bluuebunny/crossref_metadata_2025 huggingface dataset:
The use of pattern recognition for classification of stochastic specïmens by a set of standard specimens
Authors: Vollath D.
Published: August 2011

This paper describes an empirical method for the experimental assigning of specimens to a set of standard specimens. The procedure was developed especially for stochastic scenes. Typical applications of such a procedure are found in material sciences in assigning specimens to a standard series. The assignment is based on feature vectors obtained from analyses of the specimen texture by means of erosion or opening. In cases where the arrangement of particles is characteristic of the specimen, dilation or closing are used to obtain a feature vector. To increase the sensitivity of the method described the use of more than one structuring element is recommended in the analysis. Besides the classification of an unknown specimen the procedure provides information on the separability of the standard specimen and the significance of assignment.
Laser produced plasmas as intense X‐ray sources for microscopy at the Central Laser Facility
Authors: Rumsby P. T.
Published: August 2011

Some of the X‐ray microscopy work being performed at the Central Laser Facility (CLF) is reviewed. The X‐ray sources generated by the high power glass and gas lasers, Vulcan and Sprite, are discussed and compared with other sources. The instrumentation used to perform X‐ray microscopy of laser driven implosions at the CLF is outlined and the results obtained shown. Other experimental work utilizing the intense X‐ray emission properties of laser produced plasmas is reviewed and details of work involving X‐ray diffraction, EXAFS and X‐ray microscopy of biological samples is given.
Synchrotron radiation applied to the microscopical examination of solid state and biological materials in the X‐ray region
Authors: Duke P. J.
Published: August 2011

The paper describes the properties of synchrotron radiation and makes some comparison with other X‐ray sources. The use of synchrotron radiation for various types of X‐ray imaging is discussed with reference to specific examples drawn from work carried out recently at the SERC Daresbury Laboratory.
Developments in contact X‐ray microscopy in biomedical research
Authors: Davies R. Ll. , Flores N. A. , Pye J. K.
Published: August 2011

Contact X‐ray microscopy (microradiography) is a method of studying the microstructure of biological tissue. These techniques have been used to study the historadiological details of human breast tissue and sections of human ear ossicles. X‐ray microscopy can also be used to demonstrate variations in structural densities seen in histological specimens including the detection of microcalcification. A modification of existing apparatus is described which has resulted in improved image‐contrast and detail.

The ability of X‐rays to penetrate relatively thick sections of tissue makes it an ideal method for studying the morphology of biological structures, particularly in calcified tissue. The tissues may be further examined by conventional histology, elemental analysis, etc. The technique has a complementary role to alternative methods of tissue microscopy.
Absorption microanalysis with a scanning soft X‐ray microscope: mapping the distribution of calcium in bone
Authors: Kennedy J. M. , Jacobsen C. , Kirz J. , Rarback H. , Cinotti F. , Thomlinson W. , Rosser R. , Schidlovsky G.
Published: August 2011

This article describes the scanning transmission X‐ray microscope operated at the National Synchroton Light Source. The application of the instrument to elemental analysis is detailed. In particular, qualitative results on the calcium distribution in human skull tissue are presented.
X‐ray microscopy using computerized axial tomography
Authors: Elliott J. C. , Dover S.D.
Published: August 2011

Computerized axial tomography with MoKa X‐radiation has been used to study a 0.8times0.8 mm column of human femoral bone at a resolution of 15 μm. This non‐destructive X‐ray microscopic technique allowed ‘sections' to be made with 25 μm separation and the distribution of linear absorption coefficient in each section to be determined.
Recollections of the early days of SEM in the Cambridge University Engineering Department, 1948–53
Authors: McMullan D.
Published: August 2011

After a brief survey of the early development of the scanning electron microscope in Germany and the U.S.A., the paper describes the work on the subject that was carried out in the Cambridge University Engineering Department, particularly in the years 1948–53. This and later work led directly to the Stereoscan SEM marketed by the Cambridge Instrument Company in 1965. The paper is illustrated with a number of micrographs produced by early scanning electron microscopes.
High resolution electron beam fabrication: a brief review of experimental studies that began at Cambridge University in 1962
Authors: Broers A. N.
Published: August 2011

Electron beam fabrication studies with the scanning electron microscope began at Cambridge University in 1962. Early experiments demonstrated that structures could be made that were smaller than could be made by other methods, but it was not until several years later at IBM Research that useful devices with these smaller dimensions were made. It was even later (1981) before the resolution limits of electron beam lithography with conventional resist were measured. It is now understood that with conventional resists, electron/resist interaction effects limit the minimum structure size to about 10 nm. Newer methods that involve the direct sublimation of materials such as NaCl and LiF allow structures down to about 1 nm to be made.
An X‐ray diffraction analysis of rat tail tendons treated with Cupromeronic Blue
Authors: Meek K. M. , Scott J. E. , Nave C.
Published: August 2011

Cupromeronic Blue was used to stain selectively the proteoglycans in rat tail tendons under ‘critical electrolyte’ conditions. Earlier electron microscopical observations indicated that at least one type of proteoglycan filament is associated with tendon collagen fibrils at the positive staining band ‘d’. To ensure that this was not an artefact caused by specimen preparation or the subsequent positive staining of the collagen fibrils, we have analysed low angle meridional diffraction patterns from stained but not dehydrated, embedded or counterstained tissues. Axial electron density profiles of Cupromeronic Blue‐stained compared with unstained rat tail tendons revealed the axial locations and relative amounts of dye in both mature and young wet specimens.

