bluuebunny/crossref_metadata_2025_split huggingface dataset:
STM imaging of metal‐coated cell plugs of the archaeobacterium Methanospirillum hungatei GP1
Authors: XU W. , BLACKFORD B. L. , MULHERN P. J. , JERICHO M. H. , FIRTEL M. , BEVERIDGE T. J. 
Published: August 2011

SummaryScanning tunnelling microscopy (STM) images of Pt/Ir‐ and Pt/Ir/C‐coated cell plugs of Methanospirillum hungatei showed paracrystalline structures with P6 symmetry and an 18‐nm lattice constant, in agreement with electron microscopy studies. The three‐dimensional STM images unambiguously distinguished the two morphologically different proteinaceous plug assemblies and led to an improved understanding of the natural internal organization of whole plugs. Tip convolution effects and the grain size of the metal coating complicated interpretation of finer structures. We discuss possible imaging mechanisms to explain observations in which part of the film was removed but the remaining part of the structure was still imaged reproducibly.
Limits of electron probe formation
Authors: CREWE A. V. 
Published: August 2011

SUMMARYThe performance of many electron optical instruments is fundamentally limited by the dimensions of the focused probe. This is true of the scanning electron microscope and the scanning transmission electron microscope and, by inference, it may affect the transmission electron microscope. There has been very little improvement over the past few years and it seems reasonable to look for the explanation. It is possible to arrive at some simple expressions for the limiting performances of conventional instruments in a way that is independent of the design details and depends upon practical limits of field strength. Experiment and theory also appear to be in agreement with the fact that the limit for high‐voltage instruments has been reached, although there is still room for improvement for low voltages.
Environmental scanning electron microscopy of marine aggregates
Authors: LAVOIE D. M. , LITTLE B. J. , RAY R. I. , BENNETT R. H. , LAMBERT M. W. , ASPER V. , BAERWALD R. J. 
Published: August 2011

SUMMARYMarine aggregates were examined for the first time in the hydrated state using an environmental scanning electron microscope (ESEM). Sample preparation consisted of fixation followed by rinsing with distilled water to remove excess salts and fixative. Aggregates were continuously observed at resolutions comparable to conventional scanning electron microscopy through stages of hydration, from completely immersed to desiccated. Because no metallic coating is required, energy‐dispersive X‐ray spectroscopy (EDXS) can be used to analyse rapidly constituent elements occurring at low concentrations with no spectral interference. Subtle differences in mineral particles were seen in both EDXS spectra and in direct observation of relative hydration, reflecting apparent differences in mineralogy. ESEM enabled examination of effects of desiccation and rehydration on individual particles composed primarily of hydrated polymer and eliminated dehydration artefacts in delicate organisms.
Electrophoretic transfer of protein‐pigment complexes from non‐denaturing gels to electron microscopy grids
Authors: GRÄBER S. , LYON M. K. 
Published: August 2011

SUMMARYDifferent 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

SUMMARYQuantitative 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

SUMMARYSubmicrometre 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

SUMMARYIn 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

SUMMARYMicrostructural 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

SUMMARYThe 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

SUMMARYThe 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

SUMMARYA 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

SUMMARYThe 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

SUMMARYThis 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

SUMMARYTwo 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

SUMMARYA 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

SUMMARYActin 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.
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

SUMMARYThe 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

SUMMARYPowerful 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

SUMMARYA 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

SummaryA 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

SummaryThe 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

SummaryAn 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.