bluuebunny/crossref_metadata_2025 huggingface dataset:
An inexpensive device for freeze drying and plastic embedding tissues at low temperatures
Authors: Lyon R. , Appleton J. , Swindin K. J. , Abbot J. J. , Chesters J.
Published: August 2011

The preparation of biological tissues for electron microscopy by rapid freezing retains the original localization of ions and molecules. A reproducible freezing regime was established by quenching tissues in liquid propane according to the method of Elder et al. (1981). Tissue was thereafter freeze dried in a custom built freeze drying device with a liquid nitrogen cooled stage to prevent ice recrystallization during drying. The device was also designed to allow the vacuum embedding of tissue in low temperature resin such as Lowicryl® and polymerization in situ. This paper describes the design of the device and an example of its use in the freeze drying of cartilage. The results show that minimal ice damage occurs to the chondrocytes and that intracellular organelles are clearly visible. The regime described may prove a useful and pragmatic alternative to cutting tissue in the frozen state. Translocation of elements is unlikely except perhaps in the case of very labile elements such as Na and K, but this remains to be fully elucidated.
Phase separation in frozen erythrocyte membrane preparations
Authors: Finean J. B. , Hutchinson A. , Mills D.
Published: August 2011

The reversible formation of a lipid‐like phase in frozen preparations of erythrocyte membranes has been studied by X‐ray diffraction and by electron microscopy of freeze‐fracture replicas. The observations provide strong evidence for lateral migration or displacement at specific temperatures of intra‐membrane particles. This creates large areas of particle‐free membranes which fracture preferentially so as to dominate the freeze‐fracture image.
Normalization of light element X‐ray intensities for surface topography effects in frozen‐hydrated biological bulk samples
Authors: Marshall A. T. , Condron R. J.
Published: August 2011

X‐rays from light elements (Z=6–12) experience marked absorption in biological or organic matrices. Consequently surface tilt has a strong effect on X‐ray intensity. Using epoxy resin models with well‐defined surface tilt variations the effect of tilt on potassium, sodium and oxygen X‐ray intensities has been examined and methods for normalizing intensities for tilt effects have been investigated.

With an energy dispersive X‐ray detector having a high (40°) take‐off angle, samples, having local tilt variations from 20° from the horizontal towards the detector to 20° away from the detector, can be analysed for potassium using peak intensities without loss of accuracy. Little loss in accuracy is experienced for sodium over the tilt range of 10° towards the detector to 10° away from the detector. Under these conditions peak to background (P/BG) ratios offer little advantage over simple intensity values.

Sodium absorption in frozen‐hydrated biological samples is about 10% greater than in epoxy resin whilst potassium absorption is similar in both samples. It is assumed therefore that the foregoing conclusions also hold for frozen‐hydrated samples.

X‐ray peak intensity to backscattered electron (P/BE) ratios provide a means of normalizing peak intensities from light elements for surface tilt effects. This is a particularly useful method for oxygen intensities because P/BG ratios cannot be obtained with any facility at the present time. Oxygen P/BE ratios provide a means of obtaining intracellular water concentrations in fractured frozen‐hydrated biological bulk samples, where surface tilt varies. Sodium intensities can also be normalized in this way.
X‐ray microanalytical resolution in frozen‐hydrated biological bulk samples
Authors: Marshall A. T. , Condron R. J.
Published: August 2011

In coated frozen‐hydrated gelatin gels the backscattered electron yield does not increase during electron irradiation as it does in uncoated samples. Neither is the backscattered electron yield greater from coated frozen‐hydrated gels than that from more conductive organic samples. This is interpreted as indicating that a significant distortion of the electron interaction volume, due to the development of a space charge, does not occur in electron irradiated frozen‐hydrated gelatine gels when they are coated with a conducting coat.

The depth resolution as estimated from models of biological samples in the form of frozen‐hydrated photographic film and frozen‐hydrated sections of gelatine gel is consistent with that computed from X‐ray depth distribution curves, i.e. close to 2.0 μm at 15 kV. Lateral resolution was estimated from photographic film to be close to 2.0 μm also, at 15 kV.
Toughening of hot pressed beta‐alumina using zirconia additions prepared from zirconate, tartrate and carbonate precursor materials
Authors: Binner J. G. P. , Stevens R. , Tan S. R.
Published: August 2011

The development of second phase zirconia precipitates in a beta‐alumina matrix has been achieved using chemical reactions which occur during the firing process. The degree of success of each of several fabrication routes has been evaluated by use of toughness measurements and electron microscopy techniques. Approximately half of the routes yielded ∼90% retention of tetragonal zirconia, resulting in an approximate doubling of the Mode 1 critical stress intensity factor (K1c).
The characterization of interfacial structures in the oxidation of Fe and FeNiCr alloys by edge‐on transmission electron microscopy
Authors: Newcomb S. B. , Stobbs W. M.
Published: August 2011

