological samples. Results with Na were more variable and not always explainable. Success was achieved by the use of super‐dry solvents and maintenance of a dry environment at all stages. We emphasize the need to avoid water contamination not only during freeze‐substitution but also during sectioning, storage and section transfer to the electron microscope.
Polarized light microscopy of weakly birefringent biological specimens
Authors: NEWTON R. H. , HAFFEGEE J. P. , HO M. W.
Published: August 2011

A simple, but little known technique for producing marked colour contrast in images of weakly birefringent biological samples obtained with a polarized light microscope is investigated. The technique involves inserting a full wave plate at a small angle of horizontal rotation with respect to the analyser or polarizer, rather than at the conventional angle of 45° commonly employed in the field of mineralogy for examining strongly birefringent specimens. Inserting the full wave plate at a small angle (typically less than 10°) enhances the contrast between regions of tissue having different optical path differences and, in particular, different orientations of optic axes; improving the detection of structures of interest with the polarized light microscope.
Determination of localized diffusion coefficients in gels using confocal scanning laser microscopy
Authors: CUTTS L. S. , ROBERTS P. A. , ADLER J. , DAVIES M. C. , MELIA C. D.
Published: August 2011

The development of a photobleaching technique, CFMM (continuous fluorescence multipoint microphotolysis), to measur e diffusion coefficients in gel systems using a confocal scanning laser microscope is described. Diffusion coefficients (D) were determined for fluorescently labelled dextrans of varying molecular weight in agarose gels, and the results compared with two other methods. CFMM enabled diffusion coefficients to be rapidly determined from the profile across an irradiated area within a defined microscopic location of the gel. The technique was experimentally simple and produced values of D that corresponded well with classical double‐diffusion cell methods.
Stereo representation of atomic force micrographs: Optimizing the view
Authors: SHAO Z. , SOMLYO A. P.
Published: August 2011

The usefulness of stereographs for atomic force microscopy (AFM) is discussed and illustrated with AFM stereo images of biological macromolecules. The three‐dimensional nature of AFM data makes such display particularly appropriate, as it contains the information in an optimal format for creating stereographs suitable for direct evaluation by the human observer. We describe a simple procedure that employs standard functions in a commercial AFM for creating such stereographs.
Near‐atomic‐resolution EELS in silicon‐germanium alloys
Authors: BATSON P. E.
Published: August 2011

Recent advances in instrumental capabilities now allow us to combine spatially resolved EELS with annular darkfield (ADF) imaging to relate the electronic or bonding behaviour of a small region to its atomic structure. Although still limited to projections of structures at the 0·2 nm level, the ADF imaging gives excellent information about the structure and composition of crystalline interfaces and defects. Spatially resolved EELS can then obtain the local bonding and electronic structure, with a similar spatial resolution. Using the silicon 2p core excitation as a probe of the conduction band density of states, it is possible to follow changes of these bands as a function of composition and defect structure in the Ge‐Si alloy system. Effects due to local strain are also observable.
Quantification of the EELS near‐edge structures to study Mn doping in oxides
Authors: BOTTON G. A. , APPEL C. C. , HORSEWELL A. , STOBBS W. M.
Published: August 2011

The L3 to L2 white‐line intensity ratios of Mn in solid solution in yttria‐stabilized zirconia and in Mn oxide standards are measured using different approaches. We show that the L3/L2 ratio alone cannot be used as a means of deducing the oxidation state and that a combined analysis of this ratio and the normalized white‐line intensity is required to retrieve the ionicity and magnetic state of the Mn dopant with respect to reference standards. Changes as a function of composition are measured and these are discussed in terms of variations in crystal field parameters associated with possible vacancy interaction. From the oxygen near‐edge structure we analyse the localization of the extra electron charge introduced by the dopant and discuss the possible effects of the association of this extra charge with oxygen vacancies in relation to the macroscopic ion conductivity.
Atomic‐resolution electron energy‐loss spectroscopy in the scanning transmission electron microscope
Authors: BROWNING N. D. , PENNYCOOK S. J.
Published: August 2011

