bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron spectroscopic imaging of the morphology of in situ polyethylene ‘shish kebabs’
Authors: Huong D. M. , Drechsler M. , Möller M. , Cantow H.‐J.
Published: August 2011

The simultaneous imaging of the crystalline and rigid amorphous areas of semi‐crystalline polyethylene is made possible by electron spectroscopic imaging (ESI) in an energy‐filtering transmission electron microscope (EFTEM). Conventional bright‐and dark‐field imaging modes mainly provide information about the non‐crystalline areas which react chemically with classical staining agents such as ruthenium tetroxide. The ability of ESI to image structures which are inaccessible to the staining agent is shown in TEM micrographs of nascent polyethylene ‘shish kebabs’ embedded in a Lowicryl HM20 matrix.
Localization of Ca2+ ‐stores and tissue compartments with a Ca2+ ‐binding capacity in the organ of Corti of the guinea‐pig by electron energy‐loss spectroscopy
Authors: Heinrich U.‐R. , Maurer J. , Mann W. , Kreutz W.
Published: August 2011

The addition of 10 mM CaCl2 to glutaraldehyde fixative leads to the formation of small electron‐dense deposits in the organ of Corti of the guinea‐pig. These precipitates are mainly attached to cell membranes in contact with different extracellular lymphatic fluids. A higher number of precipitates is localized in the acellular parts of tectorial and basilar membrane. Electron energy‐loss spectroscopy (EELS) was used to determine the elemental composition of the deposits formed. The spectra showed a prominent signal at the Ca2+ L2,3 ionization edge. Oxygen could also be detected in all the precipitates analysed. EELS analysis of mitochondria of the inner and outer hair cells after conventional fixation (glutaraldehyde followed by post‐fixation in OsO4) revealed a small but significant calcium signal.
Three‐dimensional simulation of flow through a porous medium
Authors: Jernot J. P. , Prasad P. Bhanu , Demaleprade P.
Published: August 2011

A three‐dimensional simulated random packing of spheres is digitized and progressively densified with dilations of the initial stacking. Flow of fluid through these successive structures is then simulated with geodesic dilations inside the porous phase. The results allow a clear distinction to be made between two factors affecting the flow: the metric properties and the topological properties of the porous network.
Reduction of cross‐talk between fluorescent labels in scanning laser microscopy
Authors: Carlsson Kjell , Mossberg Karin
Published: August 2011

By using dual detectors in combination with a dichroic filter, it is possible to record simultaneously the distribution of two fluorescent labels in a specimen. It is often difficult, however, to obtain a good separation, i.e. each detector will generally detect light from more than one fluorophore. In such cases it is desirable to find image‐processing methods to improve the separation. A simple method is to form a linear combination of the recorded images. In this paper we investigate the necessary prerequisites for this method to be successful, and we also investigate to what extent these are fulfilled in some practical cases. In this context the spectral properties of the fluorophores turn out to be of crucial importance. Even when the necessary prerequisites are not strictly fulfilled, a considerable improvement in image quality can, nevertheless, be obtained.
Atomic force microscopy of conventional and unconventional nucleic acid structures
Authors: Henderson Eric
Published: August 2011

Images of conventional (Watson–Crick base paired) and unconventional (G4 RNA) nucleic acid structures have been obtained by atomic force microscopy. The images are reproducibly generated from samples deposited on freshly cleaved mica. Periodic substructural features are evident in fibres observed in both cases. In the case of G4 RNA, tip‐induced formation of large fibres is observed.
Colour thresholding and objective quantification in bioimaging
Authors: Fermin C. D. , Gerber M. A. , Torre‐Bueno J. R.
Published: August 2011

Computer imaging is rapidly becoming an indispensable tool for the quantification of variables in research and medicine. Whilst its use in medicine has largely been limited to qualitative observations, imaging in applied basic sciences, medical research and biotechnology demands objective quantification of the variables in question. In black and white densitometry (0–256 levels of intensity) the separation of subtle differences between closely related hues from stains is sometimes very difficult. True‐colour and real‐time video microscopy analysis offer choices not previously available with monochrome systems. In this paper we demonstrate the usefulness of colour thresholding, which has so far proven indispensable for proper objective quantification of the products of histochemical reactions and/or subtle differences in tissue and cells. In addition, we provide interested, but untrained readers with basic information that may assist decisions regarding the most suitable set‐up for a project under consideration. Data from projects in progress at Tulane are shown to illustrate the advantage of colour thresholding over monochrome densitometry and for objective quantification of subtle colour differences between experimental and control samples.
The application of cryo‐SEM techniques to the study of the symbiotic association in the Azolla leaf cavity
Authors: Klein Eugenia , Bari Etan , Forni Cinzia , Malkin Shmuel , Tel‐Or Elisha
Published: August 2011

