bluuebunny/crossref_metadata_2025 huggingface dataset:
Application of freeze‐etch replication for the observation of the cell‐extracellular matrix interface of cultured cells
Authors: Sawada Hajime
Published: August 2011

In order to investigate the ultrastructural three‐dimensional relationship between extracellular matrices (ECM) and the plasma membranes of cultured cells, a freeze‐etch replica method was devised.

Bovine corneal endothelial cells were cultured on a Collodion film which covered a hole punched in a plastic coverslip, and were quickly frozen with a slammer with their basal surface facing a liquid nitrogen‐cooled copper block. The cells were placed upside‐down in a Balzers freeze‐fracture machine and freeze‐etched, and then platinum‐carbon replicas were obtained.

The structure of the ECM‐plasma membrane interface was observed successfully and so this technique provides a new approach for investigating the ECM‐plasma membrane (matrix‐receptor) relationship.
Matschinski's identity and dual random tessellations
Authors: Miles R. E.
Published: August 2011

Various aspects of Matschinski's classical identity relating, in a homogeneous ergodic polygonal tessellation, the mean number of vertices, or sides, with the mean number of sides meeting at a random vertex, are discussed. It is shown how any such tessellation has a topological family of dual tessellations. An example given is that of an isotropic random tessellation, all of whose cells are quadrangles; another example has identical distributions of number of vertices and number of sides meeting at a vertex.
Affine integral geometry and convex bodies
Authors: Santaló L. A.
Published: August 2011

Given a convex body Q in Euclidean n‐dimensional space, the affine invariant measure of the set of pairs of parallel hyperplanes containing Q is an affine invariant J(Q) of Q, which, for ellipsoids, parallelepipeds and possibly for simplices of any dimensions, is proportional to V−1, where V represents the volume of Q. Consequently, for the kind of bodies mentioned, it is possible to estimate V−1 from its breadth measured in uniform random directions. If the boundary of Q is of class C2, we obtain a set of affine invariants Jh(Q) (h = any integer) which is easily calculable for ellipsoids. In particular, J‐n(Q) coincides with J(Q) and J‐(n+1)(Q) is the affine invariant measure of all pairs of parallel hyperplanes that ‘support’ Q as described by Firey(1972, 1985), Schneider (1978, 1979) and Weil (1979, 1981). For a general convex body Q the values of Jh(Q) cannot be expressed in terms of simple metric invariants (such as volume, surface area, breadth, width) and this justifies the study in the last section of certain inequalities between them.
Expectation formulas and isoperimetric properties for non‐isotropic Boolean models
Authors: Weil Wolfgang
Published: August 2011

Boolean models Y in R2 and R3 are considered and formulas connecting the quermass densities of Y with the mean values of certain functional of the primary grain are given in the non‐isotropic case. Under symmetry conditions, the mean functionals are interpreted as mixed volumes of convex bodies which are associated with Y. Extremal properties of isotropic Boolean models are deduced from the isoperimetric inequality.
On the coverage problem in higher dimensions with nonuniform density
Authors: Hüsler J. , Glauser K.
Published: August 2011

Consider the coverage of a k‐dimensional unit cube by n spheres randomly placed according to a density f. Assume that f is non‐uniform, having a unique interior minimum point m in the unit cube with f(m) = 0. We present necessary and sufficient conditions such that the probability that the unit cube is covered at least l times converges to 1 as n → ∞. These conditions depend on the local behaviour of f in the neighbourhood of m and the radii δ of the random spheres.
Morphological and elemental integrity of freeze‐fractured, freeze‐dried cultured cells during ion microscopic analysis
Authors: Ausserer Walter A. , Chandra Subhash , Morrison George H.
Published: August 2011

The effects of progressive ion beam bombardment on freeze‐fractured, freeze‐dried cultured cells during ion microscopic (SIMS) analysis were studied with scanning electron microscopy (SEM) and ion microscopy. The freeze‐fracture, freeze‐dry sample preparation method was generally found to preserve cell morphology to a level far exceeding the spatial resolution of the ion microscope, with splitting at the nuclear envelope being the most commonly observed artefact. SEM monitoring of surface topography of an NRK‐49F fibroblast after various ion bombardment doses showed relatively uniform erosion of cellular material, with some apparent selective retention of small cytoplasmic granules. Prolonged bombardment produced no detectable lateral elemental translocation. 41K+/24Mg+ signal ratios from Swiss 3T3 fibroblasts and RBL rat basophilic leukaemia cells were shown to vary generally by less than 10% during the course of extended ion bombardment. GM0415 human skin fibroblasts containing engorged lysosomes characteristic of Hurler's Syndrome were used to evaluate the effects of ion bombardment during a typical analysis session, where ion images of 39K+, 23Na+, 40Ca+ and 24Mg+ are sequentially recorded. This cell line was chosen as a worst‐case system, because these cells are often thinly spread and possess extreme surface topography. Thin cell edges were shown sometimes to sputter away during analysis, giving misleadingly low ion signals from these regions in some 24Mg+ micrographs. Various nonuniform sputtering phenomena occurring in the submicrometre spatial domain had little or no measurable impact on local intensities in ion micrographs, indicating that freeze‐dried, freeze‐fractured cells are sampled in a sufficiently uniform fashion that quantitative ion microscopic evaluations of intracellular elemental levels in the general cytoplasmic or nuclear regions are feasible.
Photometric measurement of small optical path differences
Authors: Roche E. J. , Van Kavelaar R. F.
Published: August 2011

