bluuebunny/crossref_metadata_2025 huggingface dataset:
A microscope diffusion chamber for the determination of the equilibrium and non‐equilibrium osmotic response of individual cells
Authors: McGrath J. J.
Published: August 2011

A microscope diffusion chamber has been developed which allows direct observation of the dynamic osmotic response of individual cells in micro‐volume suspensions. Continuous observation of stationary cells is possible including short experimental times while the extracellular chemical composition is changed. Multiple changes of solute type or concentration are easily imposed upon a single sample volume. Response times are a function of chamber configuration but response times as low as 1–10 s are possible with negligible solute concentration gradients within the sample region. The chamber is simple and economical to construct and use. It is the size of an ordinary glass microslide and it can be adapted easily to any common laboratory microscope. All standard optical techniques may be used with the chamber. Construction details and operating characteristics including important limitations are discussed. Example photomicrographs and osmotic data are included.
A simple method for obtaining and analysing the absorption spectra of microscopic specimens
Authors: Jennings J. B. , Fletcher C. R.
Published: August 2011

A method is described for obtaining and analysing the absorption spectra of microscopic specimens, using readily available laboratory equipment. Spectra obtained from the primary image with a hand spectroscope are recorded photographically and the negative images subsequently analysed, in terms of optical density relative to a standard spectrum, using a gel scanner densitometer and chart recorder. The system has been used to recognize and characterize the haemoglobin of a symbiotic flatworm, absorption peaks being resolved to within 5.0 nm. Separation of the recording and analytical stages confers flexibility since records can be made, if desired, in the field or by co‐workers elsewhere and analysed later in the base laboratory.
Orthogonal triplet probes: an efficient method for unbiased estimation of length and surface of objects with unknown orientation in space
Authors: Mattfeldt T. , Möbius H.‐J. , Mall G.
Published: August 2011

If the axis of anisotropy of oriented, tubular or lamellar objects is unknown, the unbiased stereological estimation of length density and surface density (Lv and Sv) requires counts on sections with isotropic uniform random (IUR) orientation. It is shown theoretically that in homogeneous, anisotropic specimens the precision of Lv and Sv estimation is considerably augmented if IUR‐oriented sets of three mutually perpendicular sections (orthogonal triplet probes=ortrips) are used instead of three directionally independent IUR sections. The mechanism of variance reduction results from a negative covariance between sections within ‘ortrips’ and corresponds to the antithetic variate principle of Monte Carlo work. Heterogeneity decreases the efficiency of the ortrip method, but this effect can often be counteracted by systematic sampling of ortrips within specimens. Practical estimation of length and surface area of the highly anisotropic, tubular myocardial capillaries per tissue volume in the left ventricles of eight normal, adult, male perfusion‐fixed Wistar rats provided estimates of excellent precision with CEs of 3.3% (Lv) and 2.1% (Sv) of the group mean. The method will hopefully allow stereologists dealing with arbitrary anisotropic structures to apply the same simple, efficient, and unbiased sampling designs that have long been used in the study of liver, lung, and kidney tissue.
A method for preparing quick‐frozen, freeze‐substituted cells for transmission electron microscopy and immunocytochemistry
Authors: Nagele R. G. , Kosciuk M. C. , Wang S. M. , Spero D. A. , Lee H.
Published: August 2011

A quick‐freeze, freeze‐substitution method is described which employs glutaraldehyde as well as osmium tetroxide (OsO4) in a ‘double‐fixation’ protocol comparable to that used for conventional transmission electron microscopy. Cultured cells are quick‐frozen in Freon 22 and freeze‐substituted in an ethanolic solution of glutaraldehyde. Specimens destined for TEM are postfixed in OsO4 in acetone, embedded in Epon‐Araldite, and sectioned. This method yielded ultrastructural preservation which was comparable to that obtained from methods employing OsO4 alone as a freeze‐substitution fixative. However, if glutaraldehyde is used alone as a freeze‐substitution fixative, specimens can be processed for immunocytochemistry without additional treatment with permeabilizing agents.
Annealing of amorphous layer in S+ implanted GaAs by electron beam inside the electron microscope
Authors: Rai A. K. , Bhattacharya R. S.
Published: August 2011

Annealing behaviour of amorphous layer in S+ implanted GaAs is studied by employing transmission electron microscopy technique. The amorphous layer is annealed inside the electron microscope by withdrawing the condenser aperture. The transformation of amorphous layer into polycrystalline layer is found to occur in the near surface region. Formation of twins is observed near the interface of amorphous crystalline region. Coarsening of grains occur on further annealing. The findings of the present experiment is compared with the explosive recrystallization behaviour of amorphous Si under laser and electron irradiation (Narayan et al., 1983). It has been concluded that both solid phase and liquid phase epitaxial regrowth of the amorphous GaAs layer have taken place upon electron irradiation.
The problem of ambiguous profiles of microvilli between apposed cell surfaces: a stereological solution
Authors: Mayhew T. M.
Published: August 2011

