bluuebunny/crossref_metadata_2025 huggingface dataset:
A convenient culture chamber for observation and embedding of macrophage monolayers for transmission electron microscopy
Authors: Hackett Charles J. , Sullivan Kate
Published: August 2011

A chamber adapted from a polypropylene test tube is described for the cultivation and processing of undisturbed monolayers of normal mouse macrophages for transmission electron microscopy (TEM). Peritoneal exudate cells are grown in the chambers on Visking dialysis membranes pretreated with Polybrene (Sigma Chemical Co.) and normal mouse serum. The cells are not further disturbed after adherence since the entire chamber, including dialysis membrane and cells, is processed for electron microscopy using standard material and protocols and the embedded dialysis membrane is readily oriented and thin‐sectioned. This method is efficient for studying adherent cells since growth can be nearly confluent and cells remain firmly attached throughout the manipulations. Since a transparent membrane is used, cells can also be observed with the light microscope at all stages prior to sectioning.
New instruments which facilitate rapid freezing at 83 K and 6 K
Authors: Escaig Jacques
Published: August 2011

We have developed three instruments to freeze different biological specimens for various purposes in the easiest and most economical way: a punching device, a rapid immersion device and a device to freeze the specimens against a copper block cooled by liquid helium. Cooling rate measurements are made by a thin flat thermocouple. Freezing against a copper block cooled down to 4 K gives cooling rates 3–5 times higher than freezing by immersion into propane at 85 K.
A low X‐ray background low temperature specimen stage for biological microanalysis in the TEM†
Authors: Nicholson W. A. P. , Biddlecombe W. H. , Elder H. Y.
Published: August 2011

The side entry specimen stage described in this paper is a development of a specimen stage for use at room temperature in the JEOL JEM 100 and 200 series transmission electron microscopes. The earlier specimen stage was designed to ensure that the extraneous instrumental contribution to thin specimen X‐ray spectra is both small and quantifiable. As well as having a low X‐ray background the new stage will also maintain the specimen at temperatures sufficiently low (down to 100 K) to minimize the loss of organic material due to electron irradiation. A new anticontaminator which provides a clean low temperature environment for the specimen stage is also described.
Thin frozen‐dried cryosections and biological X‐ray microanalysis
Authors: Barnard Tudor
Published: August 2011

The application of X‐ray microanalysis to problems of cell physiology required the development of methods to retain diffusible substances within the subcellular compartments that they occupied in vivo.

Several groups have developed methods of rapidly freezing small samples in ways that minimize mechanical traumae and ice crystal formation. This provides a narrow zone from which cryosections, believed to be representative of the in vivo distribution of electrolytes, can be cut.

The production of thin (less than 0·5 μm) cryosections that are apparently free of diffusion can be routinely performed when temperature parameters are kept below 173 K. Efficient cryosectioning requires several modifications to commercially available machines, in order to improve the ease and reliability with which various manipulations can be carried out.

Initial attempts to localize calcium at the subcellular level were disturbed by the use of mechanically damaged specimens and by insufficiently cold conditions in the cryochamber. Such sections indicated that mitochondria were calcium‐rich organelles.

When tissue freezing and cryosectioning were performed under optimized conditions, mitochondrial calcium was so low as to be quantifiable only with difficulty. Available microanalytical results show that ER‐rich cytoplasm and terminal cisternae of the sarcoplasmic reticulum seem to contain higher levels of calcium than mitochondria. Nuclei and secretory granules also contain more calcium than mitochondria.
A computer system for on‐line image capture and analysis
Authors: Skarnulis A. J.
Published: August 2011

A computer control system for electron microscopy is proposed; it consists of functionally distinct microprocessors communicating via a central microprocessor (the supervisor) by direct memory access or a common data bus. The control functions such as digital beam scan, accelerating voltage, focusing, etc., are controlled by distributed microprocessors under the direction of the supervisory microprocessor. The system includes a framestore for image storage and a special high speed processor that contains a microcode library of functions. The system is used for interactive real‐time image analysis by comparison of calculated images and processed experimental images.
Interactive image processing with an off‐line minicomputer: organization, performance and applications
Authors: Saxton W. O. , Koch T. L.
Published: August 2011

A recently established facility for interactive image processing is described, primarily from the point of view of the system's hardware organization and the special purpose software provided to support it. The performance of more general software (the ‘Semper’ system) in the new environment has been measured carefully, and found to be about 10 times its performance on our previous PDP 8/E computer; particular attention is given to the data management techniques crucial to the system's efficiency. Some current applications are illustrated, including high resolution image interpretation and image averaging of biological crystals.
A method for rapid in situ measurement of foil thickness in a TEM
Authors: Preez W. B. du , Kritzinger S.
Published: August 2011

A method is described which can be used for rapid in situ determinations of relative foil thicknesses in a transmission electron microscope, and is illustrated for the case of aluminium. A calibration curve for the foil thickness determination was obtained by measuring the relative transmission of electrons through standard thickness foils which were prepared and measured outside the electron microscope. By using this calibration curve, it was merely necessary to measure the relative transmission of electrons through an unknown specimen in order to determine its thickness.
An intercellular network artefact in glycol methacrylate dehydrated neonatal cartilage
Authors: Shepard Nora , Mitchell Nelson , Harrod John
Published: August 2011

A striking network thought to consist of glycosaminoglycan in neonatal cartilage has recently been reported following glycol methacrylate (GMA) dehydration and embedding. This study attempted to learn why the network was not seen in other preparations previously reported.