The preparation of frozen‐hydrated samples for scanning electron microscopy to observe symbionts in Azolla is described and compared to the conventional method of chemical fixation followed by critical‐point drying. The frozen‐hydrated specimens preserve the structure of the associating symbionts and the liquid phase in the Azollaleaf cavity. The leaf cavity structure and the endosymbionts in the frozen‐hydrated specimens were intact and unharmed throughout the fixation. The cyanobiont cells were distributed along the envelope of the leaf cavity, and did not aggregate around the hair cells, as reported for chemical fixation specimens. The mucilage layer removed by the chemical fixation could be observed in the cryo‐fixation specimens, especially at the lower part of the cavity.
An acoustic depth alarm device to monitor vertical translation of the microscope stage: A practical optical disector, selector and unbiased brick for routine light microscopy
Authors: Laroye Guy J. , Taylor William B.
Published: August 2011

A description is given of a simple device, which, when attached to the fine adjustment vernier of a regular light microscope, alerts the operator by sound to the vertical distances travelled by the microscope stage without losing visual contact with the specimen. Depending on whether the depth calibration is deemed to be accurate or not, this device can be used as a practical stereological disector or a selector in routine light microscopy, or as an unbiased brick in confocal microscopy.
Experimental determination of light‐element k‐factors using the extrapolation technique: Oxygen segregation in aluminium nitride
Authors: Westwood A. D. , Michael J. R. , Notis M. R.
Published: August 2011

It is shown that quantitative light‐element (Z < 10) analysis and κ‐factor determination of light elements can be routinely conducted, analogous to quantification of elements with Z >, using X‐ray energy‐dispersive spectroscopy. The extrapolation technique was used to determine the κ‐factors, κOAI = 0.88 ± 0.10 and κNAI = 1.61 ±0.22 at zero thickness. These values are found to be in good agreement with theoretically calculated κ‐factors. The limitations and applicability of the extrapolation technique for light‐element analyses are discussed. An example of light‐element quantification is provided in the analysis of oxygen segregation to inversion domain boundaries in aluminium nitride.
Beam heating in an asymmetrically illuminated thin circular film
Authors: Curzon A. E.
Published: August 2011

The method of images is used to calculate the temperature increase produced in a circular film when the film is illuminated at any point by an electron beam of circular cross‐section and constant current density over its cross‐section. Formulae are given in dimensionless form for (i) the temperature increase anywhere along the line joining the centres of the beam and of the film, (ii) the temperature increase at the centre of the beam (vc), and (iii) the position and value of the maximum temperature increase in the film vm. The relative sizes of vc and vm are discussed and it is shown that (vm – vc)/vm ≤ 0.079. The theory applies best to metallic films where power loss by means of thermal conductivity is dominant. A brief discussion is given of the cases where thermal radiation is important, sample calculations are given for carbon and copper films, and the effect of focusing the electron beam at constant current is considered.
Study of the liquid‐phase sintering process by probabilistic models: Application to the coarsening of WC‐Co cermets
Authors: Quenec‘H Jean‐Louis , Coster Michel , Chermant Jean‐Louis , Jeulini Dominique
Published: August 2011

A new method using probabilistic models is used to study the growth of WC grains in WC‐Co cermets. Two different models are tested: a Boolean model and a two‐colour mosaic model. The Boolean Poisson model is the simplest for describing and predicting material evolution, and depends only on two independent parameters. They allow the estimation of the connectivity numbers and, moreover, are used to determine the type of reaction which controls the WC‐Co coarsening kinetics.
A tilting device for three‐dimensional microscopy: Application to in situ imaging of interphase cell nuclei
Authors: Bradl Joachim , Hausmann Michael , Ehemann Volker , Komitowski Dymitr , Cremer Christoph
Published: August 2011

The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the focal plane (x‐y plane). This is true of conventional as well as confocal microscopes. For quantitative microscopy, for instance studies of the three‐dimensional (3‐D) organization of chromosomes in human interphase cell nuclei, the 3‐D image must be reconstructed by a point spread function or an optical transfer function with careful consideration of the properties of the imaging system. To alleviate the reconstruction problem, a tilting device was developed so that several data sets of the same cell nucleus under different views could be registered. The 3‐D information was obtained from a series of optical sections with a Zeiss transmission light microscope Axiomat using a stage with a computer‐controlled stepping motor for movement in the z‐axis. The tilting device on the Axiomat stage could turn a cell nucleus through any desired angle and also provide movement in the x‐y direction.

The technique was applied to 3‐D imaging of human lymphocyte cell nuclei, which were labelled by in situ hybridization with the DNA probe pUC 1.77 (mainly specific for chromosome 1). For each nucleus, 3‐D data sets were registered at viewing angles of 0°, 90° and 180°; the volumes and positions of the labelled regions (spots) were calculated. The results also confirm that, in principle, any angle of a 2p geometry can be fixed for data acquisition with a high reproducibility. This indicates the feasibility of axiotomographical microscopy of cell nuclei.
An appraisal of low‐temperature embedding by progressive lowering of temperature into Lowicryl HM20 for immunocytochemical studies
Authors: Robertson David , Monaghan Paul , Clarke Catherine , Atherton Amanda J.
Published: August 2011