A photometric method for the measurement of small optical path difference (OPD) is described. The method is based on a computer assisted calibration of the transmitted intensity associated with specific rotations of the calibration device, a Λ/10 compensator. The method has been tested on cleavages of mica and shown to be in excellent agreement with photometer assisted measurements of compensation. Application to the measurement of lateral orientation in Kevlar® 49 aramid fibres is presented.
Lattice fringe studies of (100) defects in enamel crystallites
Authors: Lee DoSuk Duke
Published: August 2011

Lattice imperfections in the structure of shark (Nataprion brevirostrais) enamel crystallites have been studied by means of lattice‐imaging in a high‐resolution transmission electron microscope. Line defects exhibiting various lengths (100–2500 Å) and widths (10–30 Å) and demonstrating phase behaviour different from that of the host crystal were observed along the fringes representing (100) lattice planes.

When defect sites were subjected to an electron beam, an annealing process was observed in which the difference in phase contrast eventually disappeared and the defect itself became an intricate part of the (100)‐type lattice planes.

A through‐focal series of the defects show a reversal in phase contrast. By examining the contrast behaviour under certain focal conditions and by adopting the Fresnel diffraction mode, the refractive index of the defect regions was determined to be lower than that of the apatite crystal. Present findings suggest that the interlayering structure along the (100) planes is responsible for the line defects observed in enamel apatites.
Elemental concentrations in air‐exposed and vacuum‐stored cryosections of rat liver cells
Authors: von Zglinicki T. , Zierold K.
Published: August 2011

Elemental concentrations in different compartments of cryosections of isolated rat liver cells cryotransferred and freeze‐dried were compared with those obtained after storage under vacuum for 12 or 60 h and after exposure to room air for 2 min. Poorer image contrast and segregation artefacts are frequently found in air‐exposed sections, together with a slight but significant decrease of the K concentration in the cytoplasm and an increase of the S concentration in the liver cell nuclei and the extracellular medium.

Extreme distortions of both ultrastructure and elemental distributions are observed if the sections are even slightly colder than the surrounding atmosphere.

While storage of frozen‐dried cryosections under vacuum for less than 12 h does not lead to alterations in the sections, gross changes are found both in morphology and elemental distribution in sections stored under vacuum for about 60 h. Long‐time vacuum storage of frozen‐dried cryosections is, therefore, not recommended.
Cryofixation of plant tissues without pretreatment
Authors: Kaeser Wulf , Koyro Hans‐Werner , Moor Hans
Published: August 2011

Root tips from Sorghum and Dahlia were frozen without cryoprotection by dipping into nitrogen slush, rapid immersion in liquid propane and by the high‐pressure method. Structural preservation of the samples was studied using freeze‐fracture (FF) and freeze‐substitution (FS) techniques for electron microscopy. It was found that most of the organelles were disrupted by freezing in nitrogen slush and that only the boundary beween the cytoplasm and the vacuole remained visible. If the samples were frozen by rapid immersion in liquid propane, small membraneous organelles, such as dictyosomes, were preserved in peripheral regions of the rhizodermal cells up to 10 μm below the surface of the tissue. Specimens frozen by the high‐pressure freezing technique showed good ultrastructural preservation throughout the tissues up to a depth of more than 100 μm.
Exact formulae for the precision of systematic sampling
Authors: Kellerer Albrecht M.
Published: August 2011

A formula is given for the variance of the intersection of two geometric objects, S and T, under uniform, i.e. translation invariant, randomness. It involves an integral of the product of the point‐pair distance distributions of S and T. In systematic sampling S is the specimen and T is the test system, for example a system of planes, lines, or dots in ℝ3 or ℝ2. The general n‐dimensional integral (or sum) is difficult to use, but for systematic sectioning, i.e. for a test system of parallel hyperplanes (planes in ℝ3 or lines in ℝ2) it can be reduced to a one‐dimensional expression: this leads to Matheron's treatment in terms of ‘covariograms’ of the specimens. Under the condition of isotropy analogous simplifications lead to equations involving the distributions of scalar point‐pair distances and to the approach developed by Matérn for sampling with point grids. The equations apply to arbitrary test systems, but they include fluctuating functions that require high precision in the numerical evaluation and make it difficult to pradict undamped variance oscillations of the volume estimator which occur for some specimen shapes but not for others. A generalized Euler method of successive partial integrations removes this difficulty and shows that, for a convex specimen, the undamped oscillations result from discontinuities in its chord‐length density. The periodicities equal the ratios of the critical chord‐lengths to the periodicities of the test system. Analogous relations apply to the covariogram. The formulae for the variance are extended also to the covariance of the volume estimators of paired specimens.
The origins of the Royal Microscopical Society
Authors: Turner G. L'E.
Published: August 2011