A wide range of scaling processes can occur during the high temperature oxidation of FeCr alloys. Here we discuss the relative merits of using alternative transmission electron microscopical (TEM) techniques, as applied to edge‐on foils, for characterizing some of the different diffusional and microstructurally limited processes which occur, in particular at oxide and metal‐oxide boundaries. We give results for interfaces in wüstite, which is formed very rapidly on pure iron, as well as for the more protective FeCr spinels and sesquioxides, as grown on FeNiCr alloys in both air and 1%C0–C02.
Resolution in low voltage scanning electron microscopy
Authors: Joy David C.
Published: August 2011

As the energy of an electron beam is reduced, the range falls and the secondary electron yield rises. A low voltage scanning electron microscope can therefore, in principle, examine without damage or charging samples such as insulators, dielectrics or beam sensitive materials. This paper investigates the way in which the choice of beam energy affects the spatial resolution of a secondary electron image. It is shown that for samples which are thin compared to the electron range, the edge resolution and contrast in the image improve with increasing beam energy. In samples that are thicker than the electron range, the resolution can be optimized at either high or low energies, but low energy operation will produce images of higher contrast. At an energy of 2 keV or less beam interaction limited resolutions of the order of 3 nm should be possible.
Point cathodes for use in virtual source electron optics
Authors: Tuggle D. W. , Li J. Z. , Swanson L. W.
Published: August 2011

A comparison is made for three point cathodes which are used in an electron gun forming a virtual rather than real source. The three cathodes operate in the field emission (FE), thermal‐field (TF) and Schottky emission (SE) regimes. A low angular intensity is chosen for this comparison, typical of an SEM as contrasted to an electron‐beam lithography system application. Energy spread, virtual source size and current fluctuations are compared.
Thermal field emission for low voltage scanning electron microscopy
Authors: Orloff J.
Published: August 2011

Low voltage scanning electron microscopy is a technique of great importance to the semiconductor industry, where inspection and testing of non‐conductive devices must be done at ∼ 1 keV beam energy. In order to perform scanning electron microscopy for inspection at high speed, improved cathodes are required in order to achieve good resolution at low energy with high beam current, the chromatic spread in the electron beam is shown to be the limiting factor in the SEM and a comparison of thermionic, field emission and Schottky cathodes is made to determine which can provide the best performance. It is concluded that a thermal field emission or Schottky, cathode is superior to either conventional field emission or thermionic cathodes.
Some recent developments in low voltage E‐beam testing of ICs
Authors: Menzel E. , Buchanan R.
Published: August 2011

The fundamental operating principles of electron beam testing of integrated circuits are reviewed. Data obtained using a recently developed secondary electron analyser located within the final probe‐forming lens in a scanning electron microscope are presented. Performance improvements in spatial resolution, energy resolution, and measurement accuracy are described.
The pair correlation function for point and fibre systems and its stereological determination by planar sections*
Authors: Hanisch Karl‐Heinz , König Dieter , Stoyan Dietrich
Published: August 2011

Traditional stereology consists nearly completely in the determination of particle size distributions and mean values such as Vv and Sv. However, for the description of the ‘inner’ structure of random structures second‐order characteristics such as the pair correlation function or reduced second moment function are useful. In the present paper stereological estimation of second‐order quantities for centres of random sphere systems and for random fibre systems are considered. In the case of sphere systems stereological formulae are given which connect the pair correlation function of the sphere centres with quantities available from planar, linear and thin sections. For random fibre systems some exact and approximate stereological methods are suggested which enable the determination of second‐order quantities from planar and thin intersections.
The stabilization of labile configurations of plant cytoplasm by freeze‐substitution
Authors: McCully M. E. , Canny M. J.
Published: August 2011

The system of elongated, vacuole‐like structures that are seen in phase‐contrast images of living plant cytoplasm, which has been called the pleomorphic canalicular system, has been shown previously to be distorted and vesiculated by conventional fixation methods. The stabilization of this labile structure in tomato hair cells by freeze‐substitution is described. Structural features of the freeze‐substituted cytoplasm as seen in whole mounts of resin‐embedded cells resemble closely those of the living cells.
The structure of the stalk surface layer of a brine pond microorganism: correlation averaging applied to a double layered lattice structure
Authors: Kessel Martin , Radermacher Michael , Frank Joachim
Published: August 2011

The surface layer of the stalk of a prosthecate halophilic microorganism is a periodic array (space group p3ml) comprised of electron dense trimers with a centre to centre spacing of 9.0nm. The structure is reminiscent of E. coli porin. We have demonstrated that the method of correlation averaging can be effectively used to separate overlapping lattices, and that this results in a higher fidelity reconstruction, when compared to the established method of Fourier filtration. Two statistical methods are used to determine the resolution of the correlation average as a function of the number of ‘windows’ averaged, (i) The phase residual of spatial frequencies in Fourier space is computed between independently obtained subaverages, and (ii) a new method of Q factor analysis examines the cumulative vector sum in Fourier space as a function of the number of windows averaged. Both methods give a resolution of 1/2.1−nm.
High resolution electron microscopy of interfaces in chlorinated phthalocyanine molecular crystals
Authors: Fryer J. R. , Smith David J.
Published: August 2011