In mature tendons, the difference electron density profile contained a broad peak centred near residue 180 along the 234‐residue D‐period. This corresponds to the electron‐optical staining band ‘d’. In young tendons a similar distribution of stain was observed although in this case there was evidence of a doublet of peaks, one centred near residue 182 (band ‘d’) and the other near residue 165 (midway between bands d and e1).

The wet proteoglycan‐Cupromeronic Blue complexes distribute over about 30 nm along the collagen fibril axis. Comparison with the images of filaments seen in the electron microscope suggests that the dye complexes collapse significantly on dehydration and embedding.
Plunge‐cooling of tissue blocks: determinants of cooling rates
Authors: Ryan Keith P. , Purse David H.
Published: August 2011

Tissue blocks have been plunged into a liquid coolant and the resultant ice‐crystal damage is discussed. Some blocks have been frozen without apparent ice‐crystal damage; they were not treated with cryo‐protectants but were mounted on streamlined foil supports which separated the specimen from the thermal mass of the support pin. Hydrated gelatin specimens of various sizes have been built around thermocouples and plunged at different speeds. Results show that cooling is relatively slow in specimens mounted on solid metal supports. In specimens mounted on foil supports cooling is related to plunge velocity in 0.25 mm diameter specimens but in 1 mm diameter specimens cooling occurs mainly after coming to rest. In 0.5 mm diameter specimens cooling is related to plunge velocity at low immersion speeds but then it decreases with increasing plunge velocity; this trend should allow monitoring of attempts to improve the efficiency of the plunge‐cooling technique.
Specimen preparation methods for the examination of surfaces and interfaces in the transmission electron microscope
Authors: Newcomb S. B. , Boothroyd C. B. , Stobbs W. M.
Published: August 2011

Various techniques for the preparation of cross‐sectional and plan view TEM specimens of surfaces and interfaces are described. Particular emphasis is given to preparative methods which are both generally applicable and which minimize differential thinning of the materials present on either side of the interface of interest, thereby improving the reliability of the approach.
Electron microscopy of oxide scales modified to resist high‐temperature oxidation
Authors: Chadwick A. T. , Taylor R. I.
Published: August 2011

The high temperature oxidation of metals and alloys is slowed to a useful extent by the presence of certain elements or their oxides in the metal or on its surface. Particles of the reactive element oxide are found in part of the oxide scale and, by an unknown mechanism, retard the diffusion of metal through oxide grain boundaries. Changes in texture are also observed, which might affect geometry of individual grain boundaries. In this study the rotational geometry of grain boundaries in nickel oxide scales has been characterized both in the presence of particles of cerium dioxide and in their absence. Proximity of the relationship of neighbouring grains to a coincidence orientation could have an important influence on diffusion rate. Ways of testing this are compared and a new method for analysing orientation data from electron diffraction is presented, which allows statistically sound conclusions to be drawn. The frequency of occurrence of coincidence orientations in the presence of CeO2 is little greater than random, so one possible mechanism for the ‘reactive element effect’ may be rejected.
Analysis of ceramic materials by electron spectroscopy
Authors: Watts J. F.
Published: August 2011

The basic principles of X‐ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy are described and their relative advantages as analysis methods for ceramic materials are reviewed. Their application in ceramics technology is illustrated by examples taken from the fields of catalysis and mineralogy. In particular, attention is drawn to recent developments of XPS which make it especially attractive for the study of ceramic materials. These include quantitative application of the X‐ray photoelectron diffraction effect to layered, single crystal minerals; the development of higher energy X‐ray sources for XPS, and the attendant study of the Sils–KLL Auger parameter as a probe of molecular and crystalline structure; and the analysis of small (≃250 μm) surface features by XPS.
Stroboscopic testing of LSIs with low voltage scanning electron microscope
Authors: Todokoro H. , Yoneda S. , Yamaguchi K. , Fukuhara S. , Komoda T.
Published: August 2011

A stroboscopic scanning‐ electron microscope (SEM) with 1 keV electrons has been developed to analyse the internal voltage waveforms in very large scale integrated circuits (VLSIs). A field emission gun and a short focal length objective lens are used to obtain a fine probe. Secondary electrons returning up through the objective lens are detected and analysed by a hemispherical retarding field energy analyser. The working distance of the objective lens is 3 mm, and a small stable probe of 0.1 μm in diameter with 1.6 nA in beam current is obtained. The linearity of the potential measurement is fairly good in spite of analysing secondary electrons which pass through the magnetic field. A minimum time resolution of 500 ps as well as a voltage resolution of 50 mV can be measured. This stroboscopic SEM has been successfully applied to evaluation of high speed waveforms in a ECL‐4K bit bipolar RAM.
Fine structure in electron channelling patterns
Authors: Madden M. C. , Hren J. J.
Published: August 2011