The origin of the Microscopical Society of London on 3 September 1839 is explained in the context of the times. The new Society immediately had an effect on the standards of scientific microscopy by acquiring a library, a set of the finest microscopes, and by publishing papers. In 1866 a Royal Charter was granted under the Presidency of James Glaisher, FRS.
Applications of electron microscopy in materials science and technology (microscopy in the modern world)*
Authors: Ralph Brian
Published: August 2011

A personal overview is presented attempting to show something of the broad range of applications of techniques associated with electron microscopy in the field of materials science and technology. The importance of electron microscopy in understanding microstructure/property relationships and the evolution of microstructure is stressed. In addition, some of the important uses of electron optical techniques in the more applied areas of materials technology are surveyed. The format adopted in this presentation is to illustrate the applications of electron microscopy in materials science and technology by citing recent studies. These include investigations of deformation, of grain growth, of precipitation and phase transformations and of the determination of the structure of interfaces.
Future trends in microscopy
Authors: Howie A.
Published: August 2011

Recent progress and current trends in microscopy are examined with particular reference to possible future developments. The scope for systematic improvement of the traditional optical or electron designs and procedures seems far from exhausted. However the introduction of novel, lens‐free microscopes which, like the scanning tunnelling microscope, produce magnification using an electronic lever arm, could expand the boundaries of the subject most dramatically. The future microscopist may find himself displaced from the more routine tasks of focusing, alignment, image recording and even simple interpretation by the electronic robot. On the other hand, he will gain access to a wider and more sophisticated range of physical and chemical phenomena governing the interaction between the specimen and the radiations he uses to probe its microscopic structure and properties.
Determination of the coordination number in disordered packings of equal spheres
Authors: Oger L. , Lichtenberg M. , Gervois A. , Guyon E.
Published: August 2011

A computation of the coordination number in a packing of equal spheres can be performed from the information obtained along a random line through the packing. It involves the distribution of the number per unit length of separators between intercepts of spheres with the line. The number of contacts per unit volume is derived from the slope of the distribution at the origin. The method is validated by computer experiments on four kinds of simulated ordered packings which are intercepted by a set of random lines (instead of a single one) in order to compensate biased results due to the small size of these packings: the comparison is then extended to a series of disordered dense packings of equal spheres. We present some possible applications of this study to stereological and physical problems.
An integrated PC‐based image analysis system for microtomography and quantitative analysis of inner micro‐object structure
Authors: Sasov A. Yu.
Published: August 2011

A new type of image processing system, including microtomographic analysis, is presented. The great majority of existing approaches to image processing and analysis can be achieved by means of software, with a minimal amount of hardware. The addition of microtomographic methods to an image processing system allows quantitative parameters of inner object microstructure to be obtained non‐destructively.
Determination of gold‐labelled surface receptors on single cells by X‐ray microanalysis
Authors: Peschke Th. , Augsten K. , Sasama F. , Gabert A.
Published: August 2011

A standardless X‐ray microanalytical procedure has been developed to determine the number of gold‐labelled surface receptors on whole single cells. The effect of the injection of K2PtCl4 into mice on gold‐labelled concanavalin A (Con A) receptors on peritoneal macrophages was examined with an energy dispersive X‐ray detector in an SEM. The numbers of gold particles seen in electron micrographs and estimated by fluorescence photometric measurements of fluorescein isothiocyanate‐labelled Con A receptors were correlated with the X‐ray microanalytical results.
In situ elemental analysis and visualization in cryofixed nervous tissues: X‐ray microanalytical investigations of embryological and mature brain, inner ear, photoreceptors, muscle and muscle spindles. Comparison of preparation methods for analysis and visualization at cellular and subcellular levels
Authors: Wróblewski Romuald
Published: August 2011

For meaningful X‐ray microanalysis (XRMA) in biology and medicine, the development of preparative and quantitative methods has been necessary. The methods need to preserve close to in vivo distribution of diffusible ions with at the same time reasonable morphological preservation of the tissue.

Analyses at low and middle microanalytical resolution are useful at the initial stages of an investigation or when data from large populations of samples have to be acquired. Cryomicrotomy, which makes it possible for the single cells within semi‐thin and thick cryosections examined by X‐ray microanalysis to be further characterized histochemi‐cally (enzyme and substrate content), has been adopted for several pathophysiological studies. The method is particularly suitable for the analysis of complex morphological tissues with many cell types as in the brain or sensory organs of the internal ear.

For microanalysis at the subcellular level, we developed a preparative procedure based on the frozen fixed preparation which is freeze‐dried in vacuo at −80°C and then at the same temperature, without breaking the vacuum, impregnated with a low‐temperature Lowicryl‐type resin. The resin is polymerized by u.v. light. This method prevents redistribution of the ions in the tissue and retains the antigenicity of the tissue. A considerable number of cells can be analysed simultaneously and the elemental composition in different cell compartments can be compared due to the similar analytical conditions within the section.

