bluuebunny/crossref_metadata_2025 huggingface dataset:
Resolution in light microscopy studied by computer simulation
Authors: Goldstein D. J.
Published: August 2011

Microscopic resolution can be characterized by K = x NA/δ, where x is the distance between two objects, or the interval of a grating, just resolved with light of wavelength λ and an objective of aperture NA. Using a computer simulation of imaging the following K values were obtained on the Sparrow resolution criterion for line and grating objects and various imaging methods (figures for the Rayleigh criterion, which assumes a finite contrast‐sensitivity of the light detector, are in parentheses). Several results appear to be novel. Due to limitations discussed in the text some data are only approximate.

With a grating object K is 1.0 (1.0) for axial coherent illumination, 0.5 (0.5) for coherent illumination at an obliquity NAobi which just enters the objective aperture, 0.5 (about 0.53) for incoherent illumination, 0.5 (about 0.52) for illumination with a condenser aperture NAc equal to NA, 0.5 (about 0.515) for transmitted‐light confocal scanning, and 0.25 (about 0.38) for fluorescent confocal scanning. If the object consists of two parallel lines K is about 0.68 (0.71) for axial coherent illumination, about 0.44 (0.5) for incoherent illumination, 0.375 (about 0.48) for optimal partially coherent illumination in which NAc may exceed NA, 0.44 (0.48) for transmitted‐light confocal scanning, and 0.32 (0.41) for fluorescent confocal scanning.

For inter‐object distances of 1, 1.5 and 2 wavelengths, respectively, NAc values of about 0.69, 0.5 and 0.375 gave optimal contrast and resolution irrespective of NA. The practice of setting NAc to about two‐thirds of the NA of a high‐power objective is supported by the fact that a condenser aperture of about 0.69 gives excellent or optimum resolution and contrast for most inter‐object distances and objective apertures tested, although with some distances and apertures reducing NAc improved contrast slightly.

The rule (sometimes attributed to Abbe) that resolving power is proportional to the mean of NA and NAc is correct for oblique coherent illumination in the case of a grating object, provided NAobl does not exceed NA. In the case of two isolated objects the rule is only approximately correct, but applies even if NAobl is greater than NA. Coherent light at an obliquity of 0.5λ/x introduces a half‐wavelength phase difference between two objects and permits their resolution (with perhaps an incorrect apparent inter‐object distance) even with objective apertures approaching zero.

In confocal scanning the width of the scanning spots has only a moderate effect on resolution, and two objects can sometimes be resolved with scanning spots wider than the inter‐object distance provided the lens apertures are neither too small nor too large.
An improved cryo‐jet freezing method
Authors: Greene William B. , Walsh Lyle G.
Published: August 2011

A new cryo‐jet freezing apparatus is described that is easy to use and gives good results using a propane‐butene mixture (3: 1). Our use of the freezer in the study of mouse spinal cord explant cultures is discussed. At the tissue surface, the quality of tissue preservation from freezing, followed by freeze substitution, rivals that of conventional electron microscopic methods. Certain intracellular structures are better visualized using our methods. There is no evidence of the tissue being distorted by the cryogen jet when the freezer is operated correctly. A new freeze substitution device is also discussed.
Investigation and use of plasmon losses in energy‐filtering transmission electron microscopy
Authors: Fromm I. , Reimer L. , Rennekamp R.
Published: August 2011

The electron spectroscopic imaging (ESI), diffraction (ESD) and different types of electron energy‐loss spectroscopy (EELS) modes in an energy‐filtering transmission electron microscope can all be used for the investigation and analytical use of plasmon losses. Shifts of plasmon losses caused by differences in composition can be detected with an accuracy of 0.1 eV by parallel‐recorded EELS (PEELS). The dispersion of plasmon losses and the cut‐off angle θc can be observed by angle‐dispersive EELS and by recording spectra at different scattering angles θ. ESD patterns with a selected energy window width of 1 eV enable the dispersion and its anisotropy to be imaged by characteristic intensity distributions between the primary beam and the first Bragg diffracted beams. The ESI mode can be used for the selective imaging of precipitates and for the investigation of the excitation volume of plasmons in small particles.
Quantification procedures for electron energy‐loss spectroscopy and imaging: the use of correspondence analysis for element determination
Authors: Gelsema E. S. , Beckers A. L. D. , Sorber C. W. J. , De Bruijn W. C.
Published: August 2011

A systematic investigation of the application of correspondence analysis to the determination of element responses in electron spectroscopic imaging is presented. The formalism of correspondence analysis has been applied to a set of artificially generated images, in which the effects of non‐homogeneous specimen thickness and noise were incorporated. The simulation experiments indicate that the results may be used for both qualitative (element distribution) and quantitative (element concentration) purposes.
A new background subtraction for low‐energy EELS core edges
Authors: Tenailleau H. , Martin J. M.
Published: August 2011

A new three‐parameter function is proposed that improves the ‘classical’ power‐law function for background subtraction processing at low energy losses in electron energy‐loss spectroscopy. The variation of the edge integrals with the width of the pre‐edge window Γ is reduced by a factor of at least five (of interest for accurate quantitative analysis), as is the variation in the height of energy‐loss near‐edge structure. The lower energy limit used for previous background functions has been cut by a factor of two or more. However, the principal advantage of this background function is that the difficulty in the choice of the pre‐edge window Γ is removed since the widest possible need only be taken.
Analysis of the calcium distribution in predentine by EELS and of the early crystal formation in dentine by ESI and ESD
Authors: Plate U. , Höhling H. J. , Reimer L. , Barckhaus R. H. , Wienecke R. , Wiesmann H. P. , Boyde A.
Published: August 2011

Predentine is a collagen‐rich extracellular matrix between the odontoblasts and the dentine with a width of about 15–20 μm. Electron energy‐loss spectroscopy of rat incisors shows a significantly higher calcium content in the predentine at the predentine‐dentine border than in the middle region of the predentine.