Comparative light and transmission electron microscopy of the network structures was carried out to determine their biological significance. We have shown the networks to be reproducible artefact, attained only following GMA dehydration of glutaraldehyde fixed neonatal cartilage.
The histochemical basis of quantitative histology*
Authors: Stoward Peter J. , Ploem Johan S.
Published: August 2011

The interaction between histochemistry and microscopy for quantifying the morphology and function of cells in sections of tissue is reviewed.

In principle, the morphological parameters of cells can be measured in suitably‐stained sections using image analysers. In practice, however, the individual cells often cannot be distinguished from each other in such systems. This problem might be overcome, at least in muscle, by visualizing types III and IV collagen in the connective tissue and basement membranes surrounding the cells.

The functional capacities of cells within tissues can be inferred by measuring the activities of their key enzymes with quantitative histochemical techniques. At present, the wrong enzymes are frequently chosen for this purpose. Flux‐generating and physiologically significant enzymes are preferable, but this requires new histochemical techniques to be devised and validated quantitatively for use with the computer‐assisted analytical microscopes that are now becoming available commercially.
Electron microscopy of frozen water and aqueous solutions
Authors: Dubochet J. , Lepault J. , Freeman R. , Berriman J. A. , Homo J.‐C.
Published: August 2011

Thin layers of pure water or aqueous solutions are frozen in the vitreous state or with the water phase in the form of hexagonal or cubic crystals, either by using a spray‐freezing method or by spreading the liquid on alkylamine treated films. The specimens are observed in a conventional and in a scanning transmission electron microscope at temperatures down to 25 K. In general, the formation of crystals and segregation of solutes during freezing, devitrification and evaporation upon warming, take place as foreseen by previous X‐ray, thermal, optical and electron microscopical studies. Electron beam damage appears in three forms. The devitrification of vitreous ice. The slow loss of material for the specimen at a rate of about one molecule of pure water for every sixty electrons. The bubbling in solutions of organic material for doses in the range of thousands of e nm−2. We propose a possible model for the mechanism of beam damage in aqueous solutions.

The structural and thermal properties of pure frozen water important for electron microscopy are summarized in an appendix.
Examination of whole cell mounts by transmission electron microscopy using cultures grown on carbon coated coverslips
Authors: Tolson Neil D. , Boothroyd Brian
Published: August 2011

Cultures of porcine granulosa cells have been grown on serum coated carbon films deposited on glass coverslips. Intact and detergent‐treated cultures were critical point dried, chromium shadowed and removed from the coverslips by dilute hydrofluoric acid. Whole cell mounts were examined by conventional transmission electron microscopy. The advantage of this system over other methods includes the ability to: (1) examine cells with a highly rounded morphology, (2) use high cell densities, (3) study cell projections extending onto the substratum, (4) maintain cultures for long periods, (5) examine large samples easily, and (6) readily use the same cultures for transmitted light and fluorescent microscopy, scanning and transmission electron microscopy (SEM and TEM).
Differential staining of polyanions according to critical electrolyte concentration principles in mixed solvents
Authors: Scott J. E. , Hughes E. W.
Published: August 2011

Three types of cationic reagent—cellulosic anion exchangers, cationic phthalocyanin dyes and detergents—formed insoluble complexes with the polyanions heparin, chondroitin‐4‐sulphate, hyaluronate, alginate and DNA. Dissociation of their complexes by MgCl2 was a critical phenomenon, occurring at a sharply defined concentration, the critical electrolyte concentration (CEC), in water and in water‐organic‐solvent mixtures. CECs of polysulphates were always higher than those of polycarboxylates or polyphosphates, especially in the presence of dimethyl sulphoxide or ethanol. The latter effect should improve biochemical fractionations and differential histochemical staining of polyanions.

A general theoretical framework is proposed. Complex formation is promoted by electrostatic attractions, and results in a loss of polymer mixing energy. Electrolytes suppress the electrostatic interaction, thus preventing complex formation. More efficient suppression of the electrostatic interaction is required in a ‘worsening’ solvent, i.e. where a rise in CEC occurs. Plots of CEC against solvent composition are interpreted on these bases. Available data on electrolyte solutions account for specific ionic effects.

The dissociation of phthalocyanin dye micelles in aqueous dimethyl sulphoxide, etc, was studied spectroscopically. The results support and extend the conclusions derived from the insoluble anion‐exchange celluloses.