When interdigitating microvilli projecting from apposed cell surfaces are sectioned randomly, it becomes impossible to specify to which of the two parent surfaces an individual microvillous profile belongs. Because of this image ambiguity, direct estimation of the ratio of microvillous surface areas by intersection counting cannot be performed. A stereological solution to this problem is proposed. It relies on counts of the numbers of bases of microvilli identified on each reference (parent) surface.
Some limitations of the use of tannic acid as a marker of damaged skeletal muscle fibres
Authors: Cottell David C. , Hooper A. C. B.
Published: August 2011

Mouse biceps brachii muscles containing single transverse incisions were treated with low molecular weight tannic acid. In the absence of fibre degeneration, intracellular spread of tannic acid was confined to a short distance along the fibres from the damaged portions of the cell membranes. Beyond this, damaged and undamaged fibres could not be distinguished from one another. Tannic acid is not a reliable marker of localized trauma to skeletal muscle fibres.
Vitrification of pure liquid water
Authors: Mayer Erwin
Published: August 2011

The methods for vitrifying pure liquid water are reviewed: jet‐freezing of water/oil emulsions, vitrification of thin water layers on an electron‐microscope specimen grid, high‐pressure jet‐freezing and rapid cooling of aerosol droplets on a cryoplate. The first three need a liquid cryomedium for heat transfer in contrast to the last one. Small sample size in at least one dimension, with a diameter in the μm range or even less, is essential for vitrification. Vitrified liquid water has been characterized by electron and X‐ray diffraction, infrared spectroscopy and by differential thermal analysis warm‐up curves. These properties are compared with those of amorphous solid water prepared by deposition of the vapour.
Low temperature embedding with Lowicryl resins: two new formulations and some applications
Authors: Carlemalm E. , Villiger W. , Hobot J. A. , Acetarin J.‐D. , Kellenberger E.
Published: August 2011

Lowicryl K4M and HM20 are methacrylate/acrylate based low temperature embedding resins for biological material which can be used in conjunction with either the progressive lowering of temperature (PLT) technique or with freeze‐substitution. K4M and HM20 are applicable over a very extended temperature range, approximately 220 K to 340 K. With two new resins, K11M and HM23, one can reach even lower temperatures, c. 200 K. Freeze‐substitution combined with low temperature embedding allows for very mild or no chemical fixation which seems to increase the sensitivity of immunocytochemical localization of antigens on sections.
Cryo‐electron microscopy of helical particles TMV and T4 polyheads
Authors: Lepault J.
Published: August 2011

Vitrified Tobacco Mosaic Virus (TMV) and T4 polyhead suspensions have been studied by cryo‐electron microscopy. The order of TMV particles is preserved to distances better than 0.3 nm when vitrified. In spite of the high beam sensitivity of unstained biological material, the resolution of images extends to 1.15 nm. Radial density distributions calculated from images of TMV and the helical aggregate of TMV protein (TMVP) show that the RNA is likely visualized in the virus. However, due to the imaging conditions of unstained vitrified specimens, the position of the RNA is erroneous. While TMV and TMVP do not show any departure from cylindrical symmetry, T4 polyheads are sensitive to the thickness of the ice layer in which they are embedded. To avoid flattening, T4 polyheads have to be embedded in an ice layer thicker than their diameter.
Light microscopy of ceramics
Authors: Roberts E. W. , Robinson P. C.
Published: August 2011

Techniques in light microscopy of ceramics are described. The information provided by transmitted light examination of thin sections is discussed with particular emphasis on polarization microscopy. The production of polarization colours and the use of retardation plates are briefly explained with reference to their use in the study of preferred orientation in alumina ceramics. The discussion relating to incident light examination of polished sections is mainly concerned with methods of disclosing microstructural details by various methods including polarization microscopy, interference contrast and etching. The final part of the discussion deals with methods of studying sub‐surface detail in polished sections of transparent ceramics, including the use of incident dark‐field and oil immersion objectives.
The importance of microscopy in studying the wear behaviour of ceramic surfaces
Authors: Czernuszka J. T. , Page T. F.
Published: August 2011

This paper demonstrates how careful microscopy of worn ceramic surfaces can be used to provide information on the mechanisms of material removal. This information is necessary as a critical complement to wear‐rate data obtainable from simple wear tests alone (e.g. lapping with diamond grits). Scanning electron microscopy has been extensively used to investigate the changing appearance of worn surfaces as plasticity and fracture processes compete as materials removal and redistribution mechanisms. Examples of the use of secondary electron imaging at different surface tilts, back‐scattered electron imaging and stereo imaging are shown. Further, transmission electron microscopy of samples specially prepared to contain the worn surface layer can reveal the presence of phase changes accompanying wear. Furthermore, observations have been made of instances whereby brittle fracture has unexpectedly occurred as a result of repeated plastic deformation of surfaces at low contact severities.