An alternative to thin plastic sections of freeze‐dried material is thin cryosections cut at −150°C and analysed at low temperatures. Although some methodological problems still exist in preparation of cryosections, this type of section is potentially the most useful in analysis of diffusible ions, especially calcium which in most biological systems is present in very low concentrations.

New preparative techniques for XRMA brought severe problems in visualization of the specimens prepared by cryomethods. Charging, low contrast, mass loss and contamination, which are often negligible in conventional electron microscopy, have still to be solved in XRMA of cryoprepared specimens. However, the methods of semi‐thin and thick cryosectioning and low‐temperature embedding were successfully used for analysis of cells and organelles and for the study of fluids in restricted biological spaces such as the inner ear, muscle spindles and ventricles of the brain in rats. Accordingly, examinations which were impossible by micropuncture and ion selective techniques could be carried out by XRMA. New data on the developing embryological organs and maturation of biological fluids in small biological cavities were obtained.
Whole‐mount preparations of mouse lens epithelium for the fluorescent cytological study of actin
Authors: Liou W.
Published: August 2011

A technique is given for the preparation of a sheet of epithelial cells from the capsule of the crystalline lens. A new method is described for fixation and staining with fluorescent phalloidin or actin antibody in order to localize the actin cytoskeleton in this tissue. Optical section of the preparation resolves such actin features as apical polygonal arrays, sequestered actin bundles, perinuclear actin aggregates, observed here for the first time, and filamentous networks in the basal region of the cell. This method is superior to previous ones in its ability to preserve actin‐abundant sectors distinctively.
Image analysis of Nissl‐stained neuronal perikarya in the primary visual cortex of the rat: Automatic detection and segmentation of neuronal profiles with nuclei and nucleoli*
Authors: Ahrens Peter , Schleicher Axel , Zilles Karl , Werner Lisa
Published: August 2011

An image analysing procedure for the morphometric characterization of cortical neurons in Nissl‐stained brain sections is described. It consists of the automatic detection of cellular profiles and their compartments: cytoplasm, nucleus and nucleolus.

The algorithm was designed to cope with the large morphological spectrum of cortical perikarya (e.g. geometrical properties of perikarya, staining intensities of cell compartments and nucleo‐plasmic area‐ratio) including pyramidal (Golgi‐category I) and non‐pyramidal (Golgi‐category II) neurons.

Clusters of cells were separated and non‐neuronal structures (e.g. glia, endothelial cells) as well as tangential, non‐nucleolated sections through neuronal perikarya recognized and excluded from further analysis without requiring interactive procedures.

The performance of the profile recognition procedure was evaluated using 426 nucleolated and non‐nucleolated profiles of different types of neurons in the primary visual cortex of the rat. Nucleolated profiles were recognized as such with a 91% accuracy, non‐nucleolated profiles were rejected correctly in 90% of cases.

After automatic segmentation and selection of nucleolated neuronal profiles from the microscopic field, a large set of quantitative morphological features including geometrical, densitometrical and textural parameters can be measured using high power light microscopy. This permits quantitative morphometric characterization of different neuronal types. This procedure is the first part of a system for the automatic classification of Nissl‐stained cortical neurons.
3‐D image formation in high‐aperture fluorescence confocal microscopy: a numerical analysis
Authors: van der Voort H. T. M. , Brakenhoff G. J.
Published: August 2011

The imaging properties of a confocal fluorescence microscope are considered on the basis of a theoretical model. The model takes into account high‐aperture objectives, the polarization state of the excitation light and a finite detector pinhole. Electromagnetic diffraction theory of the field near focus as developed by Richards and Wolf is used to compute the optical properties of the model. These are shown to be dependent on the polarization of the light. With the resulting three‐dimensional point spread function we have studied the imaging of point, line and plane objects as a function of their orientation with respect to the confocal plane. In addition, the effect of the pinhole size on the image formation of these objects is discussed.

The results indicate the necessity to take object orientation into account during image processing activities such as segmentation or analysis.
A fibre‐optic 3‐D microscope with high depth sensitivity
Authors: Fujii Y. , Yamazaki Y.
Published: August 2011

A new fibre‐optic microscope with high depth resolution is proposed and experimentally realized. In this microscope, a pair of monomode optical fibre couplers are used to form a six‐port all‐fibre Michelson interferometer. A lock‐in amplifier is used to improve the depth sensitivity and to simplify the optical and electronic circuits. The phase sensing signal is applied to a mirror at one arm of the interferometer. The optimum resolution was about 0ċ4 nm. The transverse resolution of this microscope is determined by the core diameter of the monomode fibre (9 μm). This 3‐D microscope is quite stable to ambient mechanical disturbances, and can be applied to the 3‐D microscopy of various biological objects.
Image processing techniques for 3‐D chromosome analysis
Authors: Houtsmuller A. B. , Oud J. L. , van der Voort H. T. M. , Baarslag M. W. , Krol J. J. , Mosterd B. , Mans A. , Brakenhoff G. J. , Nanninga N.
Published: August 2011

