bluuebunny/crossref_metadata_2025_split huggingface dataset:
The osmotic effect of glutaraldehyde‐based fixatives on plant storage tissue
Authors: Coetzee J. , van der Merwe C. F. 
Published: August 2011

SUMMARYMass 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

SUMMARYAs 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

SUMMARYA 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

SUMMARYA 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

SUMMARYWe 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

SUMMARYSome 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

SUMMARYComplementary 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

SUMMARYDifficulties 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

SUMMARYA 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

SUMMARYActin 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

SUMMARYMicrotubules 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

SUMMARYFrozen 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

SUMMARYHREM, 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

SUMMARYVarious 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

SUMMARYA 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

SUMMARYVital 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

SUMMARYThe 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

SUMMARYA 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

SUMMARYA 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

SUMMARYPore 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

SUMMARYThe 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.