Some conclusions are drawn regarding the influence of specimen microstructure, abrasive grit size and environment of the wear of glass‐bonded (debased) alumina and titania materials.
Transmission electron microscopy of zirconia ceramics
Authors: Butler E. P.
Published: August 2011

The relationship between microstructure and the mechanical and electrical properties is outlined for the main types of zirconia ceramic. Microstructural and microchemical characterization of these materials by TEM at 1000 kV and 100 kV accelerating potentials is demonstrated by reference to selected examples representing fully and partially stabilized zirconia and zirconia‐toughened alumina. Grain boundaries and grain boundary phases appear to be particularly important in determining the way in which zirconia ceramic microstructures evolve during sintering and/or ageing.
EXELFS of alumina, ab initio calculations and the measurement of charge transfer
Authors: Bourdillon A. J. , Tebby G. P.
Published: August 2011

EXELFS were measured for graphite, α‐alumina and a hydrated electrodeposited alumina foil. A theoretical comparison is made between EXELFS and EXAFS, and phase shifts and scattering factors calculated ab initio using curved waves. Good least squares fits to the graphite data demonstrate the applicability of calculated values to light elements in both EXAFS and EXELFS. The ab initio calculations for α‐alumina include ionic effects and a new method was devised for measuring charge transfer. Fourier transforms of the oxygen K‐edge EXELFS showed that the amorphous film, which becomes stable under the electron beam after hydration, but before crystallization, contained at the stage examined a ratio of 50% [AlO6]/[AlO4] cell units.
Microchannel plate detector for low voltage scanning electron microscopes
Authors: Russell P. E. , Mancuso J. F.
Published: August 2011

A microchannel plate detector has advantages over conventional Everhart‐Thornley detectors for low voltage SEM applications. A microchannel plate can provide symmetric SEM signal detection at the low beam energies needed for non‐destructive examination of integrated circuits. Microchannel plate detectors are effective for both secondary electron and backscattered electron imaging. Their relatively small size and ability to be mounted directly below the final pole piece give them improved performance relative to conventional detectors in applications requiring short working distances. A significant amount of laboratory and applications experience has shown that microchannel plates are reliable and sufficiently resistant to contamination for use in high volume, production environments which require low voltage SEM imaging.
Laser scanning phase modulation microscope
Authors: Hansen E. W. , Allen R. D. , Strohbehn J. W. , Chaffee M. A. , Farrington D. L. , Murray W. F. , Pillsbury T. A. , Riley M. F.
Published: August 2011

We describe the concept and first implementation of an innovative new instrument for quantitative light microscopy. Currently, it provides selective imaging of optical path differences due to birefringence; with further development, it is also possible to selectively image several optical properties, including refractive path differences, optical rotation, and linear and circular dichroism, all with diffraction‐limited resolution. An image consists of a 512×512 element array, with each pixel displaying one of 256 grey levels, linearly proportional to the specific optical property being observed. Additionally, conventional brightfield and polarized light microscopy are available, with the accompanying advantages of laser scanning and digital image processing.

The microscope consists of three subsystems, representing three distinct technologies. The laser scanning subsystem moves a focused, microspot across the specimen; the output of a photodetector is an electric signal corresponding to a scanned image. The image display subsystem digitizes this signal and displays it as an image on a video monitor. When used in conjunction with a phase modulation feedback loop, the image formed is of the specimen's birefringent retardation or other selected optical property. The digitized images are also available for computer enhancement.
A mirror electron microscope for surface analysis
Authors: Foster M. S. , Campuzano J. C. , Willis R. F. , Dupuy J. C.
Published: August 2011

The principle of mirror microscopy has been adapted to provide a relatively low resolution surface microscope (<1000 ×), a large transfer width low energy electron diffractometer and a photoelectron analyser in k|| space. A focused electron beam of ≃ 10 kV is decelerated through a Johansson lens, reflected in front of the sample and reaccelerated back through the lens to produce an electron image over a field of view of a few microns. The image can be interpreted as a micrograph of work function variations on the surface if other effects (geometry, magnetic field) are uniform. In the LEED mode, diffracted beams virtually retain their positions on the screen over the whole impact energy range used (0.160 V). Secondary electrons are preferentially focused around the lens‐gun electro‐optic axis, thus effectively filtering them out from the diffraction pattern. The design has an inherently large coherence length, of up to 104 Å. Photoelectrons can similarly be imaged in k|| space on the detector plane. The addition of energy filtering at the screen allows the two‐dimensional Fermi surface to be imaged.
A double tilting cartridge for transmission electron microscopes with maximum solid angle of exit at the specimen
Authors: Everatt P. G. , Valdrè U.
Published: August 2011

A new type of double tilting cartridge for transmission electron microscopes (CTEMs and STEMs) is described, which has been designed with the following applications and requirements in mind: (i) observation of large specimens, in particular semiconductor devices carrying electrical leads for signal exploitation; (ii) maximum (2π) solid angle of beam exit subtended by the specimen; (iii) location of the specimen as low as possible in the pole piece gap of the objective lens.

The working principle is based on the kinematics of a hemisphere resting on the end of a cylindrical tube, and can be applied to any type of electron microscope specimen holder of the top entry type.
Measurement of characteristic wavelengths in modulated microstructures by field‐ion microscopy
Authors: Miller M. K. , Burke M. G. , Brenner S. S.
Published: August 2011

A procedure to determine the characteristic wavelength of a modulated microstructure from field‐ion micrographs is described. The procedure accounts for the effects of the orientation of the modulations in the specimen and the projection of the curved FIM specimen surface by correlating the measurements from the micrograph with computer simulated micrographs generated with the same orientation. The results exhibit excellent agreement with measurements determined from the spacing of sidebands in electron diffraction patterns.
Automatic analysis of the motion of microorganisms
Authors: Gualtieri P. , Colombetti G. , Lenci F.
Published: August 2011

An automatic, interpretative, off‐line computer‐television‐microscope system for studying the behaviour of microorganisms is presented. The equipment should be available even in relatively modestly equipped laboratories.