Recent developments in the dedicated scanning transmission electron microscope (STEM) now permit probe sizes of atomic dimensions to be obtained (0·22 nm FWHM at 100 kV). When coupled with an efficient parallel detector for electron energy‐loss spectroscopy (EELS), there now exists the potential to obtain EELS with atomic resolution. This potential is further enhanced by the Z‐contrast imaging technique in the STEM, which can produce incoherent, atomic‐resolution images of crystalline structures. The Z‐contrast image, being formed from only the high‐angle thermal diffuse scattering, can thus be acquired simultaneously with the energy‐loss spectrum and used to position the probe with atomic precision. However, to obtain an atomic‐resolution energy‐loss spectrum the range of the inelastic scattering must be less than the atomic spacing. Additionally, to enable a direct correlation between the spectrum and the Z‐contrast image, i.e. a description of the spatial resolution of the spectrum in terms of a convolution of a probe intensity profile and an object function, we need to determine the conditions for incoherent imaging with inelastically scattered electrons. In this paper, a theorem for the spatial resolution of the inelastic signal first proposed by Ritchie and Howie and a hydrogenic model for the object function developed by Maslen and Rossouw are combined to calculate the object functions for several characteristic K‐edges. More generally, the collection conditions, energy range and detectability for atomic‐resolution spectra are discussed. Finally, atomic‐resolution spectroscopy is demonstrated with a cobalt L‐edge profile of an atomically abrupt CoSi2/Si (111) interface.
Probing the local structure and bonding at interfaces and defects using EELS in the TEM
Authors: BRYDSON R.
Published: August 2011

In monolithic materials the presence of, and structure associated with, defects and grain boundaries often control the overall macroscopic properties of the bulk material. Similarly in composite materials the interfaces between the various constituents may also be property‐determining. Clearly it is desirable to be able to determine the structure and chemistry of such isolated, nonperiodic features using a spectroscopic technique capable of resolving chemical‐state information from areas of atomic dimensions. Electron energy‐loss spectroscopy (EELS) conducted in the (scanning) transmission electron microscope ((S)TEM) constitutes such a spatially resolved tool. When combined with atomic‐resolution imaging and appropriate surface‐sensitive spectroscopic techniques, important information about elemental distributions, local coordinations and valence states of atomic species at interfaces and defects may be derived. Examples drawn from studies of grain boundaries, interfaces in ceramic and metal matrix composites and investigations of defects in diamond are highlighted in order to illustrate this methodology.
Energy‐filtered Fresnel contrast analysis of Fe/Cu multilayers
Authors: DUNIN‐BORKOWSKI R. E. , BOOTHROYD C. B. , LLOYD S. J. , STOBBS W. M.
Published: August 2011

Energy‐filtered imaging in the transmission electron microscope is applied to the Fresnel contrast exhibited by a fine‐period Fe/Cu multilayer. Comparisons of filtered and unfiltered image series demonstrate the effects of inelastic scattering, and it is shown that the use of energy filtering allows improved confidence in the physical origins of the contrast for a specimen, such as that described, for which the structure is still uncertain.
Spatial resolution in EFTEM elemental maps
Authors: KRIVANEK O. L. , KUNDMANN M. K. , KIMOTO K.
Published: August 2011

Imaging filters developed over the last few years permit rapid elemental mapping by energy‐filtering transmission electron microscopy (EFTEM), with resolution and sensitivity limited primarily by the sample and by the TEM. We explore the attainable spatial resolution in the elemental maps theoretically and experimentally, and suggest optimized set‐up procedures for maximizing the resolution. The chromatic aberration of the objective lens of the microscope is shown to be a major limit. Its influence can be minimized by using small energy intervals and limited collection angles, but this is done at the cost of decreased collection efficiency. Resolution of better than 1 nm and sensitivities to less than a monolayer of elements with favourable edges are readily attainable in elemental maps obtained with acquisition times of 40 s total and less. Resolution better than 0·5 nm should be attainable with further optimization of the acquisition parameters.
Segregation‐induced hole drilling at grain boundaries
Authors: ÖZKAYA D. , YUAN J. , BROWN L. M. , FLEWITT P. E. J.
Published: August 2011