Electron channelling patterns contain information from high index planes lying in the first order Laue zone. These lines are prominent in the area immediately surrounding low index poles, and can be indexed using procedures similar to those used for convergent beam electron diffraction. They reflect the lattice symmetry more clearly than the overall channelling pattern, and their exact geometry is sensitive to both lattice parameter and accelerating potential. They should also be sensitive to elastic strain.
Images of arterioles in unfixed tissue obtained by acoustic microscopy
Authors: Neild T. O. , Attal J. , Saurel J. M.
Published: August 2011

A scanning acoustic microscope operating at 600 MHz was used to observe arterioles in a thin sheet of collagenous connective tissue dissected from the submucosa of the guinea‐pig small intestine. The arterioles were clearly defined in images made using transmitted ultrasound, and the acoustic attenuation (α) of the arteriolar wall was estimated to be 120 cm−1. Images made using reflected ultrasound did not show the arterioles clearly.
Towards more precise definition of conditions for satisfactory deep etching
Authors: Haggis G. H.
Published: August 2011

Experiments were carried out to determine whether propane‐jet freezing could be as satisfactory as impact freezing in deep etch work. The material used was the intestinal brush border, previously studied by Heuser and coworkers where the 5–6 nm decoration of actin rootlet filaments, and the fine network of filaments linking these rootlets, provide good criteria by which to judge the quality of the preparation, as regards ice crystal growth and surface contamination. Propane jet freezing was indeed found satisfactory provided appropriate conditions were met (viz thin specimen, fracture near the surface). Variable results were obtained until it was realized that with a Balzers freeze‐etch unit fitted with a rotating specimen table there is a 10–15 min delay (when specimen temperature is reset) between the time the recording thermocouple shows a given temperature to have been obtained and the time the specimen block actually reaches this temperature. Appropriate allowance must be made for this lag to achieve satisfactory deep etch replicas.
Microscopic spectral imaging using a video camera
Authors: Balaban R. S. , Kurtz I. , Cascio H. E. , Smith P. D.
Published: August 2011

A technique is described which permits the simultaneous acquisition of multiple fluorescent emission and/or absorption spectra from discrete regions of a specimen under microscopic observation. The instrument consists of a modified inverted microscope, an optical diffraction grating, a silicon intensified target (SIT) camera, and a digital video image processor. Observation of the zero diffraction order of the grating with the SIT camera permits an optical slice of the specimen to be selected by positioning the region of interest over the entrance slit of the grating housing. To obtain the spectral characteristics of this optical slice, the grating is rotated to impinge the first order diffraction on the camera. The video image of this first order diffraction maintains spatial integrity along the slit's long axis and provides spectral dispersion on the perpendicular axis. Thus, each of the horizontal video lines along the long axis of the slit represents a spectral analysis of the corresponding spatial location within the specimen. The spectral resolution (0.2 nm/channel) of each video line is determined by the resolution of the camera system in conjunction with the resolution of the grating. The image processing system acquires and processes all 500 spectra in 33 ms and permits the accurate localization of the source of each spectrum in the slice. This type of topological spectral analysis permits the determination of both spatial and spectral characteristics of intrinsic or extrinsic chromophores within the specimen. In addition, this technique permits the detection of and the possible correction for photobleaching, light scattering and image plane effects. The application of this technique to the study of single cells is discussed and an example of the technique in determining the fluorescence spectra of acridine orange within the nucleus of an intact mammalian blastocyst is described.
Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy
Authors: Wouters C. H. , Koerten H. K. , Bonnet J. , Daems W. Th. , Ploem J. S.
Published: August 2011

An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi‐illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover‐glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.
Demonstration of pseudorabies virus DNA in the mouse inner ear by an in situ nucleic acid hybridization technique in plastic embedded bony material
Authors: Falser N. , Bandtlow I. , Haus M. , Wolf H.
Published: August 2011

This investigation is concerned with the possibility of identifying viral DNA using the in situ DNA hybridization method in methylmethacrylate‐embedded material. As an experimental model we chose viral labyrinthitis produced by intranasal infection of the mouse with pseudorabies virus. Fixation and embedding methods specially adapted to this procedure and bony histology preparation technique (specimens by grinding or micromilling) made it possible to identify viral DNA directly morphologically and virologically in the inner ear. Quantitative microphotometric analyses of trans‐sagittal sections of the entire skull after in situ DNA hybridization are presented and discussed here as an explicit method of investigating the path of distribution of viral DNA in the brain and the inner ear.
Surface exchange area of the porcine placenta: morphometry of anisotropic interdigitating microvilli
Authors: Hasselager Erik
Published: August 2011

The pig has a diffuse epitheliochorial placenta. The foeto‐maternal surface area (SV(F), SV(M)) is enlarged at three levels: level I: macroscopic folds of uterine mucosa covered by foetal membranes; level II: light microscopic folds of uterine epithelium covered by trophoblastic epithelium; level III: ultrastructural formation of both maternal and foetal microvilli in an interdigitating pattern.