The progressive lowering of temperature (PLT) method of embedding for electron microscope immunolabelling has been examined with the objective of formulating a standardized protocol which can be applied to a wide variety of samples. The methods described cover fixation, processing of samples by the PLT method, embedding in Lowicryl HM20 and subsequent immunolabelling. Each of the steps in the fixation and embedding protocol has been assessed for its potential to retain both morphology and antigenicity. Comparison of samples embedded in Lowicryl K4M and HM20 at −25°C indicate an increased membrane contrast in HM20 sections, and a further improvement in morphology when samples were embedded in HM20 at −50°C rather than at −25°C. The results of applying the methods described are demonstrated in a range of samples of both mammalian and botanical origin, which includes solid tissues, cells in suspension, and cells grown in vitro as a monolayer culture and embedded in situ. Samples processed by this method have been immunolabelled using a wide range of antibodies recognizing nuclear, cytoplasmic, cell membrane and extracellular matrix antigens.
Artefacts and morphological changes during chemical fixation
Authors: Kellenberger Eduard , Johansen Renata , Maeder Marlies , Bohrmann Bernd , Stauffer Esther , Villiger Werner
Published: August 2011

The normally ‘condensed’ (darkly stained) chromosomes of dinofiagellates decondense by swelling. This occurs in an increasing number of cells when the concentration of added OsO4 is decreased. With different fixatives other types of disintegration can be observed, which vary with the concentration. With cryofixation and freeze‐substitution the chromosomes are most ‘condensed’. Escherichia colt infected with bacteriophage T4, with or without active lysozyme production, were studied by optical densitometry for partial lysis and by light and electron microscopy for observing swelling. When active lysozyme is present some of the acrolein (2.5%) ‐ glutaraldehyde (2%)‐fixed cells swell at 0°C, but do not in the absence of lysozyme nor when fixed at room temperature. If OsO4 is added at concentrations ≤0.5%, partial lysis occurs when lysozyme is present. The optical density decreases, the cells lose some matter and swell slightly. The corresponding electron micrographs show gap formation by curdling and/or a decreased concentration of the cytoplasm which reveals certain phage‐related particles.
The use of Lucifer yellow, bodipy, FITC, TRITC, RITC and Texas red for dual immunofluorescence visualized with a confocal scanning laser microscope
Authors: Entwistle A. , Noble M.
Published: August 2011

A new method of comparing the relative merits of different fluorophores that undergo relatively rapid irreversible photo‐inactivation is described. This method showed that the levels of fluorescent emission seen with both fluorescein isothiocyanate (FITC) and bodipy fl conjugated to streptavidin were similar when examined under conditions where they exhibited equal rates of irreversible photo‐inactivation. Bodipy fl and FITC give lower levels of cross‐talk into images of cells immunofluorescently stained with either rhodamine isothiocyanate (RITC) or tetramethyl rhodamine isothiocyanate (TRITC) than into images of cells stained with Texas red, under conditions where the three red fluorophores exhibited an equal level of sensitivity. Furthermore, bodipy fl gave much lower levels of cross‐talk into images of RITC‐stained cells than either FITC or Lucifer yellow. TRITC, but not RITC or Texas red, gave significant levels of cross‐talk into the green band‐pass filters used to visualize FITC and bodipy fl. From these results it seems that a combination of bodipy fl and RITC provides the best contrast when visualizing dual immunofluorescence with a confocal scanning laser microscope if the 488‐nm line of an argon ion laser is used as the excitation source.
Low‐energy electron microscopy (LEEM) and mirror electron microscopy (MEM) of biological specimens: Preliminary results with a novel beam separating system
Authors: Griffith O. Hayes , Hedberg Karen K. , Desloge Denis , Rempfer Gertrude F.
Published: August 2011

Low‐energy electron microscopy (LEEM) and mirror electron microscopy (MEM) utilize a parallel beam of slow‐moving electrons backscattered from the specimen surface to form an image. If the electrons strike the surface an LEEM image is produced and if they are turned back just before reaching the surface an MEM image results. The applications thus far have been in surface physics. In the present study, applications of LEEM and MEM in the biological sciences are discussed. The preliminary results demonstrate the feasibility of forming images of uncoated cultured cells and cellular components using electrons in the threshold region (i.e. 0–10 V). The results also constitute a successful test of a novel beam‐separating system for LEEM and MEM.
Digital image acquisition in in vivo confocal microscopy
Authors: Petroll W. M. , Cavanagh H. D. , Lemp M. A. , Andrews P. M. , Jester J. V.
Published: August 2011

A flexible system for the real‐time acquisition of in vivo images has been developed. Images are generated using a tandem scanning confocal microscope interfaced to a low‐light‐level camera. The video signal from the camera is digitized and stored using a Gould image processing system with a real‐time digital disk (RTDD). The RTDD can store up to 3200 512 times 512 pixel images at video rates (30 images s−1). Images can be input directly from the camera during the study, or off‐line from a Super VHS video recorder. Once a segment of experimental interest is digitized onto the RTDD, the user can interactively step through the images, average stable sequences, and identify candidates for further processing and analysis. Examples of how this system can be used to study the physiology of various organ systems in vivo are presented.
Confocal differential interference contrast (DIC) microscopy: including a theoretical analysis of conventional and confocal DIC imaging
Authors: Cogswell C. J. , Sheppard C. J. R.
Published: August 2011