Molecular images of chlorinated copper phthalocyanine have shown the arrangement of molecules at the interface between two crystals. Mismatch does not cause distortion or recrystallization for low index junctions but for more irregular interfaces strain causes alteration of the molecular tilt angle and accommodation of the mismatch by the formation of edge dislocations whose Burger's vectors are parallel to the interface.
A sample preparation for quantitative determination of magnesium in individual lymphocytes by electron probe X‐ray microanalysis
Authors: Hook Gregory R. , Elin Ronald J. , Hosseini Jeanette M. , Swyt Carol , Fiori Charles E.
Published: August 2011

We present a sample preparation method for measuring magnesium in individual whole lymphocytes by electron probe X‐ray microanalysis. We use Burkitt's lymphoma cells in culture as the test sample and compare X‐ray microanalysis of individual cells with atomic absorption analysis of pooled cell populations. We determine the magnesium peak‐to‐local continuum X‐ray intensity ratio by electron probe X‐ray microanalysis and calculate a mean cell magnesium concentration of 39± 19 mmol/kg dry weight from analysis of 100 cells. We determine a mean cell magnesium concentration of 34 ±4 mmol/kg dry weight by atomic absorption analysis of pooled cells in three cell cultures. The mean cell magnesium concentrations determined by the two methods are not significantly different. We find a 10% coefficient of variation for both methods of analysis and a 30% coefficient of variation in magnesium concentration among individual cells by electron probe X‐ray microanalysis. We wash cells in ammonium nitrate for microanalysis or in buffered saline glucose for atomic absorption analysis. We find cells washed in either solution have the same cell viability (85%), recovery (75%), cell volume (555 μm3) and cytology. We air dry cells on thin film supports and show by magnesium X‐ray mapping that magnesium is within the cells. We conclude that: (a) our microanalysis cell preparation method preserves whole intact lymphocytes; (b) there is no systematic difference in results from the two methods of analysis; (c) electron probe X‐ray microanalysis can determine the variation in magnesium concentration among individual cells.
Measurement of the optical thickness of transparent tissue layers
Authors: Gahm Thomas , Witte Siegfried
Published: August 2011

The method of Due de Chaulnes was employed to determine the mechanical and optical thickness, as well as the refractive index, of transparent tissue layers in living specimens. To this end the reproducible accuracy of the method and its dependence on the adjustment in focusing and on the numerical aperture of the objective was evaluated on test specimens using the procedures of transmitted light, phase‐contrast (PC), and differential interference contrast (DIC) microscopy. The best working conditions were then applied to the actual measurements.
Characterization of defects in semiconductors by combined application of SEM(EBIC) and SDLTS*
Authors: Heydenreich J. , Breitenstein O.
Published: August 2011

Besides the characterization of the geometrical structure of defects in semiconductors by TEM the estimation of their electrical activity is of importance. SEM(EBIC) and SDLTS (scanning deep level transient spectroscopy) are especially suitable for this purpose; they allow the inspection of electronic properties with a spatial resolution in the micron‐range. On the one hand, SEM(EBIC) yields information on the recombination efficiency of defects in the crystal volume adjacent to a pn junction or a Schottky barrier; on the other hand, SDLTS enables the detection to be carried out of the distribution and the energetic levels of deep level defects lying in the space charge region. Accordingly, the combined application of these techniques is very promising for investigating physical processes implying an inhomogeneous incorporation of deep level defects in semiconductor crystals.

In comparison to the widely used SEM(EBIC) technique SDLTS has only rarely been applied, a fact that is due to the high detection sensitivity necessary for measuring capacity transients. The application of a highly sensitive (10—6 pF) micro‐computer‐controlled SDLTS system in combination with a conventional EBIC system allows a reliable inspection of semiconductor materials and devices, based on A3B5 compounds and on silicon. A typical application of the above technique is the investigation of the impurity distribution around extended crystal defects, like dislocations and precipitates, to study their gettering activity.
Enhanced oxygen diffusion and precipitation in silicon
Authors: Bergholz W. , Hutchison J. L. , Pirouz P.
Published: August 2011

Prolonged annealing of Cz‐silicon at 758 K results in the formation of ribbon‐like oxygen precipitates on {311} planes interpreted as the coesite phase. From this an enhancement of the oxygen diffusion coefficient by more than three orders of magnitude is inferred. Excess selfinterstitials are accommodated in extrinsic dislocation loops on {111} planes (‘loopites’) and probably also in ‘blob’‐like defects visible in high resolution micrographs with little detectable lattice strain. Evidence is presented that coesite ribbons can nucleate on {311} steps on loopites and vice versa.
Friable rock embedding in methyl methacrylate for thin‐sectioning in geological preparations
Authors: Palle Sabine , Chappard Daniel , Alexandre Christian , Orsini Jean‐Baptiste
Published: August 2011