The general concepts serve as a bridge between biochemical and histochemical fractionations (i.e. specific staining of substrates).
Problems of interpretation in high resolution electron microscopy
Authors: Howie A.
Published: August 2011

Current assumptions in wave aberration theory and specimen scattering theory are reviewed. More quantitative image simulations would be valuable as well as use of a wider range of imaging techniques, particularly STEM. The severe difficulties of high resolution three‐dimensional reconstruction are described and illustrated.
The STEM approach to the imaging of surfaces and small particles
Authors: Cowley J. M.
Published: August 2011

The characteristics of scanning transmission electron microsopy, which make it a particularly powerful tool for the study of surfaces and small crystals, include the serial nature of the image signal which makes it amenable to recording and image processing procedures, the possibility of correlating the image data with microdiffraction patterns and microanalysis of chosen small regions and the possibilities for combining several different image signals obtained simultaneously. Images obtained from surfaces using reflected diffraction beams may be very sensitive to variations of surface structure but the resolution, depth of focus and contrast of the images are strongly dependent on the nature of the surface and the aperture sizes used in the microsope. Microdiffraction patterns may be obtained from small surface features. Electron energy loss analyses of the diffracted beams provides sensitive indications on surface electron excitations. Microdiffraction patterns obtained in transmission from regions of 1–5 nm diameter provide valuable information on the structure and defects of small crystals, such as those of platinum and gold which are of interest in relation to the study of catalysts.
The high resolution electron microscopy of stacking defects in Cu–Zn–Al shape memory alloy
Authors: Cook J. M. , O'Keefe M. A. , Smith David J. , Stobbs W. M.
Published: August 2011

The characteristic defects in the martensitic phase of the memory alloy Cu–Zn–Al have been investigated using the Cambridge University 600 kV High Resolution Electron Microscope. Conditions are found under which the images can be quantitatively simulated when this involves the transfer of structural information at resolutions beyond the first zero of the contrast transfer function. Defects of sequence‐type were positively identified by the comparison of high‐resolution micrographs with full dynamical image simulations.
A transformation anomaly in a Cu–Zn–Al alloy
Authors: Stobbs W. M.
Published: August 2011

A comparison is described of the structures of an M9R Cu–Zn–Al alloy as quenched directly into the martensitic state, and as stabilized, with the structure of the material after up‐quenching into the β1 state after which treatment the martensitic memory transformation occurs thermoelastically. Preliminary results on the increased tendency for the stabilized alloy to form f.c.c. stacking sequence regions are given and discussed.
The pore geometry and structure of the eggshell of the North Island brown kiwi, Apteryx australis mantelli
Authors: Silyn‐Roberts H.
Published: August 2011

The total effective pore area of the eggshell of the North Island brown kiwi has been quantified and found to be only 27% of that allometrically predicted. This has been achieved by a reduction in the number of pores to 53% of allometric prediction and of individual pore area to 66%. A new technique for pore‐casting has been devised using a low viscosity epoxy resin designed for crack‐filling in concrete. Polished and weakly etched sections of the central layer of the shell have revealed an arrangement of calcite previously believed to be unique to the tinamou.
Effects of glutaraldehyde and critical point drying on the shape and size of erythrocytes in isotonic and hypotonic media
Authors: Eskelinen Sinikka , Saukko Pekka
Published: August 2011

Scanning electron microscopy (SEM) was applied to studies on osmotically swollen erythrocytes fixed by a method using increasing concentrations of glutaraldehyde (GA), OsO4 as a post fixative, and critical point drying (CPD). This fixation method prevented the osmotic effect of GA itself and preserved the cellular equilibrium shape intact in various media, i.e. discocytes in an isotonic medium and ellipsoids or spheres and ghosts in a hypotonic medium. The discocytes were reduced less in diameter and area than the ellipsoids and spheres during CPD, but the degree of volume shrinkage was of the same magnitude (approx. 73%) regardless of the cell shape. The shrinkage of the discocytes was advanced until the dry volume (approx. 29 μm3) was reached, while the shrinkage of the osmotically swollen cells ceased earlier. A miniaturization process coupled with solvent evaporation is suggested as an explanation for this. The osmotic swelling of erythrocytes was studied quantitatively with the aid of SEM micrographs. The increase in volume was about 60%, which is in agreement with the values obtained by light microscopy for unfixed and GA‐fixed cells in the present work and with those reported in the literature.
Isolation and preparation of colonies grown on soft agar for ultrastructural investigation
Authors: Safa Ahmad R. , Tseng Michael T.
Published: August 2011

Preparation of cell colonies grown on soft agar for electron microscopic examination is tedious. Mechanical damage to cells can occur during the process of colony removal. A procedure which permits rapid colony removal and preserves the integrity of the individual colony for ultrastructural examinations is described in this paper.
Enhancement of contrast in living and fixed specimens by the use of fibre optics
Authors: Burgess Ann M. C.
Published: August 2011