At the predentine‐dentine border in the dentine, the calcium and the phosphate groups combine to form apatite crystallites. Electron spectroscopic diffraction with zero‐loss filtering revealed that the earliest crystallites contain only Debye‐Scherrer rings of apatite, which are fewer in number and more diffuse than the diffraction rings from the mature crystallites. We therefore conclude that the early crystallites still contain lattice defects, which are annealed out to some degree with crystal growth. Electron spectroscopic imaging with zero‐loss filtering also showed that the earliest crystallites are chains of dots (or small islands); they build up strands composed of islands, which rapidly acquire a needle‐like character and coalesce laterally to form ribbon‐ or plate‐like crystallites. The parallel strands sometimes appear to reinforce the macroperiod of the collagen microfibrils (67 nm) by tiny holes without any crystal‐substance lined up perpendicular to the parallel strands of the crystallites.
The electron energy‐loss spectroscopic analysis of inhaled smoke particles
Authors: Mitchelson Brian P.
Published: August 2011

This paper details the use of electron spectroscopic imaging in the elemental analysis of smoke particles inhaled by smoke‐death victims. The results show that these particles have a varied structure and composition. Because of this, these particles may play a far more significant role in smoke inhalation injuries than has previous been recognized.
Zero‐loss energy‐filtered imaging of frozen‐hydrated proteins: model calculations and implications for future developments
Authors: Schröder R. R.
Published: August 2011

Energy‐filtered transmission electron microscopes operating in zero‐loss mode are used increasingly to study biological material in frozen‐hydrated conditions. The contrast enhancement and improved structural resolution obtainable by this method have been studied using Monte‐Carlo model calculations for the scattering processes occurring in such samples. Three models representing typical situations have been analysed, each normalized to minimal beam damage. It is shown that for proteins in thin layers of ice an optimal signal‐to‐noise ratio is achieved in the 80–120‐keV electron energy range. For proteins which have to be embedded in thicker ice layers, a considerably higher acceleration voltage is required. In particular, electron energies above 200 keV would be desirable for electron diffraction work on microcrystals.
Electron spectroscopic study (ESI, EELS) of Nanoplast‐embedded mammalian lung
Authors: Fehrenbach H. , Richter J. , Schnabel Ph. A.
Published: August 2011

The potential of Nanoplast melamine resin embedding for the study of mammalian lung parenchyma was examined by means of electron spectroscopic imaging (ESI) and electron energy‐loss spectroscopy (EELS). Samples were either fixed with glutaralde‐hyde‐paraformaldehyde or glutaraldehyde‐tannic acid, or were directly transferred to the embedding medium without prior fixation. Organic dehydrants, as well as fixatives containing heavy metals and stains, were omitted.

A very high level of ultrastructural detail of chromatin, ribosomes, mitochondria and plasma membranes was achieved by ESI from the Nanoplast‐embedded samples. The most prominent gain in ultrastructural detail was achieved when moving from an energy loss just below the L2,3 edge of phosphorus at 132 eV to an energy loss just beyond this edge. This reflects the prominent P L2,3 edge observed by EELS of Nanoplast‐embedded samples in comparison with conventionally processed samples. Thus, taking into account possible sectioning artefacts, excellent heterochromatin images which rely on the phosphorus distribution can be obtained from Nanoplast‐embedded samples by computer‐assisted analysis of electron spectroscopic images. In this respect glutaraldehyde‐paraformaldehyde fixation is preferable to glutaraldehyde‐tannic acid fixation because the presence of silicon, revealed by EELS, in tannic‐acid‐fixed samples may introduce artefacts in phosphorus distribution images obtained by the three‐window method because of the close proximity of the L2,3 edges of silicon and phosphorus.
A 360° single‐axis tilt stage for the high‐voltage electron microscope
Authors: Barnard David P. , Turner James N. , Frank Joachim , McEwen Bruce F.
Published: August 2011

A new type of specimen stage that permits more than 180° of tilting about the axis of a side‐entry rod has been developed for a high‐voltage electron microscope (HVEM). Roughly cylindrical specimens, with radial dimensions of less than a few micrometres, that can be mounted on the tip of a microneedle or micropipette are applicable. For glass micropipettes, the energy of the 1‐MeV beam of the HVEM is sufficient to image specimens through both walls. The stage employs a spindle mechanism that holds these needles or micropipettes coaxial with the tilt axis, allowing the specimen to be rotated without restriction. This arrangement, along with the cylindrical form of the specimen, is an important development for single‐axis tomography, because it permits a complete 180° set of projections to be recorded. The angular accuracy of the stage was demonstrated to be within ±0.20°, with a cumulative error of less than 1.0° over a 180° span.