In most cases, technical problems prevent the production of good geological thin‐slabs from friable rocks such as muds, sands or decomposed granites. Usual impregnation methods using Araldite or epoxies lead to incompletely impregnated blocks. Furthermore the excessive heat occurring during polymerization alters the micromorphology of heat sensitive minerals. Methyl methacrylate (MMA) is a widely used plastic for bone embedding. We have developed a new procedure using vacuum infiltration with MMA in a cold environment. Wafers from friable rocks are easily obtained and can be processed in the same way as those of firm and coherent rocks, without grain loss nor mineral heat alterations.
Metastable water at subzero temperatures
Authors: Franks Felix
Published: August 2011

The origins of physical and chemical metastability in aqueous systems are discussed with particular reference to supersaturated solutions, i.e. those where eutectic phase separation does not normally occur. Such solutions are characterized by high viscosities, and under conditions of rapid cooling to the glass temperature, freezing can be partly or completely inhibited. In such systems water acts as a plasticizer to the solute matrix but is not bound, in the correct sense of the word. At a glass temperature of 228 K, diffusion and evaporation rates of water from the amorphous solid are of the order of 1 nm/day, compared to 104 nm/s for the sublimation of ice at same temperature, despite the fact that ice has a substantially lower vapour pressure.
Low temperature light microscopy and its application to study freezing in aqueous solutions and biological cell suspensions
Authors: Körber Ch. , Englich S. , Schwindke P. , Scheiwe M. W. , Rau G. , Hubel A. , Cravalho E. G.
Published: August 2011

The freezing of biological cell suspensions can be understood in terms of ice formation in the external suspension medium and the cellular reactions to the changing environment. Cryomicroscopy allows a quantitative analysis of both categories of phenomena. Besides freezing stages of appropriate thermal design, the components used for that purpose include a microcomputer (PSI 80) based control system, an image analysis system (Intellect 100) and a spectrophotometer (MPV compact). The investigation of extracellular ice formation is focused on the following effects: The redistribution of solutes in the residual liquid and the resulting concentration profiles are determined photometrically or densitometrically. The transitions between various morphologies of the ice–liquid phase boundary (planar–cellular–dendritic) can be related to interface instability theories. With respect to solute segregation, the studies also involve the formation of bubbles from supersaturated gaseous solutes and freezing potentials resulting from the differential incorporation of cations and anions into the solid phase. The interaction between particles or cells and the advancing ice front is determined from critical interface velocities marking the transition between repulsion and entrapment. The effects of freezing on biological cells are studied mainly with blood cells, especially lymphocytes. The water efflux due to osmotical gradients across the membrane yields volume shrinkage curves which are recorded and analysed from video images for various cooling rates. Beyond a certain threshold cooling rate, intracellular ice starts to form, and different crystallization morphologies can be detected. The intracellular crystallization temperatures depend on cooling and warming rates as well as on the presence of penetrating cryoadditives. A fluorescence viability is used to determine the percentage of damaged cells immediately after thawing.
Splitting wood specimens for observation in the scanning electron microscope
Authors: Kučera Ladislav J.
Published: August 2011

This report deals with the principles of splitting wood specimens for observation in the scanning electron microscope and gives some examples in which the splitting is a crucial step in specimen preparation.
High resolution imaging of the CdTe/(100)InSb interface: A lattice‐matched hetero‐epitaxial structure
Authors: Hutchison J. L. , Waddington W. G. , Cullis A. G. , Chew N. G.
Published: August 2011

This paper reviews techniques available for obtaining images which can distinguish the two components in lattice‐matched, hetero‐epitaxial structures. From series of computer simulations it is shown that in the case of CdTe/(100)InSb the usual criteria for ‘good’ high resolution imaging (i.e. thin crystal, Scherzer focus) do not give images which differentiate the two materials. Conditions which do show different contrast features for both CdTe and InSb are found; under these conditions images are obtained which reveal the interfacial structure clearly, and show it to be essentially flat, and free of defects within the regions examined. Prospects for obtaining true ‘atomic’ images using a new 400 kV HREM are outlined.
Observation of lattice fringes of the Si(111)–7×7 structure by reflection electron microscopy*
Authors: Tanishiro Y. , Takayanagi K. , Yagi K.
Published: August 2011

Lattice fringes of Si(111)–7×7 reconstructed surface structure in reflection electron microscopy (REM) are observed for the first time, and their characteristic features are presented. Due to a glancing reflection condition in REM, the fringes with a spacing of 2.3 nm of the 7×7 surface structure lattice are seen in a region of a certain defocus range (about 6–8 μm) in a foreshortened image. The glancing reflection geometry also results in a complicated dependence of fringe directions on imaging conditions (beam alignment, crystal orientation). A shift of the fringes across the surface atomic steps and out of phase boundaries is observed.
High‐resolution electron microscopy of BaxK2–xFexTi6–xO13 crystals in polar‐glass state
Authors: Shiojiri M. , Maeda T. , Hirota Y. , Isshiki T. , Sekimoto S. , Iwauchi K. , Keda Y. I.
Published: August 2011