3‐D karyotype analysis is developing rapidly due to the availability of confocal microscopes and CCD video cameras, and the development of 3‐D processing techniques. Here, image enhancement and visualization techniques specifically designed for 3‐D karyotype analysis are described. To facilitate a good comparison between the different techniques, the same 3‐D image, obtained with a confocal scanning laser microscope (CSLM), of a mitotic prophase nucleus of a root‐tip cell of Crepis capillaris was used throughout. Besides well‐known stereoscopic presentation, another means of improving depth perception is shown, i.e. a solid modelling algorithm, which simulates the process of fluorescence.

An interactive routine to dissect objects in the image is presented as an alternative for automated segmentation algorithms, which cannot be applied to closely apposed or merging objects.

As an example of a convenient way to reduce the vast amount of data (2 Mbyte per image), a partly automated 3‐D cursor is presented in detail. This cursor is used to trace the central axes of chromosomes and record them as strings of Cartesian coordinates. The advantages of a computer graphics display, which facilitates real‐time rotation and hence is a powerful tool in studying 3‐D features of chromosomes, are also shown.
Analysing and sorting human chromosomes
Authors: Green Daryll K.
Published: August 2011

Analysing and sorting human chromosomes by flow cytometry is a powerful tool in the hands of the molecular biologist. Because of the large number of chromosomes analysed by flow, typically 105 for each sample, estimates of the distribution of DNA throughout the chromosome complement of an individual can be made to within three megabase pairs. Rearrangements of DNA in this size range can often be clearly seen and measured by flow cytometry in situations where it was not obvious by traditional cytogenetics.

The production of enriched samples of a particular chromosome by flow sorting and the subsequent construction of DNA libraries has played an important part in mapping genes to particular hereditary diseases. Techniques now exist which allow the hybridization of a few thousand sorted chromosomes with characterized or uncharacterized DNA probes to give relatively quick answers about specific chromosome genes. The process of obtaining a sorted chromosome sample from a growing population of peripheral blood lymphocytes or lymphoblastoid cells is compared with other methods of achieving similar results in molecular biological terms. Advances in flow cytometric techniques, which include slit‐scanning and hybridization of DNA probes in suspension, are likely to improve the enrichment quality of specific sorted human chromosomes.
Projection of topographic maps from curved brain surfaces into a plane
Authors: Rager Günter , Frei Bernhard , Kaup Burchard
Published: August 2011

The environment is represented in the brain by topographic maps. These maps and their interrelationship form a dominant feature in the brain, but unfortunately, they are often projected onto surfaces which are variously curved. It would be useful to be able to compare the topography on these surfaces quantitatively with the origin of the map. This comparison would be possible if the curved brain surfaces could be represented in planes. Therefore, it was necessary to develop a procedure for unfolding maps on convex structures like the retina and the optic tectum of the chick. For this purpose a unit sphere was constructed whose origin was close to the centre of gravity of the curved brain structure. The surface of this structure was then projected into the sphere. From its representation on the spherical surface it was unfolded into the plane using a polar azimuthal radially equidistant projection. Our method can safely be applied to convex structures only. However, it is not limited to the visual system. The mathematical procedure is formulated in such a way as to be easily implemented. It is exemplified by experiments on the optic tectum and the developing retina of the chick.
Assignment of two ultrastructures formed by a mixture of hexonamides using autoradiography and electron microscopy
Authors: Boettcher Christoph , Boekema Egbert J. , Fuhrhop Juergen ‐H.
Published: August 2011

The combined application of autoradiography and electron microscopy allowed the assignment of molecular components to individual micellar fibres in a mixed gel. Resolution was of the order of 0.1 μm. As a result, it was shown that bimolecular sheets of N‐dodecyl‐l‐mannonamide (= l‐Man‐12) completely separated from helical rods consisting of tritiated N‐octyl‐d‐gluconamide (= d‐Glu‐8). The method should be useful for the analysis of several other synthetic supramolecular systems.
Low‐temperature EDX microanalysis of halogenated copper phthalocyanine samples
Authors: McColgan P. , Chapman J. N. , Nicholson W. A. P.
Published: August 2011

X‐ray microanalysis at temperatures near that of liquid nitrogen is used to determine the halogen content of small volumes of halogenated copper phthalocyanine pigments. Details of the low‐temperature stage used and the benefits conferred by cooling the sample are given. The experimental procedure adopted involved recording a series of spectra from the same area and extrapolating the measured compositions to zero dose. Factors affecting the accuracy of the analyses are discussed.
Unbiased estimation of curve length in 3‐D using vertical slices
Authors: Gokhale A. M.
Published: August 2011