A method is described which enhances the contrast of living and fixed specimens examined with the stereomicroscope. It consists of immersing the ends of flexible fibre optic light sources together with the specimen in the fluid used for examination. It is reported that not only does this method increase the contrast of living specimens but that it may also be applied to specimens being prepared as thin sections or freeze fracture surfaces for examination with the transmission electron microscope. A further method of enhancement of contrast is suggested which involves the fitting of light filters of complementary colours, one to each of the fibre optic light sources, before immersion with the specimen.
Recent improvements to the Cambridge University 600 kV High Resolution Electron Microscope
Authors: Smith David J. , Camps R. A. , Freeman L. A. , Hill R. , Nixon W. C. , Smith K. C. A.
Published: August 2011

Several recent modifications to the Cambridge University 600 kV HREM have resulted in increased levels of performance and greater operating efficiency. Changes include new Permendur pole‐pieces with a reduced gap leading to improved aberration coefficients, and an attachment to the tilt/shift supplies which facilitates accurate beam alignment. Resolution figures include: (a) an axial CTF extending, without zeros, beyond 1·8 Å at 575 kV; (b) tilted beam CTFs extending beyond 1·1 Å; and (c) lattice fringes of 0·72 Å spacing.
Combining accurate defocus with low‐dose imaging in high resolution electron microscopy of biological material
Authors: Wrigley N. G. , Brown Elaine , Chillingworth R. K.
Published: August 2011

High resolution (< 2 nm) electron microscopy of biological specimens requires three exacting conditions to be met simultaneously: (a) fine specimen detail must be protected from destruction by the electron beam (low dose), (b) the electron optics must be adjusted to be capable of imaging that detail interpretably (accurate defocus), and (c) a suitable field of interest must be identified. We describe a method encompassing all three with an 80% success rate using only minor modifications to a transmission electron microscope, and no expensive on‐line computing.
HREM and STEM of defects in multiply‐twinned particles
Authors: Marks L. D. , Smith David J.
Published: August 2011

The structure of defects in multiply‐twinned particles has been studied in detail using high‐resolution lattice imaging, dark field and microdiffraction techniques. Icosahedral particles with sizes greater than about 15 nm were found to contain defects, in the form of stacking fault loops parallel with the external surface, which were extremely difficult to detect by conventional amplitude contrast techniques. Microdiffraction mappings correlated with these results, showing large rotations of the face‐centred cubic segments. Results for decahedral particles indicated the presence of stacking faults running adjacent to, and parallel with, the twin boundaries. Microdiffraction maps confirmed that the particle structure was face‐centred cubic, and also verified that the apparent epitaxy of these particles was highly variable. Models for the defects are proposed and discussed, and the relative merits of HREM and STEM for elucidating structural details in small particles is briefly considered. Finally, the potential for direct imaging at surfaces, as demonstrated by some recent images, is discussed.
Electron microscopy of frozen hydrated sections of vitreous ice and vitrified biological samples
Authors: McDowall A. W. , Chang J.‐J. , Freeman R. , Lepault J. , Walter C. A. , Dubochet J.
Published: August 2011

The preparation and high resolution observation of frozen hydrated thin sections has been studied by transmission electron microscopy (TEM and STEM) on model systems, including pure water, protein solutions, catalase crystals, myelin sheath and various tissues. The state of the ice is determined by electron diffraction. Mass measurement in the electron microscope is used to determine section thickness and control hydration. An adequate depth of vitrified material for sectioning can be obtained from many biological suspensions or untreated tissues. Frozen hydrated sections around 100 nm thick can be produced under optimal conditions from vitreous ice or from vitrified biological samples. Sectioning, transfer and observation in the electron microscope is feasible without alteration of the sample hydration or its initial vitrification. Biological structures can be preserved and observed down to 10 nm. Under favourable working conditions, specimen compression during sectioning and electron beam damage are the factors limiting high resolution observations.
TEM/STEM requirements: multipurpose or specialized instruments?
Authors: Stobbs W. M.
Published: August 2011

The relative advantages and disadvantages of high resolution and other more general imaging techniques are considered and the potential of a multipurpose, moderate resolution, TEM with EELS is assessed.
Fresnel fringe contrast of faceted voids within gem‐quality diamond
Authors: Hutchison J. L. , Bursill L. A.
Published: August 2011

The topology of faceted void‐like defects (‘voidites’) in gem‐quality type la diamonds has been revealed by high resolution phase‐contrast electron microscopy. Interpretation of the images in terms of a Fresnel contrast mechanism allows the defects to be identified as octahedral in shape. Image contrast is sensitive to defocus and other parameters, and Fresnel fringe interference effects may generate ‘superlattice’‐like contrast.
Instrumental requirements for high resolution imaging
Authors: Herrmann K.‐H.
Published: August 2011

The resolution of modern transmission electron microscopes reaches the physical limits imposed by lens aberrations and energy width. One of the many conditions to be fulfilled, the alignment of illuminating and imaging beam onto the coma‐free objective axis, is particularly discussed here since axial coma cannot be detected by the usual resolution‐checking methods. Space consumption of specimen stages prevents the full utilization of the magnetic saturation limit only in the 100 keV range. With higher energies, this handicap is obviated, and some additional advantages can be gained which promote material investigations at atomic resolution, and which are presently utilized in instrumental research projects. High resolution with biological specimens has up to now been unsuccessful because of radiation damage. Optimum utilization of all electrons scattered at the specimen must thus be given priority over optical resolution. Important instrumental requirements are minimum exposure beam control, imaging modes with high collection efficiency, and recording devices with high detection quantum efficiency connected on‐line to image processors. A remarkable decrease in beam sensitivity of organic crystals, by more than one order, has been found by cooling the specimen down to 4 K which, by the use of superconducting lenses, can be combined with both ultra high vacuum and the stability requirements for high resolution. Yet up to now, such protection has not been achieved with He cryostates in conventional lenses, perhaps because a temperature increase even of only a few degree K is harmful.