This study presents a morphometric method, using intersection counting, to estimate the surface area of these interdigitating, highly oriented microvilli at level III, i.e. the ‘true’ surface exchange area. The method is applied to different stages in pregnancy.

The epithelial surfaces are enlarged by a factor 8–11 due to the formation of microvilli. This factor varies during pregnancy, being lower early in gestation and before delivery, and for the first time it is shown to be significantly different for mother and foetus. At level III the foetal surface of placenta is larger than the maternal indicating that the two opposing surfaces are not congruent at the ultrastructural level and that membrane‐linked placental transport may be different in mother and foetus. A three‐compartment model is suggested for placental exchange with maternal, intermicrovillous and foetal compartments.
The osmotic effect of glutaraldehyde‐based fixatives on plant storage tissue
Authors: Coetzee J. , van der Merwe C. F.
Published: August 2011

Mass changes were used to monitor water gain or loss during fixation of plant tissue. Mannitol was used as reference osmoticum. Glutaraldehyde in Na‐Na‐phosphate, Na‐cacodylate or PIPES buffer exerts an osmotic effect on plant tissue. This effect is in addition to the osmotic effect of the buffer. The effective osmolarity of the fixative depends on the glutaraldehyde concentration, the buffer type, the buffer concentration and the tonicity of the tissue. Relationships among these parameters are non‐linear. Graphs are given to find the effective osmotic pressure of a buffered or unbuffered fixative containing 2% glutaraldehyde, for use with plant storage tissue with tonicity between 350 and 850 mOsm.
Pulsed laser atom probe analysis of semiconductor materials
Authors: Cerezo A. , Grovenor C. R. M. , Smith G. D. W.
Published: August 2011

As the size of semiconductor devices is reduced the active volumes of material in each device is also decreased. Under these circumstances it becomes more important to understand the microchemistry of semiconducting materials, as small fluctuations in composition can dramatically affect both the operation of the devices, and of the contacts to semiconductors. Atom probe microanalysis has been shown to be able to analyse the microchemistry of metallic materials with plane‐by‐plane resolution, and by using a pulsed laser to replace the more conventional voltage pulses the analysis of semiconducting and insulating materials becomes possible. The pulsed laser atom probe has been shown to give very accurate chemical analysis of the stoichiometry of extremely small volumes of III‐V compound semiconductors, and the composition of the interfacial layer between silicon dioxide and silicon has been identified as SiO of thickness about 0.3 nm. It has been shown to be possible to prepare specimens for analysis from thin films of semiconductors, thus allowing the microanalysis of a wide range of materials that are deposited in thin film form.
A method for observing intracellular structures of free cells by scanning electron microscopy
Authors: Fukudome Hatsuko , Tanaka Keiichi
Published: August 2011

A new method is described for scanning electron microscopic observation of cultured free cells using chitosan embedding. Intracellular structures of free cells can be clearly observed using a combination of the chitosan embedding and the Osmium–DMSO–Osmium (O–D–O) method. Chitosan, a polysaccharide, is not affected by the osmium maceration procedure central to the O–D–O method, which destroys other possible embedding media such as gelatin, agar, egg albumin and fibrin. Chitosan cracks to yield a fracture face which is featureless at low magnifications, and causes no recognizable artefacts.
Electron microscopy of unstained, freeze‐dried macromolecular assemblies
Authors: Rachel R. , Jakubowski U. , Baumeister W.
Published: August 2011

A rapid cooling/cryotransfer system was designed to achieve a high reproducibility in vitrifying thin water films containing biological specimens. In order to improve the contrast these unstained specimens were deliberately freeze‐dried in situ in the electron microscope. The preservation of the structure obtained by freeze‐drying at 180 K and subsequent microscopy at 90 K is very encouraging as has been shown with two types of test specimen. Tubular photosynthetic membranes were used to examine the effects of a variety of freeze‐drying conditions on structural preservation, as judged by flattening of the tubes. A two‐dimensional protein crystal was used to evaluate problems of low‐dose microscopy of unstained, freeze‐dried proteins, e.g. optical density of films, motif detection by cross‐correlation and transferring the accurate molecular positions from medium‐dose to corresponding low‐dose micrographs. The radiation sensitivity of the unstained, freeze‐dried protein crystal was investigated by a dose series covering a wide range.
Electronic image formation in high‐voltage TEM by sequential pixel acquisition
Authors: Fotino Mircea
Published: August 2011