An efficient sampling procedure is presented for estimation of total line length per unit volume Lv. It involves the following steps: (1) choose a vertical axis in the specimen, and cut the specimen to obtain VUR vertical slices of constant thickness Δ such that parallel planes of the slices contain the vertical direction; (2) observe the projected image of a vertical slice using transmission microscopy such that beam direction is perpendicular to the slice; (3) count the number of intersections of the projected images of the lineal features of interest with cycloid‐shaped test lines whose minor axis is perpendicular to the vertical axis. The expected value of the number of intersections per unit length prj is related to Lv as follows:

Thus, Lv can be estimated from the measurements performed on the projected images of VUR vertical slices.
Time‐resolved cryo‐electron microscopy of vitrified muscular components
Authors: Lepault Jean , Erk Inge , Nicolas Gisèle , Ranck Jean‐Luc
Published: August 2011

Biological objects may be arrested in defined stages of their activity by fast freezing and may then be structurally examined. If the time between the start of activity and freezing is controlled, structural rearrangements due to biological function can be determined. Cryo‐electron microscopy shows great potential for the study of such time‐dependent phenomena. This study examines the actin polymerization process using cryo‐electron microscopy of vitrified specimens. Actin filaments are shown to undergo a structural change during polymerization. In the early stages of the polymerization process (t < 2 min), filaments exhibit a pronounced structural variation and frequently show a central low‐density area. In the later stages of the polymerization, F‐actin‐ADP filaments have a more uniform appearance and rarely display a central low‐density area. These findings, analysed on the basis of a previously proposed polymerization model, suggest that polymerization intermediates (F‐actin‐ATP and more probably F‐actin‐ADP‐Pi) and filaments at steady state (F‐actin‐ADP) have different structures. To investigate the physiological relevance of these results at the cellular level, the potential of cryo‐substitution in preserving the structure of muscular fibre was assessed. Optical diffraction patterns of relaxed and contracted frog cutaneous muscle are similar to the corresponding X‐ray diffraction patterns. The resolution of the images extends to about 7 nm. These results show that dynamic study of muscle contraction is possible using cryo‐substitution.
The characterization of microstructures in the atom probe
Authors: Hetherington M. G. , Miller M. K.
Published: August 2011

Mathematical descriptions of typical microsctructures which are observed in the atom probe are constructed from the theory of Markov chains. Short‐range ordering and clustering, overlapping and non‐overlapping particles and spinodal decomposition can all be modelled using this formalism. The models are used to calculate the important autocorrelation function.
Atomic force microscopy of a hydrated bacterial surface protein
Authors: Wiegräbe Winfried , Nonnenmacher Martin , Guckenberger Reinhard , Wolter Olaf
Published: August 2011

The protein surface layer of the bacterium Deinococcus radiodurans (HPI layer) was examined with an atomic force microscope (AFM). The measurements on the air‐dried, but still hydrated layer were performed in the attractive imaging mode in which the forces between tip and sample are much smaller than in AFM in the repulsive mode or in scanning tunnelling microscopy (STM). The results are compared with STM and transmission electron microscopy (TEM) data.
Validation of convection‐limited cooling of samples for freeze‐fracture electron microscopy
Authors: Bailey Stuart M. , Zasadzinski Joseph A. N.
Published: August 2011

Rapid freezing is the most important step in sample preparation for freeze‐fracture and other cryotechniques for electron microscopy. A simple heat transfer model is experimentally validated to show that convection from the cryogen to the specimen is the limiting step in rapid freezing of small samples [Biot modulus, (hd/k) < 1] by measuring cooling rates in a variety of samples, materials, and cryogens. In comparison to the commonly accepted conduction‐limited model, the convection‐limited model predicts, and our experiments show, that cooling rates are proportional to the surface area to volume ratio, independent of the sample thermal conductivity, and inversely proportional to the product of sample density and heat capacity. We show that almost any material can be frozen at similar rates if the sample thickness, the cryogen, and the method and velocity of contact with cryogen are similar. Liquid ethane or propane cooled to liquid nitrogen temperature are shown to give the best results.
A preparation method of specimens of the fungus Penicillium chrysogenum for ultrastructural and immuno‐electron microscopical studies
Authors: Müller Wally H. , van der Krift Theo P. , Knoll Gerd , Smaal Erik B. , Verkleij Arie J.
Published: August 2011

A combination of cryofixation without pre‐treatment, freeze‐substitution and low‐temperature embedding was used to prepare specimens of Penicillium chrysogenum for electron microscopy. To produce specimens which are thin enough for appropriate cryofixation, the P. chrysogenum colonies were grown between dissected‐dialysis tubing on an agar plate, which in addition allowed longitudinal sectioning. In contrast to classical chemical fixation, this preparation procedure resulted in excellent preservation of ultrastructure. Furthermore, the penicillin biosynthetic enzyme acyltransferase could be unequivocally located by immunogold labelling, indicating a preservation of antigenic properties of the specimen. Labelling density was not conspicuously affected when using different freeze‐substitution media, but it was reduced after embedding in Epon 812.
Solute‐enriched surface layers and X‐ray microanalysis of thin foils of a commercial aluminium alloy
Authors: Park J. K. , Ardell A. J.
Published: August 2011