The movement of microorganisms freely swimming in the chamber of a depression slide is recorded on a video tape with the aid of a microscope and an infrared TV camera. The images are acquired by means of a frame grabber in an image controller and elaborated by a PDP 11/23 computer.

The procedures used for data processing are discussed in detail. Trajectories reconstructed by the computer are shown to be exactly superimposed to the real ones, and the motion parameters can be easily determined.
An accurate block‐trimming device for ultramicrotomy of selected cells
Authors: Gay J. L. , Faux L.
Published: August 2011

A small instrument is described which is operated on the stage of a light microscope to trim resin‐embedded specimens to precise levels for subsequent ultramicrotomy and to cut thin pilot sections for light microscopy. The operating procedure and examples of results are given.
Two‐dimensional crystallization experiments*
Authors: van Bruggen E. F. J. , van Breemen J. F. L. , Keegstra W. , Boekema J. , van Heel M. G.
Published: August 2011

Our experience in the growth of two‐dimensional crystals of different proteins is presented. Polyethylene glycol was used to produce two‐dimensional arrays of haemocyanin from O. vulgaris and of cholera toxin. The arrays showed a hexagonal close‐packed structure of only randomly oriented molecules. The increase in protein concentration probably occurred too quickly to allow complete crystallization. Different two‐dimensional arrays of hexameric haemocyanin molecules (from P. interruptus) were obtained by microdialysis through the specimen supporting film. A comparison was made with X‐ray data. Two‐dimensional tetrameric arrays of molecules, possibly rhodopsin, were seen in samples of bovine retinal rod outer segments in the presence of ammonium sulphate. Two‐dimensional crystals of complex I (from bovine mitochondria) were prepared by dialysis in the presence of ammonium sulphate. A three‐dimensional reconstruction was made from two tilt‐series by computer filtration using the direct SIRT procedure. Finally, the possibility of computer crystallization using correlation techniques in combination with correspondence analysis is discussed.
Study of the direct Fourier method for the three‐dimensional reconstruction of objects in the case of a missing angular data range
Authors: Carazo Jose María , Santisteban Antonio , Carrascosa José L.
Published: August 2011

Some practical and theoretical considerations on the three‐dimensional reconstruction of an object from its two‐dimensional projections by the direct Fourier method have been obtained using computer generated images. The procedure applied has been designed to study the specific problems derived from the collection of images in transmission electron microscopes (TEM), making special emphasis on images of biological objects. The study of the calculated point spread function (PSF) of the experimental system and the mean and variance profiles of the reconstructions have provided useful criteria to assess the quality and the interpretability of three‐dimensional reconstructions of real data.
Microscopic observation and video recording of cellular response to sudden changes in environment
Authors: Eskelinen Sinikka , Coakley W. Terence
Published: August 2011

A method to record responses of single cells to rapid changes in their environment is described. A rectangular glass microcapillary is filled with the cell suspension under investigation and placed under a light microscope. With the aid of a micromanipulator a smaller microcapillary with a narrowed tip, which was still over 10 times wider than the cell diameter, is driven into the larger, cell containing, microcapillary. The environment of the cells is changed by injecting a new medium via the smaller capillary and the cell responses are recorded using a video recording system. The usefulness of the system has been examined by studying the time course of the osmotic swelling of erythrocytes after water injection and the shape transformations caused by lysophosphatidylcholine.
Computer‐aided integrating microdensitometry and specimen imaging with the Vickers M85A
Authors: Goldstein D. J. , Lovering R. , Hartmann‐Goldstein I. J.
Published: August 2011

A Vickers M85A flying‐spot microdensitometer has been linked to a standard BBC model B microcomputer with a commercially available interface, and programs developed in which the computer controls step‐wise scanning of the specimen. At each image point thirty‐two absorbance readings are made and the average value is stored in the computer memory at 8‐bit resolution; a complete 48×48 raster takes less than 5 s. An image of the specimen displayed on the monitor uses eight different pseudo‐grey‐level characters, and the manner in which the 256 possible absorbance values are represented on the screen can be altered by the user. A mask of arbitrary shape and size, drawn on the screen under key‐board control, makes it possible to measure the integrated absorbance of even small, irregular and closely packed specimens such as individual bands in Drosophila polytene chromosomes. The coefficient of variation of repeated measurements of a Feulgen‐stained frog nucleus is typically better than 1%. Displayed images can be printed on a dot‐matrix printer, and data stored on disc for subsequent analysis. The techniques can readily be modified and adapted for other purposes, some of which are briefly discussed.
Freeze‐fracturing at defined temperatures provides information on temperature rise during fracture, and on membrane complementarity
Authors: Gruijters W. T. M. , Bullivant S.
Published: August 2011

An apparatus is described which allows freeze‐fracturing at defined temperatures, followed by immediate immersion of the specimen in liquid nitrogen. Replication is carried out in a Bullivant & Ames (1966) freeze‐fracture device.