We describe a complete hybrid imaging system for the electronic detection and manipulation of high‐voltage TEM images. The system includes a recently incorporated two‐dimensional beam deflector for dissecting the image into pixels by displacing it in a raster in front of an aperture leading to an electron detector. The characteristics of images thus formed by sequential pixel acquisition and displayed on a CRT are discussed and illustrated. Absorption or thickness profiles are also obtained by displaying the scanned pixel intensity in Y‐modulation. Similarly, the displacement of an entire diffraction pattern allows the intensities of individual diffraction spots to be quantitated directly over a wide dynamic range. The direct splitting of the image into electronically controlled pixels can be applied to local mass measurements and to energy‐loss analysis by means of a simple magnetic prism that can also provide energy filtering in lieu of a more elaborate electron‐optical, image‐preserving system.
Crystallization and vitrification in aqueous systems
Authors: Angell C. A. , Choi Y.
Published: August 2011

Some key ideas and experimental findings concerning the probability that crystallization of a liquid or its binary solutions will occur at moderate cooling rates are discussed. The use of cryoprotectants and of pressure to favourably influence these probabilities is rationalized, and some of the newer findings on small sample supercooling phenomena are reviewed.
A procedure for obtaining complementary replicas of ultra‐rapidly frozen sandwiched samples
Authors: Fetter Richard D. , Costello M. Joseph
Published: August 2011

Complementary replicas of samples prepared for electron microscopy by the freeze‐fracture/etch technique are extremely valuable in the interpretation of the exposed surfaces, the nature and location of the membrane fracture plane, and as an aid in the recognition of the potential artefacts of this technique. This paper describes a procedure for the preparation of complementary replicas of thin samples sandwiched between copper foil strips and frozen ultra‐rapidly in the absence of chemical pretreatments. In this procedure, the copper foil support bearing the replica is floated on the surface of a chromic acid solution, resulting in the controlled dissolution of the copper metal. The replica which remains at the surface of the chromic acid is then stabilized against fragmentation during subsequent cleaning and rinsing steps by placing a 50 mesh gold grid on top of the replica. To minimize agitation of the replica/grid, all cleaning steps are performed in a single depression plate well. The clean replica/grid is picked up from below on a thin Formvar film, dried, and then separated from the extra film. Careful placement of the gold grid on the replicas and low magnification electron micrograph montages of the complementary grids facilitate the location of complementary regions and simplify examination of complementary specimen areas at higher magnification.
Properties of frozen sections relevant to quantitative microanalysis
Authors: Hall T. A.
Published: August 2011

Difficulties in the quantitative X‐ray microanalysis of frozen sections may conceivably arise from ice‐crystal damage and from electron‐beam damage.

X‐ray peak‐to‐continuum ratios are commonly taken as a quantitative index of elemental concentrations. But recent reports suggest that in dehydrated frozen sections such ratios vary greatly with the scale of ice‐crystal formation existing prior to sublimation. The experiments in these reports are re‐interpreted here; it is argued that peak intensities may be affected by ice‐crystal scale but that ratios of peak to continuum should not be affected after corrections for exogenous continuum.

The accuracy of the peak‐to‐continuum method is affected by beam‐induced loss of mass from microvolumes during analysis. Mass loss can be reduced or slowed by a cold‐stage. For example, the radiation sensitivity for loss of chlorine from PVC is reduced by a factor of 1000 or more with reduction of temperature from 300 to 100 K. For sections of soft tissue the effectiveness of cooling is not nearly so striking but at 100 K, analyses of 1 μm frozen‐hydrated sections by the continuum method, with spatial resolution of the order of 1 μm, can be completed before substantial mass loss occurs. However, analysis of frozen‐hydrated sections by the continuum method at much higher resolution, say 100 nm resolution in 100 nm sections, is precluded by mass loss.

Measurements of local mass can be achieved with much lower dose by observation and calibration of the electron transmission or backscattering. But even with these methods, several problems remain in achieving quantitative X‐ray analysis at very high resolution.
The preparation and X‐ray microanalysis of bulk frozen hydrated vacuolate plant tissue
Authors: Echlin Patrick , Taylor S. Edith
Published: August 2011

A series of modifications have been devised which allow the peak to background ratio X‐ray analytical method to be used more effectively to measure elemental concentrations in large vacuolate plant cells. Planar, frozen‐hydrated fracture faces of bulk plant tissue are coated with a thin film of evaporated chromium, which prevents surface charging. Provided the film is sufficiently thin, c. 5–10 nm, there is no attenuation of the electron beam and only a small absorption of soft X‐rays. The chromium makes a small but measurable contribution to the spectral background and suitable corrections may be made to the quantitative results. An improved back‐scattered imaging system is described, which helps to overcome the problem of spurious X‐ray signals from rough surfaces. The microscope column has been modified to permit a continuous readout of beam current, sensu stricta, during X‐ray microanalysis and to allow rapid exchange of the electron gun assembly during low temperature operation. Calculations are given relating the size of the X‐ray interactive volume to electron penetration and X‐ray emission in both frozen hydrated and frozen dried cells. The problems of X‐ray microanalysis are discussed in relation to the highly vacuolate cells found in most mature plant tissues and an example given of the distribution of four major cations in tobacco leaves.
Cryo‐electron microscopy and three‐dimensional reconstruction of actin filaments
Authors: Trinick J. , Cooper T. , Seymour J. , Egelman E. H.
Published: August 2011