The new stage was tested using puffball spores mounted on a micropipette. A 180° tilt series was recorded and processed to yield a tomographic three‐dimensional reconstruction which was displayed both as a cross‐sectional view perpendicular to the tilt axis, and as a shaded surface viewed from different directions. The same computations were repeated using subsets of the tilt series to assess the effect of various amounts of missing information. Visual inspection of a selected cross‐section from these reconstructions indicated that limiting the angular range to 160° produced results nearly as good as the full data set. Limiting the range to 140°, however, produced a noticeable geometric distortion, which became increasingly severe with ranges of 120° and 100°.
Evanescent‐wave microscopy: A simple optical configuration
Authors: Murray John M. , Eshel Dan
Published: August 2011

Under suitable conditions, the region of the aqueous phase immediately adjacent to a glass‐water interface can be selectively illuminated using the evanescent wave created when total internal reflection occurs at the interface. Objects in the aqueous phase away from the glass become effectively invisible, since the intensity of the evanescent wave decays exponentially with distance from the interface. Previous methods of generating evanescent waves for light microscopy have employed accessory light sources and optical components that directed the illumination on the specimen from one side. The asymmetric illumination creates a surprising orientation dependence of visibility for some specimens. Objects such as microtubules are totally invisible unless they are orientated nearly perpendicular to the direction of illumination. An explanation of this phenomenon is provided in terms of the geometry of diffraction of light by long thin objects.

A simple method of achieving evanescent‐wave illumination is described and shown to be useful in practice for biological specimens. In contrast to previously described methods, the present arrangement has the advantage of producing circularly symmetric illumination, and of utilizing only standard optical microscope components. The system has been used for imaging specimens both by light scattering and by fluorescence. It has proved useful for following the fragmentation of flagella into isolated microtubules, observing microtubules gliding over dynein adsorbed to a surface, and also for determining the arrangement of vinculin and alpha‐actinin in cell‐substratum attachment sites and termini of growing myofibrils in cardiac cells.
The application of electron microscopy in dairy research
Authors: Schmidt D. G. , Buchheimi W.
Published: August 2011

A review is presented of the application of various electron microscopical techniques in dairy research. Special attention is given to the use of cryofixation techniques, such as freeze‐fracturing, and of cryomicroscopy. These techniques are particularly well suited for the investigation of dairy products, which are mostly characterized by a high water content and by the presence of milk fat, having a low melting point. With these techniques, fat globules in milk, cheese and cream, and casein micelles in milk and milk products have been studied in an unperturbed state. By using immunogold‐labelled cryosections it could be demonstrated that in casein micelles in milk the kappa‐casein is located at the surface of the micelles, whereas the other caseins are distributed throughout the whole micelle.
Preparation of hard particle powders for examination in the transmission electron microscope
Authors: Boswell E. , Reece M. J. , Gee M. G.
Published: August 2011

A new technique for preparing electron‐transparent specimens of hard particle powders is described. It consists of mechanically embedding the powder into a thin foil of soft metal followed by ion‐beam etching. This has numerous advantages over other methods of examining powders, such as their direct examination when supported by a carbon film, or by using epoxy resin as an embedding medium. The metal foil can be chosen to minimize overlap of the spectrographic peaks of the metal and the hard particles.
Fixation for biological ultrastructure. II. Cross‐linking of bovine serum albumin by nanosecond pulses of ionizing radiation
Authors: Luft John H.
Published: August 2011

Very large doses of ionizing radiation were delivered quickly to concentrated albumin solutions by pulses of 500 keV in air. The electrons penetrated the aluminium foil bottom of the test cell and into the solution. A dose of 4.6 Mrad (4.6 × 104 Gy) produced a gel in the albumin solution comparable to the long‐term effect of 0.36% glutaraldehyde in the same albumin solution. The cross‐links created by the radiation leading to gel formation are probably irreversible and quite different from those leading to gelation in the glutaraldehyde‐albumin reaction. Single large pulses of ionizing radiation may be useful for fast fixation of cells and tissues for microscopy.
Confocal and differential phase imaging of thin organic films
Authors: Hudson A. J. , Wilson T.
Published: August 2011

This paper discusses the imaging of thin organic films in scanning optical microscopes using both differential phase contrast and confocal modes. The Lang‐muir‐Blodgett technique is used to deposit thin films of controllable thickness. Step structures in these films are considered and theoretical models of the imaging are compared with experimental data. The model provides a measurement of film parameters such as thickness and permittivity. The differential phase contrast mode is also proposed as a simple method of assessment of film quality.
Further modification of the LKB 7800 series KnifeMaker for improved reproducibility in breaking ‘cryo’ knives
Authors: Morewood Christopher R. , Elliott Edith , Dennison Clive , Bruto Anthony G.
Published: August 2011