Using yeast as a test specimen, the following results were obtained with the apparatus: (a) It was confirmed that fracturing at temperatures between 243 K and 223 K gave undeformed volcanoes on the PF of the plasma membrane, as shown originally by Steere et al. (1980). (b) Considerable energy is released by the fracturing process, both as shown by thermocouple readings and by the fact that at relatively high fracture temperatures portions of the specimen surface were melted. A temperature rise of 50–70 K was indicated, (c) Under standard conditions, there is a lack of complementarity between the yeast plasma membrane fracture faces, trigonal point particles not being present opposite the corresponding depressions in the array. Fracturing yeast suspended in salt solutions at 203 K demonstrates these particles. Their absence in normal fractures can be explained by ‘secondary fracture’, a concept based on polymer fracture studies.
Effect of specimen preparation and section transfer techniques on the preservation of ultrastructure, lipids and elements in cryosections
Authors: Hagler H. K. , Buja L. M.
Published: August 2011

Cryofixation, cryoultramicrotomy, and proper transfer of the cryosections into the electron microscope are important for the preservation of good ultrastructure and the measurement of subcellular elemental distributions. These techniques are applicable to tissue systems which can be rapidly frozen so that minimal to no ice damage occurs during the cryofixation step. For the transfer step we have compared the cryotransfer of hydrated sections and subsequent freeze‐drying in the electron microscope with the transfer of sections into an external freeze‐dryer, followed by exposure to room temperature and humidity before introduction into the electron microscope. The use of a cryotransfer stage for section transfer from the cryoultramicrotome to the electron microscope and low temperature observation of the thin sections avoids the potential problem of rehydration damage to freeze‐dried sections as well as provides protection from the possibility of melting of the lipids in the sections. Both of these problems may lead to loss of in situ elemental distribution and morphology. In this report, observations are presented which show the damaging effects of temperatures above 273 K on ultrastructure due to lipid melting in tissues with high lipid content and the redistribution of elements which can be encountered when thin sections become inadvertantly rehydrated.
Soft X‐ray contact microscopy, using synchrotron radiation, of thin‐sectioned, lead‐contaminated chloragogenous tissue of the earthworm Dendrobaena rubida
Authors: Richards K. Sylvia , Rush A. D. , Clarke D. T. , Myring W. J.
Published: August 2011

Soft X‐ray (2.05–4.4nm) contact microscopy, using synchrotron radiation, of ultrasections of lead‐polluted chloragogenous tissue of the earthworm Dendrobaena rubida has been achieved. The positive resist, after etching to leave areas corresponding to high absorbance in the tissue, is viewed by SEM and the imaging compared with the same section viewed, after irradiation, by TEM. No radiation damage as evident and the lead deposits associated with the chloragosomes and within the debris vesicles imaged clearly. The characteristics of the broad beam have thus produced a lead ‘map’. Resolution is in the order of 70 nm. The results are compared with the markedly different imaging of control tissue containing insignificant lead levels.
Differential staining of dead and dying embryonic cells with a simple new technique
Authors: Martín‐Partido G. , Alvarez I. S. , Rodríguez‐Gallardo L. , Navascués J.
Published: August 2011

A new staining technique with toluidine blue and safranin is described which yields satisfactory results in histological sections of chick embryo tissue fixed in a mixture of glutaraldehyde and formaldehyde and embedded in Spurr's resin. In zones of embryonic cell death, this technique allows the blue‐staining of dying cell pyknotic nuclei to be clearly distinguished from red‐staining healthy cells. The possible factors underlying this differential staining of pyknotic nuclei are discussed.
A simple method for correlative light, scanning electron microscopic and X‐ray microanalytical examination of the same section
Authors: Siklós L. , Rózsa M. , Zombori J.
Published: August 2011

Thin paraffin sections, mounted on scanning specimen holders previously coated with polyester film tape (Minnesota Mining and MFG Co., Scotch film tape No. 850 gold), were processed for light microscopy (LM) in the conventional way, then covered with celloxin shellac and examined in the LM by using the upper illuminating source. After removal of the shellac from the surface of the sample by immersion in acetone, the sections were air‐dried, coated with a copper layer in a vacuum evaporator and examined in a scanning electron microscope (SEM).

The method allows: (i) high‐quality LM possibilities for establishment of the diagnosis in pathological cases; (ii) SEM examination of the same area as observed in LM; and (iii) EPMA measurements of insoluble precipitates embedded in the tissue.