Thin foils of the commercial Al alloy 7075 were subjected to X‐ray microanalysis in a Jeol 100 CX TEMSCAN using a modified double‐tilting stage. The ratio of the Cu Kx and Al Kx peaks, Icu/IAl, was found to decrease rapidly with increasing foil thickness, t, in regions thinner than about 200 nm. This was attributed to the formation of surface layers enriched in Cu during electropolishing, consistent with the findings of other investigators. An equation predicting the variation of IB/IA with t in an A‐rich alloy in which B (or additional solute elements) is dilute is derived for a sample with surface layers enriched in B. The derivation predicts that IB/IA should vary linearly with t−1, and that the intercept of such a plot should be positive. It is suggested that consistency with this prediction serves as a criterion for true surface enrichment. Our data on Cu obey these predictions, and further analysis indicates that the layers on both surfaces are enriched by a factor of about 5.6. The ratio of the intensities of the Zn Kx and Al Kx X‐ray peaks was found to be nearly independent of foil thickness to t≅400 nm. This result, combined with additional analysis, indicates that the apparent enrichment observed by other investigators is most likely due to the influence of instrumental factors.
Staining sections of water‐miscible resins 2. Effects of staining‐reagent lipophilicity on the staining of glycol‐methacrylate‐embedded tissues
Authors: Horobin Richard W. , Gerrits Peter O. , Wright David J.
Published: August 2011

Glycol methacrylate (GMA) sections of animal tissues were stained with a group of twenty‐seven reagents of very varied chemical characteristics. The artefactual background staining of the resin was found to be dependent on the hydrophilic/lipophilic character of the staining reagent, as estimated from the logarithm of its octanol‐water partition coefficient (log P). Intense background staining occurred with lipophilic stains, whose log P>2. In keeping with this, use of GMA semi‐permeable membranes for enzyme histochemistry failed to give staining when using a lipophilic substrate, probably because the substrate was trapped in the membrane. An analysis of other routine histochemical stains—in terms of the probable occurrence of high resin background staining and low tissue sensitivity—is made. A numerical guide is provided to help avoid artefacts resulting from hydrophobic and size effects. Note: small, hydrophilic reagents (log P <0; molecular weight < 550 Da) are least likely to show either type of artefact. Conversely, reagents which are lipophilic, or/and of intermediate size (log P > 2; 550 < ionic weight < 1000 Da), give strong background staining.
Extended electron energy‐loss fine structure and selected‐area electron diffraction studies of small palladium clusters
Authors: Diociaiuti M. , Picozzi P. , Santucci S. , Lozzi L. , De Crescenzi M.
Published: August 2011

Extended electron energy‐loss fine structure (EXELFS) and selected‐area electron diffraction (SAED) techniques have both been applied to the study of the crystalline structure of Pd clusters of average diameters ranging from bulk to 24 Å. The combined use of these techniques gives complementary information about the crystalline structure of Pd clusters. Both techniques show the same lattice parameter expansion, about 4% for the smallest Pd cluster, with respect to the bulk. The EXELFS analysis performed on the Pd‐M4,5 edge shows a sizeable increase of structural disorder in the smallest cluster. SAED gives additional information about the Pd bulk sample, showing the occurrence of crystalline regions about 50 Å in diameter.
The use of parallel EEL spectral imaging and elemental mapping in the rapid assessment of anti‐cancer drug localization
Authors: Huxham I. M. , Gaze M. N. , Workman P. , Mairs R. J.
Published: August 2011

Both electron spectroscopic imaging (ESI) and electron energy‐loss spectroscopy (EELS) have great potential for use in several areas of cancer research. In biologically targeted radiotherapy, cytotoxic drug therapy and boron neutron capture therapy the effectiveness of many drugs is often critically dependent upon the intracellular localization of the agent employed. We describe the use of parallel EEL spectral imaging to assess the penetration and location of the iodine‐containing drug meta‐iodobenzyl guanidine, of potential value in targeted radiotherapy, and for the rapid detection of boron within borate‐adsorbed polystyrene beads, of potential value in boron neutron capture therapy. We also describe elemental mapping of boron following low‐temperature embedding. These results show how the techniques could be applied to many forms of cancer research by discussing the validity and limitations of the techniques experimentally. We also provide an outline of other areas in this field which could benefit from the future application of ESI and EELS.
Estimation of length density Lv from vertical slices of unknown thickness
Authors: Gokhale Arun M.
Published: August 2011