This note describes further modifications to the LKB 7800 series KnifeMaker to improve the precision of scoring of glass strips and the consequent reproducibility of breaking of ‘cryo’ knives, by largely eliminating play in the mechanism, and by accurate adjustment of the cutting wheel position.
Stereo pairs of bright‐field micrographs via Wiener‐type inverse filtering
Authors: Khosla Deepak , Holmes Timothy J.
Published: August 2011

In a three‐dimensional (3‐D) image data set obtained through optical sectioning, each two‐dimensional (2‐D) segment is blurred by out‐of‐focus information from neighbouring focal planes superimposed on the in‐focus segments from that plane. Instead of attempting to remove this redundant information over the full 3‐D data set, we have developed a technique for restoring stereoscopic views. In this paper we describe the implementation of a Wiener‐type inverse filtering method for generating stereo pairs of bright‐field micrographs. A theoretical optical transfer function valid under certain simplifying approximations has been used in implementing this filtering technique. In developing this method the slice theorem of computed tomography is used. In this way the image reconstruction problem is reduced to one of processing 2‐D arrays rather than 3‐D arrays and the problem of restoring missing Fourier components within the missing‐cone region is circumvented. Limited experimentation with real micrographs shows that the approach provides images that display an effective increased depth of field and 3‐D attributes of the specimen, even though some of the underlying assumptions on which this method is based are difficult to verify explicitly. The method can be implemented with a relatively fast execution time on 386‐SX computers.
Depth discrimination in scanned heterodyne microscope systems
Authors: Somekh Michael G.
Published: August 2011

The optical transfer function of several scanning microscope systems is derived, using a physically intuitive approach. The technique allows a wide range of systems to be modelled with only minor modifications to the basic formulation.

The results are then used to determine the response of various scanning microscopes for objects both in and out of the focal plane. The possibility of performing extended‐focus phase imaging in heterodyne microscopes by scanning the sample along the optical axis is also examined. This mode of operation should allow measurements of minute topographical and phase variations on tilted or warped samples with the same lateral resolution as would be obtained when the sample is in focus throughout the entire scan.
Computer‐assisted realignment of light micrograph images from consecutive section series of cat cerebral cortex
Authors: Rydmark M. , Jansson T. , Berthold C.‐H. , Gustavsson T.
Published: August 2011

Two computer‐ and image‐analysis‐based procedures for realignment of images of consecutive light micrographs of nerve tissue (cat motor cortex) have been developed. One procedure (CENT) was interactive, employing the subjective overall ‘best fit’ of two images to each other and the other (AUTOCENT) was based on an automatic comparison of two images (image thresholding and binary comparison). Images of light micrographs of several hundred consecutive sections were realigned using the interactive and the automatic methods. The interactive procedure was easy to use; realignment of an image took only a few minutes in the hands of an experienced operator and should be easy to implement on commercially available smaller computers like PCs and workstations, but the realignment was possibly disturbed by the operator's subjective expectancy of regular forms. The automatic procedure realigned 1–4 images per hour, might be implemented on smaller computers if the programming is made more efficient, was probably objective and restored the ‘true position of images’.
Optimum recording conditions in scanning microscopes
Authors: Damm Tobias , Wilhelmi Bernd
Published: August 2011

The measuring process in the scanning microscope is analysed. The distortion of the recorded image due to finite diameter of the probe (laser spot, electron beam, nanometre tip) and due to the limited temporal response of the recording system is estimated. The influence of noise on the accuracy of the recorded image is considered. Optimum recording conditions such as choice of scan velocity and response time of the registration device are discussed with respect to minimum total error.
Experimental confirmation of super‐resolution in coherent confocal scanning microscopy using optical masks
Authors: Young M. R. , Jiang S. H. , Davies R. E. , Walker J. G. , Pike E. R. , Bertero M.
Published: August 2011

Experimental results are presented which demonstrate super‐resolution in a coherent scanning microscope. The microscope has a special optical mask, a Fourier lens and detector pin‐hole to carry out optical processing of the image. The form of the special mask was calculated using the theory of singular systems.
Optical modifications enabling simultaneous confocal imaging with dyes excited by ultraviolet‐ and visible‐wavelength light
Authors: BLITON C. , LECHLEITER J. , CLAPHAM D. E.
Published: August 2011

Optical modifications to a confocal scanning laser microscope are described which allow simultaneous fluorescence imaging of living specimens excited by ultraviolet (UV)‐ and visible‐wavelength light. Modifications to a Bio‐Rad MRC 600 Lasersharp confocal microscope include the introduction of UV‐path‐specific lenses and a specially designed UV transmitting eyepiece and tube lens. Upon UV excitation these modifications provide similar resolution and field flatness when compared with visible confocal microscopy. The UV‐path‐specific optics could be adjusted to correct for varying amounts of longitudinal chromatic aberration in commercially available objectives. Eyepiece and tube lenses were chromatically corrected for UV through visible wavelengths to minimize lateral chromatic error. With these modifications, UV‐wavelength light may be used to excite ratioing dyes to quantify intracellular ion concentrations, or as an energy source to release caged compounds in a spatially restricted volume, while simultaneously imaging with dyes excited by visible‐wavelength light.
A method for spatio‐temporal (4‐D) data representation in confocal microscopy: application to neuroanatomical plasticity
Authors: KRIETE A. , WAGNER H.‐J.
Published: August 2011