Actin filaments have been examined by electron microscopy whilst in a frozen‐hydrated state. Filaments embedded in a vitreous water layer are basically similar to those prepared by negative staining and show characteristic helical substructure, where the pitches of the helices are about 70 nm and 6 nm. Variability in spacing between long pitch helix cross‐over points has been observed, which is consistent with intrinsic angular disorder between successive filament subunits. Fourier transforms of the most ordered filaments show four strong layer lines that index as the first, fifth, sixth and seventh orders of a 35 nm repeat. A three‐dimensional helical reconstruction, calculated to a resolution of about 4 nm, shows the individual subunits to be orientated with their long axes roughly perpendicular to the filament axis. Each subunit is somewhat curved and is resolved into two domains. Most connections between successive subunits appear to be made close to the filament axis. We also report on the performance of the specimen holder (Philips PW 5699) used in this work.
Microtubule structure studied by quick freezing: Cryo‐electron microscopy and freeze fracture
Authors: Mandelkow Eva‐Maria , Rapp Roberto , Mandelkow Eckhard
Published: August 2011

Microtubules have been quickly frozen and examined by electron microscopy using several techniques: (1) freezing of a thin layer of solution by plunging into cryogen, followed by cryo‐electron microscopy of the unstained vitrified samples; (2) freezing by the propane‐jet method, followed by freeze fracturing and metal replication.

The unstained frozen‐hydrated microtubules show a structure in agreement with X‐ray diffraction data; they differ from negatively stained particles mainly by the better preservation of cylindrical shape. Secondly, they reveal a supertwist of the profilaments that is not detected reliably by other methods. This allows a determination of the number of protofilaments and the polarity. The structural resolution of unstained microtubules is similar to that of stained ones (about 2–3 nm); it is limited by low contrast and lack of crystalline order.

Propane‐jet or cryo‐block freezing followed by freeze fracturing reveals the structures of the inner and outer surfaces of the microtubule wall at a resolution of 4 nm or better. The outside is dominated by the longitudinal protofilaments whereas on the inside one observes tilted cross‐striations. Although the freezing temperatures of the two methods are different (liquid nitrogen or helium) they yield similar results for the case of thin layers of protein solution.
Electron beam radiation damage to organic inclusions in vitreous, cubic, and hexagonal ice
Authors: Talmon Yeshayahu , Adrian Marc , Dubochet Jacques
Published: August 2011

Frozen hydrated specimens of various latex spheres were used as well‐defined systems for the study of electron beam radiation damage to organic inclusions in vitreous, cubic and hexagonal ice. We found that radiolysis of organic material is modified by the presence of ice and that radiolysis in vitreous ice is different from that in crystalline ice. The pattern of damage depends also on the nature of the irradiated polymer, e.g., damage to poly(vinylchloride) is quite different from damage to polyacrylates, although in both polymers the main radiolytic process is chain scission. Some polymers such as polyacrylates were found to be much more stable in vitreous ice than in crystalline ice. The experimental results indicate that free radicals formed at the ice–organic matter interface play an important role in the radiolysis process which affects both the ice and embedded organic particles. Ice may play also a physical role in the process by limiting the diffusion of free radicals away from the interface. Although net mass loss is not much affected by ice, massive structural changes including repolymerization take place in its presence.
HREM image contrast from supported small metal particles
Authors: Gai P. L. , Goringe M. J. , Barry J. C.
Published: August 2011

HREM, image calculations and small probe diffraction/AEM have been used to characterize structure and contrast of supported small metal particles of ≤5 nm diameter. Such small particles are thought to be active species in industrial applications such as in heterogeneous catalysis where, in general, the particles employed as catalysts are supported. Image calculations (HREM and diffraction contrast) carried out at both 200 keV and 400 keV at various defoci and support thicknesses have shown that in HREM, particle images are obscured by the support contrast with the loss of edge definition and particles appear to be smaller than they actually are. The particle visibility is better at 400 keV. The calculations have also indicated that particle shape varies as a function of support thickness and defocus. The results have clear implications for identification and interpretation of surface structure of the supported small particles accurately by HREM if not performed under controlled conditions and for determining their size and shape.
Rationale for the application of thin, continuous metal films in high magnification electron microscopy
Authors: Peters Klaus‐Ruediger
Published: August 2011