A technique for obtaining differential interference contrast (DIC) imaging using a confocal microscope system is examined and its features compared to those of existing confocal differential phase contrast (DPC) techniques as well as to conventional Nomarski DIC. A theoretical treatment of DIC imaging is presented, which takes into account the vignetting effect caused by the finite size of the lens pupils. This facilitates the making of quantitative measurements in DIC and allows the user to identify and select the most appropriate system parameters, such as the bias retardation and lateral shear of the Wollaston prism.
Confocal imaging with bilateral scanning and array detectors
Authors: Brakenhoff G. J. , Visscher K.
Published: August 2011

A novel arrangement for confocal microscopy is presented, in which the key elements are the use of an array detector such as a CCD for confocal image collection and the use of one double‐sided scanning mirror element for bilaterally scanning the object and collecting the data on the CCD. The resulting arrangement is shown to be capable of confocal imaging with high photon efficiency under adjustable conditions of confocality and varying image acquisition rates, i.e. from slow speed up to real‐time imaging. Either laser or conventional light sources may be utilized. In addition to CCD registration, direct observation by eye of the confocal image in fluorescence is also possible.
In‐vivo confocal scanning laser microscopy of the cerebral microcirculation
Authors: Dirnagl Ulrich , Villringer Arno , Einhäupl Karl Max
Published: August 2011

Confocal scanning laser microscopy (CSLM) was used to study the microcirculation of the brain neocortex in anaesthetized rats. After removal of the dura mater, implantation of a closed cranial window, and intravenous injection of fluorescein, three‐dimensional reconstructions of cortical capillaries were performed down to a depth of 250 μm below the pial surface. Using a one‐dimensional approach (single line scanning), erythrocyte (negative contrast in fluorescently labelled plasma) and leucocyte (labelled with rhodamine 6 G) velocity and supply rate in cortical capillaries were measured. The effect of CO2‐inhalation on capillary blood flow dynamics was studied. Capillaries were imaged continuously for up to 1 h without changes in flow or fluorescence pattern. However, by increasing the laser power 10–100‐fold, aggregate formation was induced and capillaries were occluded, possibly due to damage to vascular endothelium. We conclude that CSLM can be used to study morphological and dynamic aspects of fluorescently labelled subsurface structures in organs of experimental animals.
Confocal microscopy of the in‐situ crystalline lens
Authors: Masters Barry R.
Published: August 2011

The fine structure of the in‐situ rabbit crystalline ocular lens from the ex‐vivo rabbit eye was observed with a confocal scanning laser microscope in the scattered light mode. The images were observed through the full thickness of the cornea and aqueous humour to a depth of 50 μm in the anterior ocular lens. The following structures were observed from optical sections of the ocular lens: two concentric regions of the lens capsule, epithelial cells, lens sutures, and surface and interior regions of individual lenticular fibres. The observed lateral resolution of the microscope objective was degraded by imaging across thick (millimetre) structures. This study shows the feasibility of obtaining high‐contrast images of transparent objects across 1.7 mm of ocular tissue (cornea and aqueous humour) using confocal light microscopy.
Effect of processing dwell‐time on the microstructure of a fibre‐reinforced composite
Authors: SUMMERSCALES J. , GREEN D. , GUILD F. J.
Published: August 2011

Carbon‐fibre/epoxy‐resin unidirectional laminates were prepared in the laboratory by vacuum‐bag processing. Three plates were produced using different ‘dwell‐times’, before pressure was applied to consolidate the laminate.

Sections from the three plates were cut perpendicular to the fibre direction and were examined with a Quantimet 570 Image Analyser. The fibre positions were recorded. The method of microstructural description is based on the size of the Voronoi cell around each fibre; the Voronoi cell around a fibre is defined as the area nearer to that fibre than any other. Thus the dimensions of the cell are a measurement of half the interfibre distance.

The measured distributions of Voronoi distances were compared with distributions calculated using the null hypothesis that the fibres are randomly distributed. Comparison showed that fibres from all three sections were clustered, but that the sample from the medium dwell time was most homogeneous. Examination of long Voronoi distances, not predicted by the null hypothesis, showed anisotropy in orthogonal directions arising from the manufacturing process. This method of microstructural analysis revealed a dependence on the plate processing method, and may now be used to evaluate more general processing problems.
The compatibility of TiB2 protective coatings with SiC fibre and Ti‐6Al‐4V
Authors: CHOY K. L. , DERBY B.
Published: August 2011

TiB2 coatings have been studied as prospective protective layers to inhibit the interfacial reaction between SiC fibres and Ti‐alloy matrices. This protective coating has been deposited onto SiC monofilament fibres using a chemical vapour deposition (CVD) technique. The fibre‐matrix compatibility of these TiB2‐coated SiC fibres in Ti‐6Al‐4V composites was evaluated by incorporating the coated fibres into Ti‐6Al‐4V using a diffusion bonding technique. The interfaces of this composite were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electron probe microanalysis, to evaluate the interfacial microstructures, chemical stability and the efficiency of TiB2 as a protective coating for SiC fibres in Ti‐alloy matrices, and to study the effects of deposition temperature on the interface of the coated fibre. Results show that stoichiometric TiB2 coatings are stable chemically to both SiC fibres and Ti‐6Al‐4V and hinder the deleterious fibre‐matrix reactions effectively. Boron‐rich TiB2 coatings should be avoided, as they lead to the formation of a needle‐like TiB phase at the fibre–matrix interface. These findings provide promising evidence for the value of further exploration of the use of stoichiometric TiB2 as a protective coating for SiC fibre in Ti‐based composites.
Fabrication of macroscopic solid models of three‐dimensional microscopic data by selective laser sintering
Authors: BARTELS K. A. , CRAWFORD R. H. , DAS S. , GUDURI S. , BOVIK A. C. , DILLER K. R. , AGGARWAL S. J.
Published: August 2011