As a trial to detect the local atomic configuration in a cellular random system, BaxK2‐xFexTi6‐xO13 crystals, in which Ba2+ and K+ or Fe3+ and Ti4+ ions are randomly distributed on different (4i) sites of the C2/m unit cell, have been observed by high‐resolution transmission electron microscopy. Contrast differences appear among the image spots reflecting the atomic columns in the Ba2Fe2Ti4O13 crystal. With the aid of image simulation, it is revealed that the differences may be ascribed to the fluctuation in the distribution of TiO6‐octahedra and FeO6‐octahedra. A series of TiO6‐octahedra cut by FeO6‐octahedra can be regarded as a region of coherent polarization or a cluster of polarized lattice ions. Similar contrast differences have been observed in the images of crystals for x=0.4 and 1.6, and the relation between the composition and the intensity of the spots is discussed.
Electron microscopic study of 5SrRNA crystals from Thermus thermophilus HB8
Authors: Morikawa K. , Fujiyoshi Y. , Ishizuka K. , Sugiyama J. , Kawakami M. , Takemura S.
Published: August 2011

Several types of crystals were grown from 5SrRNA, which was purified from the highly thermophilic bacterium, Thermus thermophilus HB8. Crystal lattice parameters were determined by X‐ray and electron diffraction. One type of crystal was suitable for electron microscopy after staining with uranyl acetate. Two projections derived from micrographs taken at different tilt angles were processed for image analysis. This result enabled us to deduce the arrangement of molecules within the crystal lattice.
Stereology of arbitrary particles*
Authors: Gundersen H. J. G.
Published: August 2011

This paper deals with isolated, countable items, often termed particles, in three‐dimensional space. Its substance is the unbiased stereological estimation of the number, height, surface and volume of such particles without any assumptions about their shape. The full range of estimators is described, some of them for the first time, some in an improved form, several in more than one version, and all of them under the single, absolute requirement that one can in fact identify what one is quantifying on sections. In terms of the minimal number of sections for the analysis, the estimators may be classified as follows:

On a single section it is possible to estimate v̄V, the mean volume of particles in the volume‐weighted or ‘sieving’‐distribution.

On two parallel sections, separated by a known distance, estimators exist of particle number and of all mean sizes (height, surface and volume) in the ordinary number distribution, as well as of SDN(v), the standard deviation in the number distribution of particle volumes. If the containing space is relatively transparent the sections may be two optical sections within one thick physical section.

On a stack of parallel sections, at least as high as the largest particle, and separated by known distances, one can get twelve mean sizes and twelve distributions of individual sizes: all combinations of three sizes: height, surface and volume in four different types of distributions: number, height, surface and volume. Fulfilling the sampling requirements of the above two estimation principles it has been shown very recently that by combining them one may even estimate mean sizes and number of arbitrary particles in a stack of sections with constant but unknown separation.

Finally, a unique, unbiased estimator of the total number of items in a specimen is described for the use of which one need not measure the distance between sections, nor their thickness, nor the volume of the specimen, nor assume anything about shrinkage/swelling, sectioning compression or lost caps. It is the fractionator.
Stereology for anisotropic cells: Application to growth cartilage*
Authors: Cruz‐Orive Luis‐M. , Hunziker Ernst B.
Published: August 2011

A number of either new or recently available stereological methods are described for estimating volume, surface area and number of anisotropic cells. The methods are illustrated with direct reference to the epiphyseal growth plate. Different estimates of a given quantity are obtained by applying alternative methods to the same set of sections, in order to compare the relative merits of the methods. For instance, the surface area of the cells is estimated via the Dimroth–Watson model (which gives a measure of the degree of anisotropy in addition to the surface area estimate) and from vertical sections using cycloid test systems. Cell number is estimated by traditional unfolding methods and by the new disector method. Also, volume‐weighted mean cell volume is estimated from vertical sections via point‐sampled intercepts using two different kinds of rulers to classify intercept lengths. Finally, nested design statistics is applied to a set of data from twelve animals in order to compare the relative impacts of biological and stereological (sampling) variations on the observed coefficient of error of a group mean estimate. The preferred methods are listed in the final section.
Developments of new Lowicryl® resins for embedding biological specimens at even lower temperatures
Authors: Acetarin Jean‐Dominique , Carlemalm Eric , Villiger Werner
Published: August 2011