Grain boundaries in metals provide preferential sites for impurity atoms to segregate, forming an area of the metal with different chemical and physical properties compared to the bulk. This also means that the electron beam damage characteristics of the grain boundary might be different from that of the bulk. Segregation can be expected to make grain boundaries particularly vulnerable to electron‐beaminduced hole drilling and beam damage, if the segregation produces a near two‐dimensional compound with increased ionicity. Here we report preferential hole drilling at grain boundaries in an Fe‐0–4 wt%P alloy. Such a phenomenon confirms evidence from energy‐loss spectroscopy that charge transfer occurs between P and Fe at the grain boundary. It also explains why it is easier to detect phosphorus using EDX compared to PEELS, and suggests that the electron beam damage may be a major limiting factor in grain‐boundary studies.
The Prion‐like Protein Doppel Enhances Ovine Spermatozoa Fertilizing Ability
Authors: Pimenta J , Dias FMV , Marques CC , Baptista MC , Vasques MI , Horta AEM , Barbas JP , Soares R , Mesquita P , Cabrita E , Fontes CMGA , Prates JA , Pereira RM
Published: August 2011

Contents

The function of prion‐like protein Doppel was suggested to be related to male fertility. In this study, the importance of ovine Doppel polypeptide on spermatozoa capacitation and fertilization was evaluated. After refolding, recombinant Doppel (rDpl) was supplemented with different concentrations (40, 80 or 190 ng/ml) to ovine spermatozoa during the capacitation process. In experiment 1, post‐thawed ovine spermatozoa were incubated with different concentrations of rDpl during 1 h for swim‐up, and changes in sperm motility, concentration, vigour, viability and capacitation were monitored (10 replicates). In experiment 2, the fertilization ability of post‐swim‐up spermatozoa incubated as above was tested through heterologous fertilization of bovinein vitromatured oocytes (n = 423, three replicates). Regardless of dosage, rDpl improved (p ≤ 0.03) spermatozoa viability. Sperm individual motility and vigour were the highest (p ≤ 0.04) for the group receiving 190 ng/ml rDpl. Sperm supplemented with the highest doses of rDpl achieved higher (p = 0.02) fertilization rates (56.0 ± 3.0%) than control (39.1 ± 2.2%) and 40 ng/ml rDpl (39.8 ± 2.7%). Preliminary data suggest that Doppel protein may enhancein vitrospermatozoa fertilizing ability.
Solitary fibrous tumor of the prostate: A report of two cases
Authors: Talvitie Heidi , Åström Kristina , Larsson Olle , Åhlén Jan , Bergh Anders , Egevad Lars
Published: August 2011

We here report two cases of solitary fibrous tumor (SFT) arising in the prostate. Two men, 66 and 69 years old, with urinary tract symptoms were diagnosed with SFT on transrectal needle biopsy and transurethral resection of the prostate, respectively. The tumors were removed by a low anterior resection including tumor, prostate and rectum en bloc and cystoprostatectomy, respectively. Both tumors were well‐circumscribed but also showed some infiltration of the prostate glands. They were composed of storiform bundles of bland spindle cells that stained strongly for CD34 and vimentin but negative for muscle markers. Although rare, SFT should be considered as differential diagnosis of spindle cell lesions on prostate biopsies.
Correlation between transient tachypnea of the newborn and wheezing attack
Authors: Çakan Mustafa , Nalbantoğlu Burçin , Nalbantoğlu Ayşin , Demırsoy Uğur , Say Aysu
Published: August 2011

Background:  Transient tachypnea of the newborn (TTN) is the most common cause of respiratory distress in newborns. Although associated with some morbidity, it is generally believed that once TTN resolves, there is no further increased risk for respiratory disease. However, in limited studies frequency of wheezing attacks is found to be increased in patients who had TTN diagnosis during the newborn period, in comparison to patients who had no respiratory problem. Thus, the question arises as to whether TTN is an innocent disease.

Methods:  This study was done retrospectively. We recorded the demographic characteristics of 103 infants born between 17 October 2003 and 17 October 2004 at Zeynep Kamil Hospital and hospitalized because of TTN in the neonatal intensive care unit. In the second phase, we telephoned the parents of the 103 infants and asked about wheezing attacks. A total of 103 other infants, born during the same period, with no health problems during the newborn period, were included in the study as the control group and the same procedures were applied to them.