Various metal films of different thicknesses were deposited on to a particle test specimen and their effects on topographic contrast generation and specimen preservation were determined. Tobacco mosaic virus adsorbed on to thin carbon supports or silicon chips was imaged in TEM or high resolution SE‐I SEM at a magnification of 350,000×. Tantalum films of 1–2 nm (average mass) thickness produced best contrasts and prevented volume loss of the particles from electron beam damage. Excessively thick films of 5–10 nm thickness blanketed fine structures and caused severe volume losses. Discontinuous 2 nm thick films of gold or platinum decorated the surfaces, caused a loss in topographic contrasts and induced very high volume losses. Thin continuous metal films were necessary to generate high topographic contrast and to prevent volume loss from beam damage by providing sufficient mechanical stability for small topographic features and increased thermal conductivity of the specimen surface.
A method for TEM visualization of the extracellular matrix three‐dimensional organization in tissues
Authors: Cidadāo António J. , David‐Ferreira José F.
Published: August 2011

A method for obtaining high‐resolution three‐dimensional images of the extracellular matrix organization in tissues is described. It consists of TEM observation of rotary‐shadowed platinum–carbon replicas obtained from critical‐point dried resinless sections of polyethylene glycol‐embedded specimens. The procedure is simple and rapid, with high rates of sample recovery. An example of its application to EM immunocytochemistry (fibronectin localization) is presented. The utilization of the method to demonstrate cell‐extracellular matrix relationships, and its limitations in the study of cells are discussed.
Locating water‐soluble vital stains in plant tissues by freeze‐substitution and resin‐embedding
Authors: Canny M. J. , McCully M. E.
Published: August 2011

Vital stains, moving in the transpiration stream in leaf apoplast, may be kept in place through freezing, freeze‐substitution, embedding and sectioning, to reveal their position in the living plant. This technique has been used to study the details of movement of water out of the veins of leaves, and has wide application in histochemistry with water‐labile dyes, and for following dye movements in protoplasm. Patterns of water movement in the leaf of Zea mays L. are presented as an example.
Extraction of proteins and membrane lipids during low temperature embedding of biological material for electron microscopy
Authors: Weibull C. , Christiansson A.
Published: August 2011

The extraction of proteins and membrane lipids from biological materials during embedding procedures for electron microscopy carried out at temperatures down to 223 K was studied. Glutaraldehyde‐fixed cells of Acholeplasma laidlawii mainly served as test material. More than 99% of the protein and 88% of the lipid of these cells were retained after dehydration with ethanol or acetone between 277 and 223 K and infiltration with methacrylate at 223 K. When methanol was used for dehydration, only 54% of the lipid was retained. The amount of extracted lipid was essentially independent of the ratio between volume of extraction liquid and amount of material subjected to extraction.

The cytoplasmic membrane of sectioned Acholeplasma‐cclls dehydrated and infiltrated as described above appeared more diffuse than that of cells fixed with glutaraldehyde and osmium tetroxide in epoxy resin at room temperature.

Glutaraldehyde‐fixed erythrocyte ghosts retained 85% of their phospholipid content when dehydrated with ethanol between 277 and 223 K and infiltrated with methacrylate at 223 K. Spinach chloroplasts and thylakoid vesicles retained 61% and 35%, respectively, of their cholorophyll content.
A stereological analysis of the effect of buffer osmolarity on a salt secreting epithelium, under variable physiological conditions
Authors: Cowan F. B. M.
Published: August 2011

A stereological analysis of salt gland epithelium showed differences in the size of the intracellular space and the volume fraction of some cellular organelles, according to the salinity in which the animals were kept. These changes were similar to earlier non‐quantitative work on several salt secreting epithelia in various states of activity.

However, alteration of one variable in the processing technique, osmolarity of the fixative buffer, was shown to produce the same stereological result in salt glands held at constant activity. Most experimental manipulations used in previous experiments to alter glandular activity, e.g. salt loading, altered perfusate, or inclusion of pump inhibitors, probably also alter intracellular ion contents. It is therefore difficult to decide whether changes in lateral intercellular space geometry represent physiological mechanisms in vivo or differences in osmotic behaviour during processing for electron microscopy.
Consistent and efficient delineation of reference spaces for light microscopical stereology using a laser microbeam system
Authors: Hunziker Ernst B. , Cruz‐Orive Luis‐M.
Published: August 2011

A serious problem in stereology is to ensure a consistent definition of reference spaces at different levels of magnification whenever the boundaries of such reference spaces are either fuzzy or non‐existent, and hence they have to be defined artificially. (It is well known that inconsistent definitions of the reference space leads to unknown amounts of bias in stereological results.)