Length density of lineal features, Lv, is an important stereological parameter. The efficient stereological procedure for the estimation of Lv from the counting measurement performed on the projected images of the vertical slices (foils) is modified and improved: Lv can be now estimated from vertical slices of unknown thickness, and the slices need not be of the same thickness. The required assumption‐free stereological relationship is

image

is the average number of intersections of straight test lines parallel to the vertical axis with the projected images of the lineal features in the vertical slices, per unit test line length. is the average number of intersections of the lineal features with the parallel planes of the vertical slices, per unit area. Note that there are two parallel planes in a slice, and therefore their total area is twice the area of the observed projected image frame. is the average number of intersections of orientated cycloid‐shaped test lines (minor axis perpendicular to vertical axis) with the projected images of the lineal features in the vertical slices, per unit length. For practical applications of this result, it is necessary uniquely to identify the points of intersections of the lineal features with the parallel planes of the slices in the projected images, so that can be estimated unambiguously. However, in practice, this is not a problem in biological, as well as materials microstructures.
A fast multi‐level decomposition algorithm for n‐dimensional discrete Gabor transforms
Authors: Zhu Zhi Yuan , Barba Joseph
Published: August 2011

Recently, joint spatial and spatial‐frequency representations have been used in signal processing of non‐stationary signals due to their natural local property and high joint resolution in both the spatial and spatial‐frequency domain. However, a major obstacle to their implementation is their large computation requirements. This paper presents a fast n‐dimensional Gabor transform and signal reconstruction algorithm employing multi‐level parallel decomposition and fast Fourier transform techniques. The algorithm structure lends itself to implementation using VLSI/ASIC technology. Examples of two‐dimensional Gabor transform and reconstruction performed on a AT computer demonstrate the substantial computational saving that can be achieved using the fast Gabor transform.
High‐conductivity amorphous TiSi substrates for low‐temperature electron microscopy
Authors: Rader R. S. , Lamvik M. K.
Published: August 2011

To avoid problems with electrostatic charging, electrically conductive specimen support films or coatings must be used when observing non‐conductive organic specimens by electron microscopy. At room temperature, microscopists typically use either carbon films or carbon‐coated plastic films as specimen substrates. Such films have also been adopted for low‐temperature microscopy. Recent measurements have shown that conventional carbon films have high electrical resistivity at low temperature. Thin titanium films have been used to provide improved electrical conductivity, but the structure of titanium films is excessively granular for high‐resolution electron microscopy. With the hope of solving both problems, thin films of Ti88Si12 have been produced using a standard evaporation system. The deposited films are amorphous, as shown by electron microscopy, electron diffraction and differential scanning calorimetry. The electrical conductivity of the binary alloy films is nearly as high as that of pure titanium films. Therefore, these films appear to be appropriate substrates for low‐temperature electron microscopy.
Prospects for confocal imaging based on nanosecond fluorescence decay time
Authors: Morgan C. G. , Mitchell A. C. , Murray J. G.
Published: August 2011

A fluorescence microscope has been developed to give contrast on the basis of fluorescence decay time. Two different detection schemes are used, one based on an imaging single photon detector equipped with an external photon correlator and the other using an RF‐modulated intensified CCD camera. Examples of the new contrast are shown, and the possible routes to development of a confocal version of the microscope are discussed.
An FFT‐based method for attenuation correction in fluorescence confocal microscopy
Authors: ROERDINK J. B. T. M. , BAKKER M.
Published: August 2011

A problem in three‐dimensional imaging using a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. A new method is proposed to correct for these effects. The approach, valid for weak attenuation, consists of multiplying the measured fluorescence intensity by a correction factor involving a convolution integral of the measured signal, which can be computed efficiently by the fast Fourier transform. Analytical and numerical estimates are given for the degree of attenuation under which the method is valid, and the method is applied to various test images. A real CSLM image is restored. Finally, the method is compared with a recent iterative method with regard to numerical accuracy and computational efficiency.
A method to extend the application of microdroplet techniques in the SEM
Authors: CASSID M. F. , ALLEN D. E.
Published: August 2011

A thin‐film method of microdroplet analysis is described. The method differs from those published previously. First, uniformly distributed amorphous deposits of chemicals were obtained through the use of a non‐aqueous anti‐crystallization agent, ethylene glycol, which has not been described previously. Second, films of celloidin were used; these could be prepared routinely at 60–80 nm and contained no interfering elements. Initially, these films were too unstable to be of use, but heating them at 60°C greatly improved their stability. A nebulizer was used to deliver microdroplets to these supports, which were, in turn, supported by a specially constructed carbon holder. Minimum detectable concentrations were improved with this method.
Estimation of gold‐labelled macromolecules attached to cell surfaces
Authors: THOMAS N. , GWYNN I. AP
Published: August 2011

A method for obtaining a semi‐quantitative estimation of the amount of colloidal gold label attached to a cell surface is described. The X‐ray emission, in a scanning electron microscope, from an even metal coating applied by diode sputter coating is used as an internal standard. The emission from the standard is used to correct for errors which would have arisen due to factors such as variable specimen surface topography. Examples of the semiquantitative estimation of 10‐nm gold‐labelled wheat‐germ agglutinin to L929 murine fibroblast cells are given.
Promises and problems of biological atomic force microscopy
Authors: YANG J. , TAMM L. K. , SOMLYO A. P. , SHAO Z.
Published: August 2011

Recent developments in the biological applications of atomic force microscopy are reviewed, and the great potential of this novel, high‐resolution imaging technique, including its limitations and possible future directions for biological research, are discussed.