This paper describes a new method for data representation and visualization in four dimensions (three dimensions plus time). Sequential volumes, exhibiting morphological activity, are acquired non‐invasively with a confocal scanning laser microscope, where each data set corresponds to a time sample. A pipelined processing includes packing of volumes and specific volume rendering techniques. Subsequent processing in HIS (hue, intensity, saturation) colour space combines functional, packed images with shaded three‐dimensional views. As a result, even subtle changes in morphology become visible and computational time is saved. Experimental findings obtained from investigations of synaptic plasticity in cultured retinal tissue are reported.
A synchrotron X‐ray study of the changes occurring in the corneal stroma during processing for electron microscopy
Authors: FULLWOOD N. J. , MEEK K. M.
Published: August 2011

Using a high‐intensity synchrotron X‐ray source, the structural changes occurring in the corneal stroma were monitored during each stage of several different processing runs for the transmission electron microscope (TEM) and scanning electron microscope (SEM). The parameters studied were interfibrillar spacing, intermolecular spacing, D‐periodicity and fibril diameter.

The processing schedule that produced the least changes in spacings for TEM specimens involved extended fixation in glutaraldehyde followed by low‐temperature embedding in Lowicryl K4M resin. However, interfibrillar material was better preserved after embedding in LR White resin or Nanoplast.

Almost every processing stage for electron microscopy produced significant changes in one or more structural parameters in the cornea. Glutaraldehyde fixation significantly increased the intermolecular spacings, while resin infiltration and resin polymerization each resulted in shrinkage of all the spacings monitored. Critical‐point drying for SEM specimens resulted in considerable shrinkage in all three spacings, but was still preferable to air drying, which caused reduction in the order of the fibril packing, resulting in loss of the interfibrillar X‐ray pattern. Perhaps the most drastic effect was caused by post‐fixation in osmium tetroxide, which resulted in loss of the intermolecular pattern, and also increased the amount of shrinkage in the interfibrillar spacings and the D‐periodicity which occurred during later stages of processing.
Glucose alone does not completely hydrate bacteriorhodopsin in glucose‐embedded purple membrane
Authors: PERKINS G. A. , BURKARD F. , LIU E. , GLAESER R. M.
Published: August 2011

Glucose embedding is a simple and highly effective method for preparing biological macromolecules for high‐resolution electron microscopy. The investigation of conditions that can trap the M‐state intermediate in the bacteriorhodopsin (bR) photocycle has revealed, however, that when glucose‐embedded bR is prepared at ambient humidity, it does not fully retain the capability to execute a proper photocycle. However, ‘native’ photocycle properties are returned after glucose‐embedded samples are equilibrated at 81% relative humidity. Equilibration at relative humidities significantly higher than 81% causes glucose to dissolve in its own water of hydration, resulting in samples that may be too thick to be suitable for electron microscopy. The results obtained with bR indicate that caution should be taken with other biological specimens, and it cannot be assumed that glucose‐embedded biological macromolecules retain completely their native, hydrated structure, even when high‐resolution electron diffraction patterns are obtained. Equilibration of such samples at high humidity may generally be a worthwhile precaution when using the glucose‐embedding technique.
Grid‐specimen interactions: factors affecting copper nanocluster formation on polymer thin films
Authors: BURNS J. L. , SPONTAK R. J.
Published: August 2011

All transmission electron microscopy (TEM) studies require the use of grids to support thin films or sections of specimens within the microscope. These grids are presumed to remain inert during sample loading and analysis, an assumption which becomes questionable when on‐the‐grid processing methods are employed to probe the response of a specimen to a particular stimulus, such as temperature increase. Uniform dispersions of nanoclusters were observed to develop on and undergo chemical complexation with thin cast films of a poly(siloxaneimide) copolymer supported on various copper grids during thermal annealing at temperatures of up to 200°C. The thermal conditions which promoted nanocluster formation were investigated. Experiments designed to elucidate the mechanism by which these nanoclusters develop revealed that cuprous oxide (Cu2O) from the copper support grids is deposited on these imide‐bearing films from the vapour phase.
Scan speed limit in atomic force microscopy
Authors: BUTT H.‐J. , SIEDLE P. , SEIFERT K. , FENDLER K. , SEEGER T. , BAMBERG E. , WEISENHORN A. L. , GOLDIE K. , ENGEL A.
Published: August 2011

The scan speed limit of atomic force microscopes has been calculated. It is determined by the spring constant of the cantilever k, its effective mass m, the damping constant D of the cantilever in the surrounding medium and the stiffness of the sample. Techniques to measure k, k/m and D/m are described. In liquids the damping constant and the effective mass of the cantilever increase. A consequence of this is that the transfer function always depends on the scan speed when imaging in liquids. The practical scan speed limit for atomic resolution in vacuum is 0·1 μm/s while in water it increases to about 2 μm/s due to the additional damping of cantilever movements. Sample stiffness or damping of cantilever movements by the sample increase these limits. For soft biological materials imaged in water at a desired resolution of 1 nm the scan speed should not exceed 2 μm/s.
A simple modification to the LKB 7800 series Knifemaker and a balanced‐break method to prepare glass knives for cryosectioning
Authors: WEBSTER P.
Published: August 2011