We discuss and give examples of the use of selective laser sintering to fabricate solid macroscopic models of microscopic specimens that have been imaged with a confocal microscope. The digital image processing necessary to create structurally sound models of both translucent and opaque specimens is presented. The fabricated models offer the ultimate in data visualization since they can be physically handled and manipulated to investigate the shape and features of the specimen. Such a powerful visualization tool is useful in both research and educational environments.
Vertical LM sectioning and parallel CT scanning designs for stereology: application to human lung
Authors: PACHE J.‐C. , ROBERTS N. , VOCK P. , ZIMMERMANN A. , CRUZ‐ORIVE L. M.
Published: August 2011

Practical, unbiased stereological methods are described to estimate lung volume and external surface area, and total volume and surface area of relatively large and anisotropic structures (bronchi and arteries) inside the lung.

The volume of each of five lung strata was estimated first by fluid displacement and then by computed tomography (CT) using Cavalieri's method; the reliability of CT was assessed through a calibration procedure, and image thresholding criteria for an accurate volume estimation using CT were established. The parallel, perfectly registered CT section images were also used to estimate the external surface area of each stratum by the spatial grid method.

Unbiased estimation of internal surface areas in lung is a long‐standing problem: since the structures are large and essentially void, large sections are needed; to facilitate identification, thin sections have to be used for light microscopy, and since such structures are anisotropic, the sections should be vertical. A practical stereological design is demonstrated here on an infant lung, which fulfils all these requirements.

This study illustrates the potential of using unbiased stereology to characterize infant pulmonary hypoplasia.
The rotator
Authors: JENSEN E. B. VEDEL , GUNDERSEN H. J. G.
Published: August 2011

The mean particle volume can be stereologically estimated using the nucleator principle. In the present paper, we discuss another principle for estimating mean particle volume, namely the rotator. The vertical rotator has already been previously described and is supplemented in the present paper by the isotropic rotator. For a collection of particle profiles, simulations show that the variance of the rotator is smaller than that of the nucleator.
Quantitative parallel electron energy‐loss spectrometry of Cu—Be alloys
Authors: STRUTT A. J. , WILLIAMS D. B.
Published: August 2011

A technique is described for the quantitative microanalysis of a Cu‐Be alloy using parallel electron energy‐loss spectrometry. Spectrum acquisition and processing variables were optimized specifically for Cu—Be alloy spectra in an attempt to ensure that signal collection was equally efficient for both Be and Cu edges and that reliable, consistent results could be obtained from the subsequent quantification procedure. Analyses of the Cu Be γ–phase showed that the nominal composition of 50 at.% Be is not constant, and that Cu solubility increases slightly with increasing ageing temperature.
Measurements of wet metaphase chromosomes in the scanning transmission X‐ray microscope
Authors: WILLIAMS S. , ZHANG X. , JACOBSEN C. , KIRZ J. , LINDAAS S. , VAN'T HOF J. , LAMM S. S.
Published: August 2011

Radiation damage to Vicia faba chromosome structure, as measured by the mass loss, was determined in the scanning transmission X‐ray microscope for unstained specimens in both the wet and dry states. Dried specimens remain undamaged after either single or multiple images at doses up to 2400 Mrad at wavelengths of 3·15 or 3·64nm. In contrast, wet specimens are damaged irrespective of the imaging protocol. The damage induced by multiple exposures is greater than that seen in a single exposure of the same total dose. Thus, the rate of data collection is greater than or equal to the rate of damage. The damage during multiple exposures of wet chromosomes is influenced by several factors. First, the fixative used influences the extent of radiation damage. Wet chromosomes fixed with glutaraldehyde are more resistant than those fixed with formaldehyde or osmium tetroxide. A second factor is ionic strength. Damage to wet chromosomes increases if the ionic strength decreases below that at which chromatin undergoes a conformational transition. The mass of wet and dry chromosomes is the same, and consequently quantitative measurements can be made on wet specimens. Such measurements give a DNA mass fraction of 39 ± 8% for V. faba chromosomes.
Surface studies in UHV SEM and STEM
Authors: MILNE R. H. , HEMBREE G. G. , DRUCKER J. S. , HARLAND C. J. , VENABLES J. A.
Published: August 2011