Purely magnetic imaging energy filters are about to be developed to a high optical quality but have been employed so far in only a few high resolution instruments. Such filters allow removal of the inelastic background and thus improvement of contrast of images of low‐Z specimens, particularly in the dark field mode.

Finally, some ‘non‐conventional’ projects have made progress. Correction of spherical and chromatic aberration by multipole lenses offers a chance to improve remarkably the resolution in the 100 keV range, to extend the bandwidth of phase contrast transfer and to obtain highly resolved information about inelastic images when an energy filter is also applied. Electron holography provides possibly useful large area phase contrast, particularly if the electron energy is decreased, which may be of great benefit in investigations of unstained specimens.
Air‐drying of human leucocytes for scanning electron microscopy using the GTGO procedure
Authors: Gamliel Haim , Gurfel Dorit , Leizerowitz Rachel , Polliack Aaron
Published: August 2011

The utilization of tannic acid and guanidine hydrochloride as mordants for better osmium binding has been shown to serve as an excellent alternative to metal coating of organ tissue specimens for scanning electron microscopy (SEM). The present report describes the GTGO procedure, a modification of the TAO technique introduced by Murakami et al. (1977, 1978), which we have found successful for the preparation of air dried peripheral blood leucocytes for SEM studies. Air dried, GTGO‐treated leucocytes show excellent preservation of surface features with minimal cell shrinkage. When critical point dried, GTGO‐treated cells are examined, they also show less shrinkage than cells prepared with standard glutaraldehyde fixation and critical point drying. The potential application of this air drying procedure (GTGO‐AD) to other soft biological specimens is currently under investigation. This technique is recommended as a new and effective air drying procedure for the successful preparation of cells for SEM.
Visualization of skin appendages by scanning electron microscopy
Authors: Tsuji Takuo
Published: August 2011

A method for SEM visualization of skin appendages and elastic fibres around them is described. Skin sections were autoclaved for 4 h and followed by fixation, dehydration and xylol‐air drying. Using this method, the three‐dimensional architectural details of each type of skin appendage and related elastic fibre network were demonstrated.
A method for the detection of amyloid in human tissue embedded in epon using a combination of polarizing light microscopy and electron microscopy
Authors: Cottell David C.
Published: August 2011

This paper describes a method for demonstrating amyloid in human tissue embedded in epon using a combination of congo red staining and polarizing microscopy. The method involves the removal of the embedding medium from semi‐thin sections prior to staining with congo red and examination in the polarizing microscope. Typical apple green birefringence was detected in areas of amyloid deposition; these areas were mapped for selective trimming and ultra‐thin sections taken serially were examined in the electron microscope. The method was found to be reliable, easy to use and particularly of value as a sampling procedure prior to investigation in the electron microscope.
Morphometric studies of the spleen of the hypoxic mouse
Authors: Clegg E. J.
Published: August 2011

Adult male mice were exposed for periods of up to 7 days at atmospheric pressures of either 72 or 52 kPa (540 or 390 mm mercury). Changes in body weights, spleen weights and red and white splenic pulp weights were compared with those in normoxic controls.

Contrary to expectation, spleen weights and amounts of red pulp were greater (in terms of experimental‐control differences) in the animals kept at 72 kPa, suggesting that at 52 kPa loss of overall fitness reduced the adaptive erythropoietic response. However, in view of the fact that increased erythropoiesis has disadvantages as well as advantages, the lesser response at the lower pressure may not be wholly disadvantageous.

Coefficients of variation of the spleen variables examined showed a tendency to decrease in the animals at 72 kPa, but rose markedly at 52 kPa. This finding was interpreted as indicating that at 72 kPa spleen and red pulp changes are adaptive, but at 52 kPa they indicate an overall relative failure of adaptive mechanisms, with consequent reduced somatic fitness.
Commercial image analysers and the characterization of microscopical images
Authors: Bradbury S.
Published: August 2011

This review surveys some of the current approaches to the characterization of microscopical images using commercially available image analysers. The role of image pre‐processing and segmentation is considered, together with the characterization of images by optical density, as well as size and shape.