A novel method of using the LKB 7800 series Knifemaker to produce glass knives using a balanced break is described. The method produces knives of sufficient quality to section aldehyde‐fixed, sucrose‐cryoprotected, frozen biological material in a cooled cryochamber of an ultramicrotome. The modifications to the Knifemaker are minimal and, if required, the machine can be returned easily to its normal state after use.
Numerical apertures of light microscope objectives
Authors: LEHMANN W. , WACHTEL A.
Published: August 2011

The numerical aperture of light‐microscope objectives is measured via the exit angle of the rear lens towards the image space, and the magnification of the objective. The method is reliable because of its simplicity and is independent of special instrumentation such as apertometers. Results from eight commercially available objectives indicate fair agreement of nominal data with measured data for the magnifications, but not for all numerical apertures.
Microstructure‐property relationships in silicate‐matrix composites
Authors: LEWIS M. H. , DANIEL A. M. , CHAMBERLAIN A. , PHARAOH M. W. , CAIN M. G.
Published: August 2011

Electron microscopy and associated analytical spectroscopic techniques have been used to characterize interfaces in SiC‐fibre/silicate‐matrix composites. Interface structure, formed via reaction during hot‐press fabrication, is a function of time, temperature, matrix composition and fibre type. Interfaces with Nicalon or Tyranno fibres vary from amorphous carbon in fine precipitated form to continuous graphitic layers.

Interface behaviour in a stressed composite, and hence the matrix microcracking stress, is a sensitive function of microstructure. Interface debond and shear properties have been assessed using indent‐based ‘push‐down’ and ‘push‐through’ tests using a specially developed instrument within a scanning electron microscope. This uses piezoload measurement and translation, and is capable of dynamic image recording of the indentation sequence.

Interface micromechanical (indent) measurements have been correlated with structure and macromechanical response in bend testing for a range of fibre/matrix types, processing and post‐processing thermal treatments. An example is also given of interfaces prepared by fibre precoating.
Acoustic microscopy of ceramic‐fibre composites
Authors: BRIGGS G. A. D. , LAWRENCE C. W. , SCRUBY C. B.
Published: August 2011

A variety of ceramic‐fibre composites has been studied by acoustic microscopy, at 1·9 GHz with a resolution of 0·8 μm. The materials studied were Nicalon‐reinforced borosilicate glass, SiC fibres in a magnesium–aluminosilicate matrix and a calcium‐aluminosilicate matrix, and SiC monofilaments in a Ti–6Al–4V matrix. In all the specimens the contrast was dominated by strong excitation of Rayleigh waves in the surface. This gave strong contrast from different phases, and revealed interfaces and cracks by characteristic crack patterns. Quantitative agreement between observed and calculated fringe patterns was found, and values of shear and Young's modulus were measured. In the SiC monofilament specimens, various stages of progressive deterioration as a result of thermal ageing treatments were observed.
TEM observations of SiC‐SiC composites with a carbon interphase layer annealed in air at high temperatures
Authors: SEBIRE‐LHERMITTE I. , GOMINA M. , VICENS J.
Published: August 2011

SiC–SiC composites with a carbon interphase layer have been annealed in air at high temperature (800–1400°C) in order to determine the oxidation mechanism. Transmission electron microscopy and high‐resolution electron microscopy observations have been performed on thin slices of oxidized specimens. Observations show that, with oxidation above 800°C, the carbon interphase can be replaced totally by a thick silica layer, in agreement with previous work. However, in many cases the modified interphase is heterogeneous. The turbostratic carbon structure disappears and the carbon interphase layer is partially replaced by an amorphous silica layer found preferentially at the contact zone between the fibres and the carbon layer. A very thin silica layer can also be observed on the matrix side at the matrix‐fibre interfaces. Some decohesion appears in areas where carbon is still present. For short‐duration exposures, decohesion occurs between the fibres and the carbon interphase layer.
The microstructure of silicon carbide (Nicalon) fibre reinforced silicon carbide ceramic‐matrix composites heat treated in vacuo and in pure oxygen
Authors: VINE W. J. , STEEDS J. W.
Published: August 2011

Silicon carbide (Nicalon) fibre reinforced SiC composites have been heat treated in vacuo and in pure oxygen environments at 1400°C for 100 h. The response of the microstructure and, in particular, of the interface between fibre, carbon interlayer and SiC matrix components has been studied. Microstructural modifications were observed by transmission electron microscopy, using imaging, electron energy‐loss spectroscopy and electron diffraction techniques, and fibre stoichiometries were determined using a scanning Auger microprobe.

Recrystallization of Nicalon fibres within composites heat treated in vacuo was found to result from decomposition of the metastable silicon oxy‐carbide phase found in the fibres. No significant changes to the pyrolytic carbon interlayer were observed. Fibre recrystallization was considered to embrittle the composite.