Some recent advances in surface analyses with scanning electron microscopy techniques are reviewed. It is shown that secondary electron microscopy can image monatomic steps, emergent dislocations and the early growth of thin films and oxide nuclei. The chemical composition of inhomogeneous surfaces can, in some cases, be determined with nanometre resolution with Auger signals. Overlayer coverages can occasionally be monitored with the secondary electron signal if the change of work function with coverage has been determined previously. The interpretation of these secondary and Auger signals is discussed. The combination of angular electron spectroscopy with electron microscopy is potentially a powerful technique for determining atomic positions of surface atoms and one method for obtaining the experimental data is described. Several substrate/overlayer systems are used to illustrate the amount of information that can be obtained about surface processes with scanning electron microscopy.
Three‐dimensional imaging of corneal cells using in vivo confocal microscopy
Authors: PETROLL W. M. , CAVANAGH H. D. , JESTER J. V.
Published: August 2011

Confocal microscopy is a unique and powerful imaging paradigm which allows optical sectioning through intact tissue. Real‐time tandem scanning confocal microscopy has previously been used to generate high‐magnification two‐dimensional (2‐D) images of cells in living organ systems. Inherent problems with movement, however, have prevented the in vivo acquisition of complete 3‐D datasets. The development of a new objective lens, used in combination with specialized real‐time image acquisition procedures, has allowed sequential serial sections to be obtained in vivo from the rabbit cornea for the first time. These sections can be digitially registered and stacked on the computer to provide a 3‐D reconstruction of the corneal cells. This technique should serve as a useful method for studying 3‐D structures and analysing 4‐D phenomena at the cellular level in living animals. Three‐dimensional images of a stromal nerve in normal rabbit cornea and of fibroblasts within a rabbit corneal wound are presented as examples of current capabilities.
A scanning tunnelling microscopy study of the formation and chemical activation of step defects on the basal plane of pyrolytic graphite
Authors: MOROZOV V. N. , SHERMAN J. , KALLENBACH N. R. , DU SHOU MING , SEEMAN N. C.
Published: August 2011

Scanning tunnelling microscopy is used to monitor etching of the basal plane of highly orientated pyrolytic graphite by ozone, oxygen and nitric acid. These treatments are seen to produce numerous single and multilayer step defects. Subsequent modification of the graphite sheet edges flanking these cavities by cyanuric chloride, TiCl4 and other reagents is shown to activate the edges, thereby making them capable of covalently binding various molecules.
The number of glomerular capillaries estimated by an unbiased and efficient stereological method
Authors: NYENGAARD J. R. , MARCUSSEN N.
Published: August 2011

A method for unbiased capillary number estimation based on the estimation of the Euler–Poincaré characteristic or Euler number with the disector is evaluated. The generation of a new capillary creates a new loop in the capillary network corresponding to a change of exactly one unit in the Euler number of the network. In this way the Euler number has a one‐to‐one relationship to the number of capillaries in a network. In this report rat glomerular capillaries are counted.

Three tissue blocks from each of 14 perfusion‐fixed rat kidneys, aged between 5 days and 18 months, were uniformly sampled and embedded in Epon. Two complete glomerular profiles were sampled per block. Three consecutive sections were studied per glomerulus using the middle one to evaluate the topological events of the capillaries in the sampled glomerulus. The use of complete glomerular profiles eliminated problems with the edges, whereas the disector ensured unbiased sampling in the third dimension. Estimates per animal were weighted averages over blocks, the weights being half the number of glomerular profiles in a section from a block. The number of capillaries in a glomerulus is the mean Euler number per disector volume multiplied by the mean volume of glomeruli obtained by the fractionator.

The observed coefficient of variation between animals was 18.3% for the estimated number of capillaries per glomerulus, and the observed coefficient of error at the level of blocks within animals was 14.7%. The exact capillary number from the node‐branch network of two reconstructed glomeruli equalled the capillary number obtained by the total Euler number from the same glomeruli. This shows in an applied example, as would be expected, that the estimation of capillary number using the Euler number is unbiased and independent of the direction of sectioning.
Measurements of fibre direction in reinforced polymer composites
Authors: CLARKE A. , DAVIDSON N. , ARCHENHOLD G.
Published: August 2011

High‐quality data on the three‐dimensional (3‐D) spatial distributions of glass and carbon fibres in fibre‐reinforced polymer composites are important for both process control and the modelling of the mechanical and thermal properties of these composites. The advent of economical, high‐speed, image analyser systems has enabled numerous research groups to measure directional distributions of fibre samples. Specimens are microtomed and polished and, using optical reflection microscopy, thousands of elliptical fibre images may be analysed within a short period of time. From the eccentricity of the fibre images, estimation of the angles (θ, φ) of each fibre relative to the vertical axis and within the measurement plane is deduced. However, this measurement is subject to considerable error. The confocal scanning laser microscope (CSLM), operating in fluorescence mode or reflection mode, is capable of improving the angular resolution (δθ, δφ) for all fibre directions. The ability of the CSLM to optically section glass and carbon fibre‐reinforced polymer composites down to depths of 20 or 30 μm allows the user to determine accurate fibre directions from the apparent movement of fibre profiles. The CSLM has the potential for standardizing measurements of 3‐D fibre directions in polymer composites and providing the quality directional data which are required for the theoretical modelling of composite processing and composite strength.
Near‐field optical microscopy in transmission and reflection modes in combination with force microscopy
Authors: VAN HULST N. F. , MOERS M. H. P. , BÖLGER B.
Published: August 2011