Two new Lowicryl resins have been developed for embedding biological materials at temperatures down to 210K (hydrophilic K11M) and to 190K (hydrophobic HM23). They have similar properties to Lowicryl K4M and HM20. The new resins were first tested for low temperature applications by the ‘progressive lowering of temperature’ procedure and this shows that the low viscosity of K11M and HM23 is favourable for the infiltration of biological specimens. Hardening is achieved through photo‐polymerization at these lower temperatures. These properties make K11M and HM23 suitable for cryosubstitution of rapidly frozen material and it is speculated that the preservation of antigenicity may be further improved.
The comparison of paired compartments by autoradiography: a methodological note
Authors: Reddingius J. , Vries O. M. H. de
Published: August 2011

summary

Biochemical activities in adjacent cells, or nuclei, or other ‘compartments’, are sometimes compared on the basis of the number of silver grains visible on autoradiographs. In this report, the problem of how to test statistically the null hypothesis that the differences between the numbers of grains in two adjacent compartments are due to chance only, if one has available a sample of absolute values of such differences is discussed. The Kolmogorov test for discrete distributions is difficult to apply. More simple tests using approximations by means of the normal and chi square distributions are described. These tests are valid if, and only if, it may be assumed that the numbers of grains in the compartments are independent random variables with Poisson distributions. In autoradiographic data on RNA synthesis rates of the partner nuclei in dikaryotic cells of Schizophyllum commune, the differences appeared in general to be much smaller than expected on the basis of this assumption and the null hypothesis. This suggests that the numbers of grains in these autoradiographs follow a regular distribution rather than a Poisson one; however, it is shown that this is not necessarily true. Theoretically, the autoradiographic process might lead to regular as well as to clustered distributions. It is concluded that assuming Poisson distributions, although reasonable at first sight, may be wrong. The theoretical basis for a statistical analysis of grain counts, therefore, is insufficient.
The use of lead methacrylate 2‐ethylhexanoate to create a radio‐opaque resin suitable for microvascular corrosion casting
Authors: Burton G. J. , Ingram S. C. , Palmer M. E.
Published: August 2011

summary

A radio‐opaque methacrylate‐based resin suitable for microvascular corrosion casting can be prepared by substituting lead methacrylate 2‐ethylhexanoate for a proportion of the methyl methacrylate monomer normally used. Best results are achieved when conventional methacrylate resin is injected first to fill the vascular tree, and this is chased with the radio‐opaque resin. The pattern of arterial and arteriolar distribution can then subsequently be followed by means of a radiographic survey of the cast, and this is of value prior to sampling from vascular systems which show functional subdivisions that cannot be correlated with any surface features. The radio‐opacity of the resin and the quantity injected can be varied easily to suit individual requirements.
Automatic description of microstructures
Authors: Powley G. , Kulick J. , Gallerneault C. E.
Published: August 2011

Present automatic image analysis techniques quantify a materials morphology in terms of the standard stereological parameters such as constituent size and distribution. While useful this information lacks the ‘depth’ available from qualitative examination. In this first of three papers it is suggested that automatic methods can be developed to capture and describe image structure in the descriptive terminology of a metallographer. The goal of such work is to permit quantification of formerly descriptive parameters. A preliminary stage in image analysis is the segmentation of an image into homogeneous regions in preparation for further processing. It is demonstrated that metallographic images (in this case binary eutectic alloys of aluminium) can be segmented using measures of the spatial organization, or texture, present in an image. A weighted average of the Hadamard transform of an image is shown to give a local measure of the texture suitable for segmentation of the images into homogeneous regions of microstructure.
Automatic description of microstructures
Authors: Gallerneault C. E. , Kulick J. , Powley G.
Published: August 2011

This is the second in a series of three papers concerned with developing automatic analysis and classification techniques for metallographic images (specifically binary eutectic alloys of aluminium). The goal of the developmental work was to capture and describe image structure, in the descriptive terminology of a metallographer, and to permit quantification of formerly descriptive parameters. The first paper dealt with the preprocessing stage of image analysis and entailed a study of two segmentation techniques. This second paper reports on work demonstrating the use of texture analysis to characterize structural aspects of metallic microstructures. It is shown that the morphological classes can be related to textural features present in digitized images.
Cryo‐transfer revised
Authors: Frederik P. M. , Busing W. M.
Published: August 2011

Vapour deposition of water is a major problem in cryo‐tranfer and cryo‐observation of frozen specimens. The use of (sliding) shields to protect the specimen during transfer has been evaluated and was found to be a fail‐safe procedure. Shielding the specimen with solidified coolant is proposed as an alternative or additional protection. The protective layer (ethane, Freon 22) can be evaporated in the high vacuum of the microscope. The evaporating coolant will carry away water films deposited on top. The temperature of coolant evaporation is low enough to prevent possible devitrification of water in the specimen itself.
Elemental mapping with elastically scattered electrons
Authors: Pennycook S. J. , Berger S. D. , Culbertson R. J.
Published: August 2011