Samples heat treated in oxygen showed no appreciable fibre recrystallization. Study of interlayers in such aged samples often revealed decohesions, holes and narrow silica layers. In the most extreme cases, complete displacement of carbon by SiO2 was found and such interfaces were identified as silica and α‐cristobalite. Interfacial modifications were considered to be responsible for the retention of the small β‐SiC grain size in Nicalon fibres and were also considered to be deleterious to the mechanical properties.
A microstructural investigation of deformation in fine SiC particulate‐ and whisker‐reinforced aluminium‐matrix composites
Authors: BARLOW C. Y. , HANSEN N. , LIU Y. L.
Published: August 2011

Aluminium‐based composites, reinforced with low volume fractions of whiskers and small particles, have been formed by a powder route. The materials have been tested in tension, and the microstructures examined using transmission electron microscopy. The whisker composites showed an improvement in flow stress over the particulate composites, and this was linked to an initially enhanced work‐hardening rate in the whisker composites. The overall dislocation densities were estimated to be somewhat higher in the whisker composites than the particulate composites, but in the early stages of deformation the distribution was rather different, with deformation in the whisker material being far more localized and inhomogeneous. This factor, together with differences in the internal stress distribution in the materials, is used to explain the difference in mechanical properties.
The microstructures of locally reinforced squeeze‐cast Al‐alloy metal‐matrix composites
Authors: KANG H. G. , ZHANG D. L. , CANTOR B.
Published: August 2011

Locally reinforced metal‐matrix composites (MMCs) have been manufactured by squeeze casting an A357 Al alloy into a die containing a reinforcement insert of either steel, an A356/10 vol.% SiCp MMC, or a 20 vol.% Al2O3 short‐fibre preform. The temperatures of the Al‐alloy matrix, reinforcement insert and die were monitored continuously during squeeze casting, and the microstructures of the ascast MMCs were examined. Application of high pressure caused intimate contact along the insert‐matrix interface with refinement of the matrix microstructure. There was little bonding between the matrix and the steel insert, while good bonding was obtained between the matrix and the A356/SiCp MMC insert, which was partially melted at the interface. The Al2O3 short‐fibre preform insert was fully infiltrated at high pressure, with a band of high Si content in the matrix microstructure near the interface.
Heterogeneous fibre microstructures and their influence on failure
Authors: ZYWICZ E. , KINNEY J. H. , SATTLER M. L. , BREUNIG T. M. , NICHOLS M. C.
Published: August 2011

A detailed microscopic, experimental and micromechanical evaluation of fibre damage initiation in a unidirectional aluminium matrix‐silicon carbide fibre (SCS‐8TM) composite has been performed for a monotonic load sequence. The salient fibre features include a 33‐μm‐diameter monofilament turbostratic carbon (C) core, a ∼ 1·5‐μm pyrolytic C layer, an interior sheath of β‐phase silicon carbide (SiC) crystallites imbedded in an amorphous C matrix, and an exterior sheath of radially orientated β‐SiC. Quantitative microscopy shows that the interior sheath's surplus C varies smoothly from ∼ 35% by volume near the core to zero at the mid‐radius. The multi‐phase structure of the fibre produces an internal mechanical stiffness that increases with distance from the core, and thus peak stresses result in the exterior sheath. X‐ray tomographic microscopy (XTM) reveals that cores fracture randomly, without failure of the surrounding SiC, at stress levels above half the ultimate strength of the composite. Three‐dimensional XTM reconstructions show planar, non‐planar and spiral cracks in the failed fibre, suggesting multiple and competing initiation mechanisms. Qualitative fracture assessments suggest that flaws near the C core grow outward in a curved manner through the SiC–C and planar beyond the mid‐radius, whereas cracks originating near the fibre–matrix interface favour planar trajectories inward across the whole fibre.
The microstructure of spray‐formed Ti‐6Al‐4V/SiCf metal‐matrix composites
Authors: ZHAO Y. Y. , GRANT P. S. , CANTOR B.
Published: August 2011

Spray‐forming is a possible manufacturing route for the fabrication of Ti alloy fibre‐reinforced metal‐matrix composites (MMCs) because high rates of alloy‐droplet cooling on impact with the fibres prevent excessive fibre‐matrix reaction. Ti–6Al–4V matrix MMC monotapes containing TiB2‐coated SiC fibres have been manufactured by electric‐arc spray‐forming, and the key MMC microstructural characteristics in the as‐sprayed monotapes have been investigated by optical and scanning electron microscopy. Fibre infiltration increases with decreasing spraying distance, decreasing atomizing gas pressure and increasing arc current, because of higher temperatures in the Ti alloy spray droplets on impact with the fibres. Too much binder in the fibre preform leads to poor fibre–matrix contact, while removing the binder leads to the fibres becoming misaligned during spraying.
Comparison of interfaces in Ti composites reinforced with uncoated and TiB2/C‐coated SiC fibres
Authors: GUO Z. X. , DERBY B. , CANTOR B.
Published: August 2011

Interfaces play an important role in determining the mechanical properties of composite materials. The interfaces established between a titanium‐alloy matrix (Ti‐6Al‐4V) and uncoated and TiB2/C‐coated SiC fibres are analysed by scanning electron microscopy, transmission electron microscopy and X‐ray techniques. Emphasis is placed upon the interfacial morphology and microstructure, identification of reaction products, and the stability of the coating layer. Complex multi‐reaction layers are observed frequently in the interfacial zones. Previous, often contradictory, reports about the interlayers are reviewed. Experimental observation demonstrates that the type and distribution of interlayers vary in a given system, due to prolonged treatment of the samples at temperature. The formation and distribution of the interlayers are discussed further, with respect to these and previous findings. Methods of reducing interfacial reactivity are discussed.
Modelling of three‐dimensional fluorescence images of muscle fibres: an application of the three‐dimensional optical transfer function
Authors: SHEPPARD C. J. R. , GU M.
Published: August 2011