The usefulness of the proposed method is obvious in cases where the composition of a precipitate on LM scale is to be compared with the LM appearance of the surrounding tissue.
Fine structure of the images of atoms in 011‐gold crystals in various Bragg reflecting conditions
Authors: Hashimoto H. , Takai Y.
Published: August 2011

Fine structures with petal and horseshoe shape contrast have been observed in the high resolution electron microscope images of atoms in a 011 gold crystal in deviated Bragg reflecting conditions. The images with such fine structures have been recorded by using only the beams of 000, four 111 and two 200 reflections with axial illumination. The contrast of the images has been discussed by the many beam dynamical theory of electron diffraction and the image formation theory considering the spatial and temporal coherence of the electron waves. It has been noted that the temporal coherence has much more effect on the change of image contrast than the spatial coherence. The imaging conditions have been analysed in terms of Fourier images and the aberration free focus conditions.
High resolution transmission electron microscopy of Sm‐Co based permanent magnets
Authors: Hiraga K. , Hirabayashi M. , Ishigaki N.
Published: August 2011

Interface microstructures between rhombohedral Sm2Co17 and hexagonal SmCo5 phases in a sintered permanent magnet of Sm(Co0.675Fe0.175Ni0.05Cu0.1)7 have been investigated by edge‐on observations with the many‐beam imaging technique. In the specimen annealed at 800°C for 4 h which shows a maximum magnetic coercivity, the interface boundaries were coherent accompanying the lattice distortion; the habit planes were parallel to the {101}2:17 and {111}1:5 planes with the orientation relationship of (0001)2:17//(0001)1:5 and [1010]2:17//[1120]1:5. In the specimen annealed for 150 h which shows a lower coercivity, the interface boundaries were characterized with regular alignments of interfacial edge dislocations. The observed interface structures were discussed in connection with the change in magnetic properties.
Post‐embedding cytochemistry with gold‐labelled reagents: a review
Authors: Roth J.
Published: August 2011

The detection of antigens and glycoconjugates with the protein A–gold and the lectin–gold techniques, respectively, is reviewed. Special attention is directed to the necessary conditions for fixation and embedding as well as to the staining procedures of tissue sections for light and electron microscopy.
Light microscopical detection of single 5 and 20 nm gold particles used for immunolabelling of plasma membrane antigens with silver enhancement and reflection contrast
Authors: Hoefsmit E. C. M. , Korn C. , Blijleven N. , Ploem J. S.
Published: August 2011

To detect plasma membrane antigens, cytocentrifuge preparations of the macrophage‐like cell line P388D1 were incubated with monoclonal antibodies and labelled with 5 and 20 nm gold particles conjugated to immunoglobulins or protein A. The 20 nm, but not the 5 nm, particles could be observed by reflection‐contrast light microscopy. Single 5 nm particles, however, were clearly demonstrable with silver contrast enhancement, both in absorption contrast and in reflection contrast. The method proved to be compatible with cytochemical methods such as acid phosphatase.
An extrapolation method for the determination of Cliff‐Lorimer kAB factors at zero foil thickness
Authors: Horita Zenji , Sano Takeshi , Nemoto Minoru
Published: August 2011

An extrapolation method is proposed to be useful for the determination of the Cliff‐Lorimer kAB factor at zero foil thickness. The method consists of measuring kAB factors as a function of the measured foil thickness, tM, and extrapolating the relationship toward tM=0. The intersection between the extrapolated line and the ordinate of tM=0 gives (kAB)0 which is free from the effect of absorption. The straight line extrapolation that can be achieved by a linear‐least squares method is particularly developed to eliminate arbitrariness introduced in the extrapolation process.

The extrapolation method is applied to data available in the literature. It is shown that the method yields the (kAB)0 factors compatible with those predicted from the theoretical calculation. It is also shown that this method can circumvent several problems which make it complex and difficult to determine accurate values of the absorption‐free kAB factors.

Using the straight line extrapolatioin, it is possible to estimate the degree of the thickness overestimation which arises when the foil thickness is measured by the contamination spot separation (CSS) method. Validity of the straight line extrapolation is further discussed.
The interpretation of EBIC images using Monte Carlo simulations
Authors: Joy David C.
Published: August 2011

summary

Charge collection microscopy, usually known by the acronym EBIC (Electron Beam Induced Current) imaging, is a powerful technique for the observation and characterization of semiconductor materials and devices in the scanning electron microscope. Quantitative interpretation of EBIC images is often difficult because of the problem of accurately representing the electron‐beam interaction with the semiconductor. This paper uses a Monte Carlo technique to simulate the electron‐beam interaction, and it is shown that this permits simple analytical point‐source solutions to be generalized to fully represent the experimental situation of an extended, non‐uniform, carrier source. The model is demonstrated by application to EBIC imaging in the Schottky barrier geometry.
Simple SEM stereophotogrammetric method for three‐dimensional evaluation of features on flat substrates
Authors: Boyde A. , Howell P. G. T. , Franc F.
Published: August 2011

summary

A semi‐automated method was developed for the measurement of the volume of a calcified tissue substrate which can be resorbed by single, isolated resorptive cells (osteoclasts). The procedure would, however, be applicable to the study of any pit in, or projection from, a flat surfaced specimen. Stereo‐pair images are recorded with an SEM. 3‐D co‐ordinates of relevant surface features are derived using a stereo‐comparator which outputs data directly to a microcomputer. Algorithms adopted have been chosen to give the best compromise between accuracy and speed of data acquisition.
Study of the conditions necessary for propane‐jet freezing of fresh biological tissues without detectable ice formation
Authors: Haggis G. H.
Published: August 2011