Near‐field optical microscopy is the optical alternative of the various types of scanning probe microscopes. The technique overcomes the classical diffraction limit in conventional optical microscopy. In this paper the concepts of near‐field optics (NFO) are introduced, followed by a short review of current trends in NFO microscopy. Specifically, developments concerning the efficiency and versatility of both aperture and dielectric probe types are discussed. We present our advances in NFO microscopy, using both fibres and integrated silicon nitride (SiN) structures as dielectric probes. The use of an SiN probe as a combined optical and force sensor is shown to be advantageous, as it provides a feedback mechanism and allows direct comparison between topography and dielectric effects. Images of technical and biological samples are presented with a lateral resolution down to 20 nm, depending on the microscopical arrangement used.
Silver‐enhanced colloidal‐gold labelling of rabbit kidney collecting‐duct cell surfaces imaged by scanning electron microscopy
Authors: HERTER P. , LAUBE G. , GRONCZEWSKI J. , MINUTH W. W.
Published: August 2011

The luminal cell surfaces of rabbit kidney cortical collecting‐duct cells were labelled with peanut lectin (PNA) and investigated by scanning electron microscopy. Labelling was performed either on 20‐μm‐thick cryostat sections from prefixed and cryoprotected rabbit kidney tissue or on cultured collecting‐duct epithelium using biotinylated PNA and a 6‐nm colloidal‐gold‐coupled antibody against biotin. Colloidal‐gold labels were detected at low magnification (2000–4000 x) using silver enhancement. Coating with chromium allowed simultaneous imaging of both cell‐surface morphology and labelling topography in the backscattered electron imaging mode. Our results show that PNA binding is specific for a subtype of intercalated cells equipped with microvilli on the luminal surface. The presented method promises to be useful for the identification of specific cell types in heterogeneous tissues.
Variation in maceral chemistry within and between coals of varying rank: An electron microprobe and micro‐Fourier transform infra‐red investigation
Authors: MASTALERZ M. , BUSTIN R. M.
Published: August 2011

Electron microprobe and reflectance micro‐Fourier infra‐red techniques have been used to document the evolution of maceral chemistry from lignite to anthracite as well as to determine chemical variations between macerals and within macerals in iso‐rank coals. This study shows that the evolution of elemental composition and functional groups in vitrinite differs from those of liptinite. In vitrinite, there is a decrease in the absorbance of both aliphatic stretching bands (2800–3000 cm—1) and the aromatic band (1600 cm—1) with an increase in rank from high‐volatile bituminous to anthracitic coals. The change in functional groups is associated with an increase in C content from about 86 up to 94% and a decrease in O content from 7 to 1%. In sporinite, carboxyl/carbonyl bands (1710 cm—1), distinct in sub‐bituminous and high‐volatile bituminous ranks, disappear at higher rank. The 1600 cm—1 band is relatively weak in liptinite and increases dramatically when liptinite and vitrinite coalification paths merge. These changes in sporinite functional groups are accompanied by an increase in C content from 73 to 88–89%, decrease in O from 14 to 4% and decrease in H from more than 9 to less than 4%.

Variations in chemistry between macerals and within macerals depend on coal rank. Up to high‐volatile bituminous rank, liptinite macerals have higher absorbances of aliphatic stretching bands and lower absorbances of the aromatic 1600 cm—1 band than any other macerals. At medium‐volatile bituminous rank, aliphatic stretching bands are distinct in vitrinite and semifusinite and very weak to absent in other inertinite macerals. At anthracitic rank, variations both in elemental composition and in functional groups between maceral groups are of the same magnitude as within maceral groups.
Scanning tunnelling microscopy of conjugated polymers
Authors: BOND S. F. , FRIEND R. H. , HOWIE A.
Published: August 2011

Images of the conjugated polymers poly(p‐phenylene vinylene) and poly(3‐hexylthienylene) deposited on gold, silver and graphite have been obtained using a Nanoscope II STM in air at room temperature. Areas of semi‐crystalline structure have been observed, with local alignment of chains on the surface.
Euler‐Poincaré characteristic of a randomly filled three‐dimensional network
Authors: JERNOT J. P. , JOUANNOT P.
Published: August 2011

The random filling of a three‐dimensional face‐centred cubic network is simulated on a computer at increasing densities between 0 and 1. The Euler‐Poincaré characteristic (for the spaces ℝ0, ℝ1, ℝ2 and ℝ3) is then measured on all the simulated structures. A quantitative description of this simple densification process is obtained and the maxima observed correspond to the highest values practically attainable for the stereological parameters.
The cryopuncher: A pneumatic cryofixation device for X‐ray microanalysis of tissue specimens
Authors: ZIEROLD K.
Published: August 2011

A cryopunching device is described which allows cryofixation of tissue specimens by quick contact with a precooled copper surface during excision. The advantage of the cryopuncher for analytical electron microscopy of cells and tissues in defined functional states is illustrated by electron probe X‐ray microanalysis of freeze‐dried cryosections from rat liver and dogfish kidney. In comparison with results obtained from specimens plunged into liquid propane, cryopunching in situ results in similar preservation of morphology and remarkably improved intracellular K/Na ratio.
Random models for morphological analysis of powders
Authors: JEULIN D.
Published: August 2011