The newer analysers are capable of investigating relationships between grey‐levels in small areas of the image (‘texture‐analysis’); some of the descriptive approaches in this latter field are briefly outlined.
Standards for quantitative energy dispersive X‐ray microanalysis of biological cryosections: validation and application to studies of myocardium
Authors: Hagler Herbert K. , Lopez Linda E. , Flores Jesse S. , Lundswick Rebecca J. , Buja L. Maximilian
Published: August 2011

The preparation of a comprehensive set of gelatin‐glycerol‐salt standards for quantitation of diffusible ions in ultrathin cryosectioned tissue is described. This set of standards serves two basic functions. The first is to calibrate the X‐ray analytical system so that peak‐to‐continuum ratios obtained from test specimens may be converted to elemental concentrations. The second is to provide a known set of elemental concentrations which can be used to check the performance of the preparative and analytical steps involved in thin section biological X‐ray microanalysis. The application of these standards and techniques for cryoultramicrotomy and analysis of myocardial tissue also is described.
Vascular smooth muscle orientation in straight portions of human cerebral arteries
Authors: Walmsley James G.
Published: August 2011

A technique of embedding, sectioning and analysis has been developed for studying the orientation and proportional composition of smooth muscle in the straight portions of human major cerebral arteries. Various distortions, which occurred during processing and sectioning, were measured quantitatively. Nerve fibres were implanted as a reference frame within the paraffin block containing an arterial bifurcation. The nuclei of smooth muscle have been treated as three‐dimensional vectors of cellular orientation. The projected length in the plane of the section and the section thickness were used to define section pitch. To relate these vectors to the overall geometry of cerebral arteries they were transformed such that the resultant pitch would be the same as that observed if the sections were cut normal to the arterial longitudinal axis. Matrix transformations of nuclear vectors were of the expansion and Eulerian forms. The average pitch for the five sections from three straight portions was −0.22° ± 2.36 (SD of five means) with a range between −2.8° and 3.2°. Because this pitch is small it is possible to use 6.9 μm thick longitudinal and mid‐plane sections of straight arteries to obtain estimates of proportional composition. Stereological point counting was used to determine that smooth muscle comprised 72.0% ± 4.76 (SD for ten segments) of the tunica media in cerebral arteries.
Indentation analysis: applications in the strength and wear of brittle materials
Authors: Lawn B. R. , Hockey B. J. , Richter H.
Published: August 2011

Some recent developments in the principles and applications of indentation fracture in brittle materials are surveyed. Attention is focused on ‘sharp’ indenters, for which precursor ‘plasticity’ is an essential element of the crack development. A major consequence of this plasticity is a residual contact stress field which exerts a dominant influence on ensuing mechanical behaviour. This influence is discussed in relation to strength and wear properties of brittle ceramics. Emphasis is placed on the advantages of the indentation method as a means of producing controlled cracks for evaluating material fracture parameters and for gaining insight into flaw micromechanics.
Field emission, developments and possibilities
Authors: van der Mast K. D.
Published: August 2011

The overall performance of any electron microscope is to a great extent determined by the electron source. For example, the current acceptance of the STEM concept is due to the stimulation provided by field emission sources, which made it possible to image single atoms in the STEM.

The field emission source remains the electron source with the highest brightness and the lowest energy spread. As a consequence, considerable research and engineering work has been and is still being conducted in a number of important areas. These areas include: (i) the mechanisms which determine the beam current stability; (ii) the electron‐electron interactions which lead to beam spreading and an increase of the energy spread; (iii) various types of emitters such as bare tungsten tips, oxygen processed tips, zirconium coated tips, carbon emitters, carbon coated tungsten emitters and even solid state emitters; (iv) the lifetime limiting factors, e.g. inclusions, ion bombardment and flashovers effects; (v) different types of optical systems for field emission guns, e.g. choice of magnetic and/or electrostatic lenses at high voltage potential in UHV systems, and special magnetic lenses just below the anode; (vi) field emission guns operating at higher kV's, up to 1·6 MeV.

Because analytical work in the smallest possible volumes is a growing area of scientific interest, the demand for field emission sources which are reliable, easy to operate and stable will continue.
The use of a digitizing frame store in microscopy
Authors: Agar A. W.
Published: August 2011

A digitizing frame store giving real time read out of the image for TV or slow scan rates is now available. This equipment has a number of applications in both optical and electron microscopy. Examples of results obtained by linkage to a low light level TV system are given showing that use of the frame store yields image information not otherwise available. Other possibilities of the system are indicated.
The role of specimen and instrumental parameters in STEM‐EDS X‐ray microanalysis of thin foils
Authors: Doig P. , Flewitt P. E. J.
Published: August 2011

The optimization of STEM‐EDS X‐ray microanalysis within thin metal foils can be achieved by control of the instrumental and specimen variables. The contribution of incident electron probe size, accelerating voltage and sampling frequency together with the specimen variable of foil thickness are discussed with reference to typically enountered microstructural features.
Microscopy of sclerotinites in the coal beds of the central part of the Appalachian coal field, U.S.A
Authors: Thompson Carolyn L. , Lyons Paul C. , Finkelman Robert B. , Brown Floyd W. , Hatcher Patrick G.
Published: August 2011