summary

The performance of a commercial double‐propane‐jet freezer (Balzers QFD 101) has been assessed, for rapid freezing of fresh tissues in freeze‐etch work. Samples of diaphragm muscle and intestinal villi were frozen between copper sheets, with a spacer to give 20–30μm thickness of tissue. Fracture cuts were made with the Balzers BAF 400 freeze‐etch microtome within 5–10μm of a freezing face (i.e. a tissue face in contact with the copper sheets of the frozen sandwich). After some modifications to the QFD 101, replicas showing no evidence of ice were obtained of muscle cells, although for intestinal epithelial cells some evidence of ice formation was found. Infiltration with 5% glycerol or dimethylsulphoxide improves the depth of good freezing. Results and problems arising from such infiltration are briefly discussed.
Visualization of biological specimens by cryoHVEM
Authors: Fotino Mircea
Published: August 2011

summary

This article describes the operation and the characteristics of cryoHVEM imaging of biological specimens using a top‐entry cryostage. The procedure for inserting frozen specimens into the microscope column is also presented. Whole mounts were thus observed under optimal imaging conditions by combining: (i) fixation by fast freezing for structure preservation without exposure to chemicals, (ii) observation in the hydrated (frozen) state or in the dried state without exposure to the atmosphere after the initial fixation by freezing, and (iii) ultrastructural visualization with the key imaging factors of resolution, penetration and beam‐induced damage at their best by high‐voltage electron microscopy.
Temperature control in Lowicryl K4M and glycol methacrylate during polymerization: is there a low‐temperature embedding method?
Authors: Ashford A. E. , Allaway W. G. , Gubler F. , Lennon A. , Sleegers J.
Published: August 2011

An apparatus for embedding tissues at resin temperatures down to 228 K is described. By placing thermocouples in the resin the temperature has been monitored during embedding at low temperature with glycol methacrylate (GMA) and Lowicryl K4M. Even in this apparatus with a liquid cooling bath the heat of polymerization is not dissipated and the resin temperature rises. This rise is directly proportional to the resin temperature at the onset of polymerization and is higher in Lowicryl K4M than GMA. The initial resin temperature also affects the time taken for polymerization. The time to the onset of the peak and its duration are both increased as the temperature is lowered. This effect is more pronounced with GMA than Lowicryl K4M and polymerization of GMA is inhibited at the lowest temperature used. When Lowicryl K4M, polymerized at low temperature, is warmed up to ambient a further exothermic reaction occurs, which causes the resin temperature to rise well above ambient. Both this temperature peak and that during polymerization are reduced, but not totally eliminated, by reducing the resin volume. Aircooled systems are inefficient compared with the low‐temperature apparatus used here and the resin temperature rise is consequently greater and, even with small resin volumes, it can be very high. It is therefore unlikely for published methods that the temperature specified has been maintained in the resin during polymerization. The implications of these findings are discussed in relation to enzyme and antigen survival. Recommendations include use of very small volumes of resin, refrigerated liquid‐bath rather than air‐cooled systems and contact with a heat sink when specimens are warmed up to ambient temperature. Examples of enzyme reaction, antigen survival and structural preservation obtained with the method are presented.
Quantitative microlocalization of diffusible ions in normal and galactose cataractous rat lens by secondary ion mass spectrometry
Authors: Burns Margaret S. , File David M.
Published: August 2011

Secondary ion mass spectrometry (SIMS) is a surface analytical technique with high sensitivity for elemental detection and microlocalization capabilities within the micrometre range. Quantitative analysis of epoxy resins and gelatin have been reported (Burns‐Bellhorn & File, 1979).
The application of a microwave technique for the preparation of cell blocks from sputum
Authors: Kok L. P. , Boon M. E. , Ouwerkerk‐Noordam E. , Gerrits P. O.
Published: August 2011

Microwaves stimulate diffusion in tissue and affect the proteins and membrane of the cell. These actions can be used to shorten the time required to fix and prepare cell blocks from sputum. The fixative containing ethyl alcohol and polyethylene glycol is particularly suitable for microwave enhanced fixation. Applying a commercially available domestic microwave oven, in which the temperature and power level can be set, cell blocks can be prepared from fresh sputum in 35 min. The microscopical results are of a superior quality because of the microwave‐induced homogeneous distribution of the effects of the applied immersion fluids on the cells. The method is very successful for all specimens containing a large amount of mucous. Health hazards for laboratory personnel are nil in this cell blocking method; in contrast to when fresh sputum containing infectious agents is used for smear preparation.
The use of malachite green during fixation of plant tissues for the preservation of lipids
Authors: Lawton June R.
Published: August 2011