Results:  The rate of wheezing attack among patients with TTN diagnosis was found to be significantly higher than that in patients who had no TTN diagnosis (P < 0.01). TTN was found to be an independent risk factor for wheezing attack (OR, 2.378; 95% CI, 1.20–4.70).

Conclusion:  In conclusion, we established that TTN is an independent risk factor for wheezing. In addition we also hypothesized that genetic and environmental interactions synergistically predisposed these children for future wheezing.
Environmental evidence for net methane production and oxidation in putative ANaerobic MEthanotrophic (ANME) archaea
Authors: Lloyd Karen G. , Alperin Marc J. , Teske Andreas
Published: August 2011

Uncultured ANaerobic MEthanotrophic (ANME) archaea are often assumed to be obligate methanotrophs that are incapable of net methanogenesis, and are therefore used as proxies for anaerobic methane oxidation in many environments in spite of uncertainty regarding their metabolic capabilities. Anaerobic methane oxidation regulates methane emissions in marine sediments and appears to occur through a reversal of a methane‐producing metabolism. We tested the assumption that ANME are obligate methanotrophs by detecting and quantifying gene transcription of ANME‐1 across zones of methane oxidation versus methane production in sediments from the White Oak River estuary, North Carolina. ANME‐1 consistently transcribe 16S rRNA and mRNA of methyl coenzyme M reductase (mcrA), the key gene for methanogenesis, up to 45 cm into methanogenic sediments. CARD‐FISH shows that ANME‐1 exist as single rod‐shaped cells or pairs of cells. Integrating normalized depth distributions of 16S rDNA and rRNA (measured with qPCR and RT‐qPCR respectively) shows that 26–77% of the rDNA (a proxy for ANME‐1 cell numbers), and 18–76% of the rRNA (a proxy for ANME‐1 activity) occurs within methane‐producing sediments. These results, along with a re‐assessment of the published Iiterature, change the perspective to ANME‐1 as methanogens that are also capable of methane oxidation.
Psychological and psychiatric factors as predictors for success in sacral neuromodulation treatment
Authors: Marcelissen Tom A.T. , Leong Randall K. , Nieman Fred H.M. , van Lankveld Jacques J.D.M. , van Kerrebroeck Philip E.V. , de Wachter Stefan G.G.
Published: August 2011

Study Type – Therapy (preference)

Level of Evidence 4

What’s known on the subject? and What does the study add?

Sacral neuromodulation (SNM) is a well‐established treatment for patients with chronic LUTS. The selection of eligible candidates could be improved by identifying factors that can predict a successful response. In the present study, we evaluated the role of various psychological and psychiatric factors in relation to SNM treatment.

OBJECTIVE

• To evaluate if psychological and psychiatric factors can predict the outcome of test stimulation or permanent treatment with sacral neuromodulation (SNM).

PATIENTS AND METHODS

• Between 2006 and 2009, patients with overactive bladder syndrome or non‐obstructive urinary retention who were eligible for test stimulation were included.

• All patients completed the Amsterdam Biographic Questionnaire (ABQ), which measures the personality traits of the patient, and the Symptom Check‐List‐90‐Revised (SCL‐90‐R), which is a screening instrument for neuroticism, and for current level of complaints.

• The results of the questionnaires were compared with the outcomes of test stimulation and permanent treatment.

• In addition to the questionnaires, we also included the psychiatric history as a potential predictive factor.

RESULTS

• On univariate analysis there was no relationship between the psychological characteristics and the outcome of test stimulation or the occurrence of adverse events (AEs) with permanent treatment.

• A history of psychiatric disease was not related to the outcome of test stimulation, but was shown to be a positive predictor for the occurrence of AEs with permanent SNM treatment.

CONCLUSIONS

• In the present study there was no evidence that psychological screening with the ABQ or SCL‐90‐R can predict the outcome of SNM treatment.

• Patients with a medical history of psychiatric disease appear to be more likely to encounter AEs with permanent SNM treatment.