Models to estimate powder size distributions or fractions of components in a mixture of powders were developed and tested for various specimens. The models derived from the Boolean model and from the dead leaves models can be implemented on rough secondary electron scanning electron microscope images, obtained after minimal sample preparation. With the dead leaves tessellation, the size distributions of spherical particles or short fibres can be estimated either from the size distribution of intact grains, or from the area fraction measurement after binary erosions. With the dead leaves random function, there is no need for image segmentation. It provides data on the size and shape of a population of particles from an estimation of the distribution of grey‐level images after erosions by convex structuring elements of increasing size. A version of these models for long fibres is developed for estimating their diameter distribution. Allowing for superposition of particles, the proposed methods enable an unbiased estimation of the size distribution and characterization of the shape of complex particles. The approach is illustrated by applications to spherical particles obtained by simulations and from scanning electron microscope micrographs.
Vitrification depth can be increased more than 10‐fold by high‐pressure freezing
Authors: SARTORI N. , RICHTER K. , DUBOCHET J.
Published: August 2011

Biological specimens prepared for cryoelectron microscopy seem to suffer less damage when they are frozen under 2 kbar pressure rather than under normal conditions. The volume that can be well preserved is larger. This fact has been illustrated in a number of publications on a number of different samples. However, there is a lack of quantitative data concerning the depth of this good specimen preservation.

Catalase crystals in various sugar solutions have been used as test objects and vitrification, as determined by electron diffraction, has been used as the criterion for good freezing. Keeping all other conditions equal, the depth of vitrification is approximately 10 times larger with freezing at high, rather than normal, pressure. The high‐pressure vitrification depth in a 15–20% sugar solution averages 200 μm. Fully vitrified specimens up to 700 μm in thickness are obtained. When crystalline water is observed it is frequently in the form of high‐density ice II, III or IX.

These results are probably also relevant for typical biological specimens. The advantage of high‐pressure freezing must be balanced by the possible consequences of a considerably increased cooling time and by the damage that may be induced by the pressure.
Computer simulation of a mirror STEM
Authors: CREWE A. V.
Published: August 2011

The results of a computer simulation indicate that it is possible to design and build an STEM that is free from spherical aberration and should therefore have a very high resolution. The computer program was written in APL. The calculations include the effects of apertures and, consequently, mimic a realistic situation.
High‐spatial‐resolution maps of sulphur from human hair sections: An EELS study
Authors: HALLEGOT P. , CORCUFF P.
Published: August 2011

High‐resolution sulphur maps have been acquired from human hair using a Zeiss CEM 902A transmission electron microscope equipped with an energy filter. Analysis by electron energy‐loss spectroscopy (EELS) was performed on ultrathin sections of hair shafts embedded in three different types of resin: Nanoplast (water‐soluble), Spurr (epoxy) and Lowicryl (low‐temperature resin). Good‐quality energy‐loss images have been obtained with the three resins, although it was found that Nanoplast gave the best image contrast. For the first time, the results obtained for the detection of sulphur by silver staining of hair sections, which until now has been the only way to map sulphur at the electron microscopic level, have been confirmed. The results are compared with local sulphur concentrations from bulk analysis.
Remapping disparate images for coincidence
Authors: GALBRAITH W. , FARKAS D. L.
Published: August 2011

With the development of complex multimode computerized microscope systems, it is possible and necessary to obtain images of the same area of the microscopical preparation by several methods of microscopy, such as differential interference contrast, reflection interference microscopy, several wavelengths of fluorescence microscopy, laser scanning and confocal modes. Thus, varied information may be obtained about a single field, in the form of a set of images, taken at different ports of the microscope, using different digitizing cameras, each appropriate to certain tasks. For comparative purposes, the images should be superimposable, pixel by pixel, but in general they are not — they differ in image shape and size, magnification, distortion, centration and orientation. This paper shows how the problem may be approached, using an extension of the remapping procedures described in a previous paper, in which images of a separate grid reference slide are used to detect, quantify and correct the image errors. Affine remapping, without the use of grid images, is also described.
Simplified nerve cell counting in the rat brainstem with the physical disector using a drawing‐microscope
Authors: GUNTINAS‐LICHIUS O. , MOCKENHAUPT J. , STENNERT E. , NEISS W. F.
Published: August 2011

A simple modification of the physical disector is presented, which is used to count the number of neurons in the hypoglossal nucleus of the rat in a series of paraffin sections. One disector consists of two adjacent sections (6 μm thick) that have been Nissl‐stained with cresyl fast violet. In the first step of the procedure each of the two sections is investigated separately with a drawing‐microscope. The boundary of the hypoglossal nucleus and the position of neurons devoid of, or containing a part of, the cell nucleus in the plane of the section are marked on transparent paper. In the second step, these two drawings are placed one upon another, aligned and the number of cell profiles that show a cell nucleus in one but not in both drawings counted. This modification of the disector method for cell counting needs no specialized equipment, simply a light microscope with drawing apparatus, and can be combined with histochemical studies of other sections from the same tissue block.