Osmiophilic droplets are shown in chloroplasts of leaves of ryegrass after the addition of malachite green to glutaraldehyde during primary fixation. Similar droplets are shown in leaves of bean and maize but not in the roots of these plants. These droplets are not shown in tissue fixed in glutaraldehyde in the absence of malachite green. Evidence is presented which indicates that these droplets contain a high proportion of lipids, particularly phospholipids. This evidence includes the use of other fixation methods which retain lipids, e.g. formal calcium, potassium dichromate. The droplets give a positive reaction with the silver hydroxylamine reaction for ester linkages. Tests for aldehydes by treatment with thiocarbohydrazide and silver proteinate, and proteins by digestion with protease were ambiguous. Digestion with solvents before fixation indicate that the droplets are lipids. TLC of chloroform: methanol extracts of fresh and fixed tissue and of the fixative solvents support the idea that malachite green aids retention of lipids during fixation. Phospholipids are also demonstrated by silver hydroxylamine in plant cell nuclei, myelin of rat brain and vesicles in bean root tips.
Selective contrast‐enhancement of the cochlear duct glycocalyx after OsO4/K4Ru(CN)6 post‐fixation
Authors: Groot J. C. M. J. de
Published: August 2011

Fixation‐dependent selective contrast‐enhancement of the cochlear duct (or endolymphatic) glycocalyx was observed in the guinea pig inner ear after post‐fixation with osmium tetroxide‐potassium rutheniumcyanide. Contrast of the perilymphatic glycocalyx, however, was not significantly enhanced. The possible factors underlying this phenomenon are discussed.
Beam induced mass loss in high resolution biological microanalysis
Authors: Cantino M. E. , Wilkinson L. E. , Goddard M. K. , Johnson D. E.
Published: August 2011

Electron beam induced loss of mass from the organic matrix and from higher Z constituents of biological samples was measured by monitoring bremsstrahlung and peak changes in EDS spectra. When any effects of contamination, extraneous X‐rays, beam current drift, specimen drift, and specimen shrinkage were monitored and corrected for, the three types of samples gave consistent and similar results at 296 K. Bremsstrahlung losses averaged 45%, 46% and 50% respectively for muscle homogenate, salivary gland sections and albumin. Sulphur losses average 74%, 72% and 86% for the same three sample types. No other elements suffered significant losses. Dl/e for bremsstrahlung averaged 0·14 C/cm2. Bremsstrahlung loss at 93 K began approximately one order of magnitude higher in dose, and the extent of loss varied. Sulphur losses, however, were greatly reduced at low temperatures.
A simple photographic grid system for the correlative examination of cell smears by light and scanning electron microscopy
Authors: Chang Alexander R.
Published: August 2011

A simple inexpensive grid system reproduced photographically on black‐and‐white film provides a support system that allows the same cells to be examined by light and scanning electron microscopy.
Diffraction pattern indexing in lithium niobate
Authors: Twigg M. E. , Nakahara S. , Maher D. M. , Sheng T. T. , Homes R. J.
Published: August 2011

Crucial to the interpretation of diffraction contrast experiments conducted in the transmission electron microscope is the self consistent indexing of zone‐axis patterns for the crystal under study. Because indexing the diffraction patterns of a crystal as complicated as lithium niobate is not a trivial exercise, computer simulation of diffraction patterns, extinction distance calculations, and consideration of several unit cell representations are used in order to make diffraction pattern interpretation more tractable. It was found that the zone‐axes were most easily indexed by considering the pseudo‐cubic representation of rhombohedral perovskite crystals. The pseudo‐cubic system also facilitates the indexing of Bragg reflections and the visualization of the major features of the reciprocal lattice. Ultimately, however, hexagonal indices must be used to understand a particular lattice defect, since the symmetries associated with the hexagonal lattice are real symmetries, unlike the apparent symmetries that are implied by a pseudo‐cubic crystal. For this reason, we have tabulated the matrices for transforming from the hexagonal system to the pseudo cubic system. For the sake of completeness, transformation matrices relating all four relevant lattice systems (hexagonal, pseudo‐cubic, primitive rhombohedral, and orthohexagonal), as well as the atomic positions in each system, are tabulated.
Particles and pits matched in native membranes
Authors: Ting‐Beall H. P. , Burgess Florence M. , Robertson J. David
Published: August 2011

We have obtained high resolution electron microscopic images of complementary membrane surface replicas of purified microsomal vesicles from pig kidney outer medulla containing Na+, K+‐ATPase. Ultra‐rapid freezing of a membrane suspension was followed by fracturing and replicating of the liquid helium cooled specimen under ultra‐high vacuum conditions free of hydrocarbon contaminants. The protoplasmic fracture faces are populated with intra‐membrane particles while the external fracture faces reveal complementary pits. This is the first demonstration of extended precise matching of individual intramembrane particles and their corresponding pits in biological membranes containing transmembrane proteins. The data are also consistent with the theory that the majority of Na+, K+‐ATPase mass is located at the protoplasmic half of the membrane.
Environmental scanning electron microscopy in colour
Authors: Danilatos G. D.
Published: August 2011

The environmental SEM has been developed to a stage where colour can be introduced during imaging. A new approach to production of colour micrographs is demonstrated together with some typical uses of the environmental mode of SEM. A combination of the outputs of two backscattered electron (BSE) detectors alone or in combination with the outputs from a gaseous detector device can form images corresponding to particular aspects of a given specimen. Two or more of these images can be superimposed on to the same colour print to produce a colour micrograph.