Rodlets from fusain and carbominerite bands or lenses in bituminous coal beds of the central part of the Appalachian coal field in the eastern United States were studied in three dimensions using both light microscopy and scanning‐electron microscopy (SEM). Infrared studies and chemical analyses complemented reflectance measurements. The rodlets are found strati‐graphically from the Pocahontas no. 3 coal bed (Pocahontas Formation, Lower Pennsylvanian or Namurian B & C) to the Washington coal bed (Washington Formation, Lower Permian). The most common rodlets are noncellular, range in diameter from 60 to 440 μm, and, when polished, have the relief and high reflectances typical of the inertinite maceral group. They have characteristic notches, ovoid shapes, distinctive fracture patterns, dense (oxidized) rims, vesicles, cavities and canals, some of which contain minerals (tentatively identified by SEM semiquantitative X‐ray and infrared analyses). Some rodlets show a cellular cast on their longitudinal surfaces. Due to different exposures in variously‐oriented polished sections, this type of rodlet is classified as sclerotinite of the inertinite maceral group. Comparison of the sclerotinites from the central part of the Appalachian coal field with the fusinitized resin rodlets from coal and coal balls of the Illinois basin (Kosanke & Harrison, 1957) and with the ‘sclerotioids’ of the St Rose no. 5 coal of Nova Scotia indicates that they represent the same bodies. All are interpreted to be rodlets of resinous origin belonging to one or more of the genera of medullosan seed ferns of Carboniferous and Permian age. We suggest using the existing terms ‘resino‐sclerotinite’ and ‘fungo‐sclerotinite’ for two distinct maceral varieties of the inertinite maceral group because of their distinguishing properties and their different paleobotanical origins. Two types of cellular rodlets, woody splinters and sclerenchyma strands, are also documented in this study because of their close association with the resino‐sclerotinites of the central part of the Appalachian coal field.
A comparison of optical properties of carbonized sporinite and vitrinite concentrates of coals of the same rank
Authors: Goodarzi F.
Published: August 2011

This paper reports a systematic study of variation of morphological and optical properties of low and medium rank sporinites (carbon = 830 and 87.1% d.a.f.) and vitrinites (carbon = 79.6 and 86.6% d.a.f.) of same coals carbonized over temperatures ranging from 300 to 950°C for 1 h at a heating rate of 5°C/min in an atmosphere of nitrogen. Contrasts are drawn between the molecular structure of carbonized residues of sporinites and vitrinites of the same coal based on variation of morphological and optical properties. For the same carbonization conditions, carbonized residues of sporinites eventually develop better ordered molecular structures than do the carbonized vitrinites, due to the greater level of fluidity of sporinite than vitrinite.
Graphitizability of carbonaceous materials as studied by TEM and X‐ray diffraction
Authors: Oberlin A. , Oberlin M.
Published: August 2011

Progressive graphitization is measured from X‐ray patterns by the probability P for finding a carbon layer pair reaching the graphite order in the solid. TEM studies show that all carbons (whether graphitizing or not) contain small basic structural units less than 10 Å both in thickness and in diameter which acquire, locally, a preferred parallel orientation during the carbonization process, i.e. at the end of tar release. However, tilt and twist boundaries remain between adjacent units which create wrinkled layers. All the materials are porous; from microporous if the orientation is less than 50–100 Å (non‐graphitizing carbons) to macroporous if the preferred orientation is due to coalesced mesophase spheres (graphitizing carbons). For graphitization to occur it is necessary to have small basic structural units distributed in large areas of preferred orientation. Graphitization occurs only after a sudden elimination of the defects situated at the boundaries between two adjacent structural units, i.e. after development of perfect two dimensional order by dewrinkling and stiffening of the carbon layers (turbostratic structure made of large layers). If both Lamax and Lcmax are too small (micropores) graphitization is impossible. There is a continuous range of materials extending between those with the smallest regions possessing the same molecular orientations (non‐graphitizing carbons) and the largest (graphitizing carbons). A nearly continuous increase of P (from P = 0 to P = 0.8) was found by studying a great variety of kerogens, coals and asphalts. For a given sample, the maximum possible value of P depends only on the size of the oriented areas which itself depends only on the elemental composition at the very moment of its production. There is an opposite influence of the hydrogenated functional groups and the cross‐linking functional groups such as oxygen, sulphur and nitrogen.
A new, less toxic polymerization system for the embedding of soft tissues in glycol methacrylate and subsequent preparing of serial sections
Authors: Gerrits P. O. , Smid L.
Published: August 2011

The paper describes a new polymerization system for embedding soft tissues in glycol methacrylate (GMA). The polymerization of GMA is initiated by means of a barbituric acid derivative in combination with chloride ions and dibenzoyl peroxide. The catalyst system contains no aromatic amines which constitutes a toxicological advantage over the commonly employed system of peroxide/aromatic amine. Clear blocks are obtained from which 1–2 μm sections are easy to cut. In combination with an appropriate softener, polyethylene glycol 400, serial sectioning may be practised.
Microtopographical analysis of surface structures in a scanning electron microscope
Authors: Knoesen D. , Kritzinger S.
Published: August 2011