We describe a technique for efficient, quantitative, standardless elemental mapping using a high‐angle annular detector in a scanning transmission electron microscope (STEM) to collect elastically scattered electrons. With a single crystal specimen, contrast due to thickness variations, diffraction, and channelling effects can be avoided, so that the resulting image contrast quantitatively reflects variations in impurity concentration. We compare a number of simple analytical approximations to the elastic scattering cross sections and show that a standardless analysis is possible over a wide range of atomic number and inner detector angle to an absolute accuracy of better than 20%.
A combined electron microscope and optical metallographic study of microstructures in high‐strength, low‐alloy (HSLA) steels after welding
Authors: Handeland T. , Erkkilä E. , Gjønnes J.
Published: August 2011

A combined TEM and optical metallographic technique has been applied to microstructures in the heat affected zone in two high‐strength, low‐alloy steels with carbon equivalent 0·37 and microalloyed with Nb (0·024%), Ti (0·015%) and Nb (0·025%), V (0·05%) respectively. Volume fraction, size distribution and composition of the carbonitrides of microalloying elements were determined as function of distance from the fusion line and compared with other microstructural features, such as primary austenite grain size. Differences in microstructure are attributed to the higher stability of the Ti, Nb particles during the weld thermal cycle.
Quantitative image analysis of finite porous media
Authors: Macdonald I. F. , Kaufmann P. , Dullien F. A. L.
Published: August 2011

Software for the reconstruction of branch‐node charts from serial sections is tested with a simple cubic lattice, and is applied to determine the genus per unit volume (specific genus) of a Berea sandstone sample from seventy‐eight serial sections.

The genus is found analytically for the cubic lattice as a function of depth, cross‐section and volume. These results enable us to draw conclusions about the relative importance of depth and cross‐section and provide useful information on how to treat finite sample data to obtain the specific genus of an infinite homogeneous porous medium. It is shown that it is more important to have a deep sample (many serial sections) than a sample of large cross‐section.

For samples which are too small in the direction of sectioning, the genus and the rate of change of genus with volume are too low because some of the large loops are not completed within the sample boundaries. For the Berea sample, this ‘shallow‐sample depth’ is about 2·2 grain diameters (forty‐five serial sections). Only data points at depths in excess of this value are used in determining the genus per unit volume. The slope of the genus versus volume curve is the better predictor of the genus per unit volume of the unbounded medium, of which the sample is a small part, than the value of genus/volume of the sample.

For samples of finite cross‐section, the Gmin and Gmax versus volume curves are divergent and they only become essentially parallel when the dimensions in the plane of sectioning are large multiples of the grain size. It is shown that it is reasonable to assume that the arithmetic mean slope found for a small cross‐sectional area is a good estimate of the common slope that would be attained at high cross‐sectional areas. Therefore, samples of small cross‐section can be used, resulting in a reduction in the amount of data to be processed and an enhancement of the resolution of small features.

The Berea sample data, beyond the ‘shallow‐sample’ region, show the same qualitative features as predicted analytically for the cubic lattice. The ratio of the slopes of the Gmax and Gmin curves is 1·22 and the genus per unit volume from the mean slope is 52·6 × 10−8 μm−3.
Estimation of surface area from vertical sections
Authors: Baddeley A. J. , Gundersen H. J. G. , Cruz‐Orive L. M.
Published: August 2011

‘Vertical’ sections are plane sections longitudinal to a fixed (but arbitrary) axial direction. Examples are sections of a cylinder parallel to the central axis; and sections of a flat slab normal to the plane of the slab. Vertical sections of any object can be generated by placing the object on a table and taking sections perpendicular to the plane of the table.

The standard methods of stereology assume isotropic random sections, and are not applicable to this kind of biased sampling. However, by using specially designed test systems, one can obtain an unbiased estimate of surface area.

General principles of stereology for vertical sections are outlined. No assumptions are necessary about the shape or orientation distribution of the structure. Vertical section stereology is valid on the same terms as standard stereological methods for isotropic random sections. The range of structural quantities that can be estimated from vertical sections includes Vv, Nv, Sv and the volume‐weighted mean particle volume v̄v, but not Lv.

There is complete freedom to choose the vertical axis direction, which makes the sampling procedure simple and ‘natural’. Practical sampling procedures for implementation of the ideas are described, and illustrated by examples.
Averaging of periodic images using a microcomputer
Authors: Squire John M. , Luther Pradeep K. , Agnew Gerard D.
Published: August 2011

A system based on a commercial microcomputer and a commercial image‐capturing TV interface is described which carries out both real space and conventional Fourier transform‐based image averaging procedures. Various real space manipulations are described for handling images with either one‐dimensional or two‐dimensional periodic features. In certain cases, real space averaging procedures have many advantages and are recommended to those not familiar with the mathematics of methods based on Fourier transformation. In other cases Fourier transformation processes are necessary and these also have been implemented on the microcomputer. They can be used to determine periodicities, to produce two‐dimensional average images or as a basis for three‐dimensional reconstruction. Examples showing the principles of application of these methods use appropriate images from electron micrographs of muscle and collagen.
A new method for the two‐dimensional analysis of bone structure in human iliac crest biopsies
Authors: Garrahan N. J. , Mellish R. W. E. , Compston J. E.
Published: August 2011

A new method is described for the analysis of the two‐dimensional structural pattern of trabecular bone in human iliac crest biopsies. 8 μm thick undecalcified sections stained with the von Kossa technique were examined at a magnification of ×9. Using an Ibas II image analyser, the ratio of nodes to free ends and the length of different strut types (cortex to free end, node or cortex, free end to free end and node to node, loop or free end) expressed as a percentage of total strut length were assessed.