Striated muscle fibres can be modelled by a simple geometry, which has allowed three‐dimensional (3‐D) images in conventional and confocal microscopes to be calculated. This model is useful for comparing different imaging methods and represents a simple example of an application of the 3‐D optical transfer function (OTF) for the system. The rejection of out‐of‐focus blur is demonstrated, and the effects of fibre thickness and confocal pinhole size on image contrast are investigated. The effects of using a simple filter for image enhancement are studied, elucidating the characteristics of the OTF.
Fluorescence photobleaching recovery in the confocal scanning light microscope
Authors: BLONK J. C. G. , DON A. , Van AALST H. , BIRMINGHAM J. J.
Published: August 2011

Measurement of mobilities of species in liquid systems is of great importance for understanding a number of dynamic phenomena. A well known method for measuring mobilities driven by diffusion is fluorescence photobleaching recovery (FPR), also known as fluorescence recovery after photobleaching (FRAP). New FPR recovery equations for three‐dimensional (3‐D) apertured scanning using a Gaussian approximation for the axial beam profile have been successfully developed and found to provide a solid basis for extraction of the lateral diffusion coefficient from confocal scanning light microscopy (CSLM)‐FPR experimental data. The 2‐D diffusion coefficients of fluorescent species can be successfully measured by FPR in the CSLM, which has the great advantage that bleaching can be targeted at a well‐defined volume element in bulk samples. Two‐dimensional diffusion coefficients of 45‐nm latex spheres, of FITC molecules and of a 2·45‐nm protein‐FITC complex in water‐glycerol mixtures, measured by FPR in the CSLM, are in close agreement with those calculated from the size of the diffusing species and viscosity of the medium. Diffusion coefficients as high as 2 times 10−6 cm2/s can be measured.
Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index
Authors: HELL S. , REINER G. , CREMER C. , STELZER E. H. K.
Published: August 2011

The effect of refractive‐index mismatch, as encountered in the observation of biological specimens, on the image acquisition process in confocal fluorescence microscopy is investigated theoretically. The analysis takes the vectorial properties of light into account and is valid for high numerical apertures. Quantitative predictions on the decrease of resolution, intensity drop and shift of focus are given for practical situations. When observing with a numerical aperture of 1·3 (oil immersion) and an excitation wavelength of 514 nm the centre of the focus shifts 1·7 μm per 10 μm of axial displacement in an aqueous medium, thus yielding an image that is scaled by a factor of 1·2 in the axial direction. Furthermore, it can be expected that for a fluorescent plane 20 μm deep inside an aqueous medium the peak intensity is 40% less than for a plane which is 10 μm deep. In addition, the axial resolution is decreased by a factor of 1·4. The theory was experimentally verified for test samples with different refractive indices.
Utility of the horizontal slice for stereological characterization of lineal features
Authors: GOKHALE A. M.
Published: August 2011

The efficient stereological procedure for estimating the length density Lv of lineal microstructural features is further improved: the length density of long or short (or those of any arbitrary geometry) lineal features can now be estimated from the measurements performed on the projected images of vertical and horizontal slices of unknown and variable thickness. It is not necessary to identify uniquely the intersections of the lineal features with the parallel faces of the slices in the projected images and distinguish them from the images of the feature end points contained in the slices. The new technique does not involve any assumptions, and it is applicable to the lineal features of any arbitrary geometry and anisotropy. As a ‘bonus’, the same input measurements also yield an assumption‐free estimate of the total number of true lineal‐feature end points per unit volume Ev, and the average slice thickness.
Stereological estimation based on isotropic slices through fixed points
Authors: KIEU K. , JENSEN E. B. VEDEL
Published: August 2011

Stereological estimators of length and surface area based on measurements in an isotropic slice through a fixed point are described. Measurements of three‐dimensional angles are not needed. The estimators depend only on distance measurements. The variance of the length estimator is studied in some detail. Applications to particle analysis and second‐order stereology are discussed.
Contact distances and random free paths
Authors: SAXL I.
Published: August 2011

The first part of the present paper is aimed at a unified introduction of various random distances in a system of disjoint particles in ℝd or in its bounded subset. A general formula for the distribution function of distances is given and applied to several particular situations.

In the second part, the detailed results for three selected types of random as well as regular structures are presented.
On the computation of the Minkowski dimension using morphological operations
Authors: KANMANI S. , RAO C. BABU , RAJ B.
Published: August 2011

The Minkowski dimension as a complexity measure, and its relation to the other dimensions in the case of self‐similar fractal objects, is discussed. An implementation of the Minkowski dimension, using morphological operations on a square grid, is presented. The effect of resolution, orientation and the symmetry of the structuring element used on the estimation of the Minkowski dimension is studied. The corrections required for free end points are presented.