In this paper a new application is found for the laser microbeam system used in microdissection, whereby the required boundaries can be easily and neatly traced (in fact, cut) directly onto uncovered sections for light microscopy. The dangers of bias inherent from inconsistencies of definition are thereby eliminated completely, and alternative, very expensive procedures requiring direct marking of paper prints with a pen are no longer necessary.
Preparation of serial sections in dry snow specimens
Authors: Perla R. , Dozier J. , Davis R. E.
Published: August 2011

Pore space in a dry snow specimen is filled with a water‐insoluble liquid which is frozen solid. The specimen is microtomed, polished with carbon dust, and micrographed through a photomicroscope mounted directly over the microtome. Serial sections are prepared and micrographed at the rate of about ten per hour. The micrographs are automatically digitized, and converted to binary images for microstructural analysis.
Comparison of the Diffraction Contrast and the Slice Method for Image Computation
Authors: Howie A. , Hutchison J. L.
Published: August 2011

The relation between the diffraction contrast theory and the slice method for image contrast calculations is discussed with specific reference to small voids, twin boundary images and weak beam images of stacking faults. The difference between the deformable ion and the rigid ion model for the scattering potential is explored.
Intensity analysis of boron and nitrogen K‐edge spectra for hexagonal boron nitride by EELS
Authors: Saito S. , Higeta K. , Ichinokawa T.
Published: August 2011

Boron and nitrogen K‐edge spectra for hexagonal boron nitride (h‐BN) were measured by electron energy loss spectroscopy (EELS) at orientations of a momentum transfer q parallel and perpendicular to the c‐axis in order to investigate anisotropic unoccupied states π* and σ*, separately. Differences between intensity profiles for BK and NK spectra are attributed to different transition probabilities at BK and NK edges from Is states to the π* and σ* states. The intensity of the first peak at q∥c in the BK spectrum, which is ascribed to the transition from B‐1s to the Q—2 state in the π* band, is 10 times as large as that in the NK spectrum. Quantitative calculation of transition probabilities for BK and NK spectra at q∥c shows that the contribution of B‐2pz orbital to the Q—2 state is 1–7 times as large as that of N‐2pz. This value suggests that the enhanced peak in the BK spectrum is explained by means of single electron excitations, disregarding the electron‐electron interactions for the exciton reported by several workers. The contributions of 2px and 2py orbitals of B and N atoms to σ* states are also estimated by intensity analysis of BK and NK spectra at q⊥c.
Measurement of translation component at ordered interfaces by weak‐beam α‐fringe electron microscopy
Authors: Miyazawa Kun‐ichi , Ishida Yoichi
Published: August 2011

Lattice translations at a stacking fault and a twin boundary were measured from fringe shifts at a set of common weak‐beam diffraction conditions (weak‐beam α‐fringe imaging) in stainless steel SUS316. It is suggested that the present technique is applicable in determining the translation component between neighbouring crystals with coincidence related boundaries.
High resolution electron microscopy of grain boundaries in Nb3Ge‐films
Authors: Nissen H.‐U. , Kitano Y. , Wessicken R.
Published: August 2011

Nb3Ge‐films synthesized on Mo‐substrates by the co‐evaporation method show a characteristic fabric with a quasi‐twelvefold symmetry around the [001]‐axis of the A15‐type cubic structure. Among approximate +30°‐ and ±60°‐rotation boundaries, grain boundaries having rotation angles between 28° and 29° are observed most frequently in the Nb3Ge‐films. Using the high resolution electron micrographs, a Σ17‐boundary having an ideal rotation angle of 28.07° is analysed and plausible boundary models on the atomic level are proposed.
Some aspects of liquid metal ion sources
Authors: Mair G. L. R. , Mulvey T.
Published: August 2011

Liquid metal ion sources (LMIS), in view of their very high brightness, have opened new horizons in the field of ion beam technology and microscopy. In this paper some basic ion‐optical problems of LMIS‐based systems are discussed, and also some of the fundamental mechanisms concerning the operation of LMIS. While a type of field‐evaporation is a strong candidate for the main ion emission mechanism, a space‐charge based theoretical treatment successfully predicts the current‐voltage curves of LMIS of low liquid flow impedance. These conclusions are based on the most detailed measurements to date of the ‘peak energy deficit' of the ion energy distribution, and on the comparison with theory of the current‐voltage curves of a variety of LMIS. Finally, from data on gallium LMIS it is shown that an optimum operating current exists around 2 μA.
Structure and composition analysis of silicon aluminium oxynitride polytypes by combined use of structure imaging and microanalysis
Authors: Bando Y. , Mitomo M. , Kitami Y. , Izumi F.
Published: August 2011

The relationship between the chemical composition and the crystal structure of the 12H and the 15R polytypes of silicon aluminium oxynitride (sialon) is studied by combined techniques of crystal structure imaging and spectroscopic microanalysis using a new 400 kV high‐resolution analytical electron microscope. The chemical compositions of 12H and 15R are determined to be SiAl5O2N5 and SiAl4O2N4, respectively, from quantitative analysis with energy dispersive X‐ray spectroscopy and electron energy loss spectroscopy. The space groups are determined uniquely to be non‐centrosymmetric P63mc and R3m for the 12H and the 15R, respectively, by observing symmetries appearing in both convergent beam electron diffraction patterns and structure images. The structure images observed are interpreted on the basis of calculated images for proposed structure models, in which the structures consist of stacking sequences of the octahedral and the tetrahedral layers. The structure models are confirmed by calculation of the X‐ray diffraction pattern.