The reproducibility of the method was good for most of the measured indices but inter‐observer and inter‐section variation were greater. Comparison of biopsy sections obtained from eleven young healthy control subjects and eleven patients with hepatic osteoporosis revealed a significantly higher node to free end ratio, node to loop and node to node strut length and significantly lower cortex to free end and free end to free end strut length in the controls. No significant differences were seen in node to free end, cortex to cortex or cortex to node strut length. This approach to trabecular bone structure analysis should prove useful in determining patterns of bone loss in health and disease and in examining the effects of treatment on bone structure in osteoporosis.
A versatile light microscope heating stage for biological temperatures
Authors: Lowndes R. H. , Hallett M. B.
Published: August 2011

A light microscope heating stage suitable for biological temperatures is described. A novel feature of the design is the use of cupro‐nickel foil heaters (0.1 mm thick). With a temperature sensor mounted on the slide clip and a temperature controller unit, this apparatus maintains a temperature of 310 K with a precision of 0.1 K. The apparatus is inexpensive, reliable, not confined to use with one microscope and does not interfere significantly with the stage movement or optical alignment.
Generalized associated point methods for sampling planar objects
Authors: Jensen E. B. , Sundberg R.
Published: August 2011

For estimation of the mean number of objects per unit area from observations in a bounded region (sampling window) T in the plane, a generalized associated point method is discussed. By this method, m points are associated with each object, and an object with k associated points inside T is counted with weight k/m. A general formula for the variance of the estimator is derived and explicit expressions are given for Poisson and hard‐core models for the spatial distribution of objects in the plane.
Correction of window size distortion of crack distributions on plane sections
Authors: Attiogbe Emmanuel K. , Darwin David
Published: August 2011

The scanning electron microscope (SEM) has been used to measure crack distributions on the surfaces of cement paste and mortar. However, true distributions of cracks (or straws, needles, etc.) on a plane section may not be obtained directly if only a portion of the plane section is visible within the field of view (window), as is the case with an SEM. The procedures used to estimate the true distributions are presented. These procedures are based on the concepts of geometrical probability and statistics. Relationships are established between the observed and the true distributions, and iterative techniques are employed to estimate the true distributions. For a given window height, a guideline is provided to determine if the window size distortion needs to be corrected. It is shown that the total length of cracks per unit area on a plane section is not affected by the window size.
Information recovery in missing angular data cases: an approach by the convex projections method in three dimensions
Authors: Carazo José María , Carrascosa José L.
Published: August 2011

We have studied the method of projections on to convex sets (POCS) for the restoration of three‐dimensional signals, especially applied for the degraded information obtained in three‐dimensional (3‐D) reconstructions by electron microscopy due to the limited tilting angular range of the sample that is obtainable in practice. A computer generated specimen has been used as a test object. Several noise levels were added to the specimen, obtaining signal‐to‐noise ratios (signal power/noise power) of between 12 and 0·5 dB. Different missing data angular regions of 30° and 10° zenithal angle have been considered. Our results illustrate the possibilities of POCS to restore incomplete 3‐D data in the absence and the presence of noise, and offer ways for further applications to improve the quality of 3‐D reconstructions based on real data.
Determining nucleolar multiplicity and cell number from sectional data
Authors: Gramsbergen A. , Kok L. P. , Poortema K. , Schaafsma W.
Published: August 2011

The occurrence of more than one nucleolus within the cellular nucleus (polynucleolarity) is a well‐known phenomenon during the proliferative cell cycle, both under normal and pathological conditions (e.g. neoplasia). It can also be observed in neuronal nuclei at early stages of their maturation. Polynucleolarity merits investigation for cytological reasons.

In an histological section, the observed number of nucleoli in a nucleus may be smaller than the actual number. In order to estimate the true distribution of the number of nucleoli per nucleus from the observed distribution, the mathematical relation between these distributions is derived (Section 4) on the basis of rather restrictive (Sections 3 and 11) stereological assumptions (Section 2). It is indicated how these distributions can be estimated from the data available and how the statistical uncertainties involved can be expressed (Sections 5, 6 and 7).

This paper arose from making cell counts (Section 1). Two methods may be applied: (1) all visible nuclear profiles are counted, (2) nuclear profiles are only included if at least one nucleolus is visible in the section. We recommend a combination of these two methods (Section 8). An advantage of our theory for determining cell number (Section 9) is that one can often manage without the rather restrictive stereological assumptions needed hitherto.

The advantage of expressing statistical uncertainties in estimated nucleolar multiplicity probabilities and cell numbers is indicated (Section 10).