A stereographic‐based method, utilizing both tilt and rotation, is demonstrated, whereby the underlying crystallography of three‐dimensional planes and directions on fine grained and highly deformed metallic specimens can be determined in a scanning electron microscope (SEM). It requires the recording of two micrographs of the same area (or three‐dimensional object) at different orientations, obtained by tilting and rotating the specimen holder. By measuring the projected lengths and angles of lines with respect to the tilt axis on both micrographs, the interangles between directions can be found, and eventually the crystallographic planes and directions can be identified.
Simple procedure for the production of carbon‐polymer support grids for use in X‐ray microanalysis
Authors: Smits H. T. J. , Linders P. W. J. , Stols A. L. H. , Stadhouders A. M.
Published: August 2011

A simple and rapid procedure for the production of low‐cost carbon‐polymer support grids is described. These grids are particularly useful for X‐ray microanalysis of thin specimens because of their low X‐ray background properties. The grids can be prepared for immediate use, which guarantees constant quality.
Precise and reproducible deposition of thin and ultrathin carbon films by flash evaporation of carbon yarn in high vacuum
Authors: Peters Klaus‐Rüdiger
Published: August 2011

A simple, small device and its use for reproducible flash evaporation of carbon yarn in high vacuum are characterized. Using this flash evaporator, carbon films of any thickness up to 20 nm can be deposited without spark generation under minimized photon radiation, and with an accuracy better than ±0·2 nm. The films have less background structure (imaged in phase contrast) than conventionally rod evaporated films and are therefore suitable for many kinds of thin and ultrathin carbon film applications in electron microscopy, e.g. as backing for formvar films or sections, as isolating carbon layers for autoradiography, as ultrathin films (floated from mica) for support of macromolecules to be metal shadowed and as support and cover for negative staining of various specimens by the sandwich technique.
Differential phase contrast in scanning optical microscopy
Authors: Hamilton D. K. , Sheppard C. J. R.
Published: August 2011

High‐quality high‐resolution transmission and reflection images produced using a scanning optical microscope and the split‐detector technique are presented. These images exhibit differential phase contrast, the method avoiding some drawbacks of the usual Nomarski DIC arrangement. Imaging is treated theoretically and compared with the Nomarski method.
An empirical formulation relating boundary lengths to resolution in specimens showing ‘non‐ideally fractal’ dimensions
Authors: Rigaut J. P.
Published: August 2011

The concept of fractal dimension offers an elegant basis for the understanding of changes in boundary lengths due to different resolutions. However, in many cases, the usual log‐log ‘fractal plot’ does not quite fit the observed data, with most biological structures for instance.

An automated boundary following algorithm has been used with an IBAS image analyser on various objects, to evaluate boundary lengths at different magnifications and with a wide range of discrete divider stride lengths on each digitized image. A formula which relates the boundary length to the divider stride length and fits very closely the experimental data has been empirically obtained.

Examples are shown with various object boundaries observed by TV‐camera, macroscopically or with a light microscope. At different magnifications, the same constants are obtained for a given object, notably a theoretical maximum boundary length (Bm) when the divider stride tends towards zero. The formula allows to calculate a variation of the log‐log plot slope which fits very closely the upper convex plots observed when the boundaries do not display an ideally fractal behaviour. Plots derived from the Lineweaver‐Burk and Hill ones (used in enzymology) allow a relatively easy graphic determination of the parameters of the proposed formula, which may offer useful criteria for object shape classification, as demonstrated by a biological example.

Instead of a true (constant) fractal dimension, it seems that, in many situations, exists a ‘continuous fractal dimensional transition’, inside which an apparently linear segment in a given range of resolution of the usual log‐log plot could be explained by asymptotic limits at higher and lower resolutions. The ideal situation with a constant fractal dimension over all resolution ranges could be considered as a limit, achieved when the maximum boundary length (Bm) tends towards infinity.
Stereoscopic representation of complex overlapping elemental maps in electron spectroscopic images
Authors: Arsenault A. L. , Ottensmeyer F. P.
Published: August 2011

A stereoscopic method is described enabling the direct visualization of the spatial relationships of elemental maps generated by electron spectroscopic imaging. The cartilage growth plate in early stages of mineralization was used to illustrate elemental maps of calcium, phosphorus, and sulphur. Stereopsis is achieved by creating a stereo‐pair which in one image of the pair the elemental maps are superpositioned as they occur naturally while in the second image the maps are laterally shifted. In a stereo perspective the maps appear superpositioned in distinct planes.
Improved staining of microfilament bundles in plant cells for high voltage electron microscopy
Authors: Pesacreta T. C. , Parthasarathy M. V.
Published: August 2011

A post‐polymerization en bloc staining method for high voltage electron microscopy is described. Embedded specimens were initially trimmed to an area close to the point of interest. Trimmed blocks were stained in 5% uranyl acetate/75% ethanol solutions for 36 h at 333 K. This procedure allowed specimens to be stained without the necessity of exposing Formvar films to damaging solutions. After such staining, both the contrast and fine detail of structures such as microfilament bundles were superior to that seen in material stained with aqueous solutions of uranyl salts.