bluuebunny/crossref_metadata_2025 huggingface dataset:
Three‐dimensional reconstruction from images of tilted specimens: the paramyosin filament
Authors: Dover S. D. , Elliott A. , Kernaghan A. K.
Published: August 2011

The three‐dimensional structure of the paramyosin filament has been reconstructed from images obtained when the specimen is tilted at different angles about its long axis. The micrographs were correlated spatially using gold marker beads and digitized. The different views contained in each micrograph were combined using a computational technique involving Fouri***er transforms and the resulting three‐dimensional images are presented as various sets of serial sections. The results confirm that the Bear‐Selby (1956) net can be seen at all levels in the specimen.
Morphometric studies on mouse bone using a computer‐based image‐analysis system
Authors: Green D. , Howells G. R. , Thorne M. C.
Published: August 2011

The morphological structure of the ilium, femur, third lumbar vetrebra and a central caudal vertebra of the female CBA mouse has been studied using 5 μm thick, plastic embedded, transverse and longitudinal sections. The sections were analysed on a Quantimet 720, system 30, image analyser connected on‐line to a PDP11 computer. Separate endosteal and periosteal surface to volume ratios were calculated for each position of sampling in each bone. For this calculation the anisotropy of the bone was estimated from measurements of mean chord lengths in longitudinal sections of the bone using a new analytical technique. Chord length distributions in transverse sections of bone were also measured and the relevance of such measurements to the study of morphological changes in the bone and its included marrow are briefly discussed.
The use of a computerized algorithm to determine single cardiac cell volumes
Authors: Marino Thomas A. , Cook Larry , Cook P. Nong , Dwyer Samuel J.
Published: August 2011

Single cardiac muscle cell volume data have been difficult to obtain, especially because the shape of a cell is quite complex. With the aid of a surface reconstruction method, a cell volume estimation algorithm has been developed that can be used on serial sections of cells. The cell surface is reconstructed by means of triangular tiles so that the cell is represented as a polyhedron. When this algorithm was tested on computer generated surfaces of a known volume, the difference was less than 1·6%. Serial sections of two phantoms of a known volume were also reconstructed and a comparison of the mathematically derived volumes and the computed volume estimations gave a per cent difference of between 2·8% and 4·1%. Finally cell volumes derived using conventional methods and volumes calculated using the algorithm were compared. The mean atrial muscle cell volume derived using conventional methods was 7752·7 ± 644·7 μm3, while the mean computerized algorithm estimated atrial muscle cell volume was 7110·6 ± 625·5 μm3. For AV bundle cells the mean cell volume obtained by conventional methods was 484·4 ± 88·8 μm3 and the volume derived from the computer algorithm was 506·0 ± 78·5 μm3. The differences between the volumes calculated using conventional methods and the algorithm were not significantly different.
Sectioning and imaging hard mineral fibres in biological tissues
Authors: Johnson N. F. , Ibe K.
Published: August 2011

A technique is described which overcomes the problems associated with sectioning biological tissue containing hard mineral fibres. 0·2–0·5 μm thick sections were cut with a diamond knife, placed in a folding grid, conventionally stained with uranyl acetate and lead citrate and viewed at an accelerating voltage of 200 kV in the scanning transmission mode.
Stereological estimation of the radial distribution function of centres of spheres
Authors: Hanisch K.‐H. , Stoyan D.
Published: August 2011

The usual stereological formulae yield mean values or one‐dimensional distributions. However, for the description of correlations in particle systems, second‐order characteristics are useful, for example the radial distribution function or the second‐order product density of the point process of particle centres. For the case of spherical particles some exact and approximate stereological formulae are given. These connect the spatial second‐order quantities with quantities available from planar or linear intersections.
Focused apparent image position with convergent, parallel binocular and monocular microscopes
Authors: Richards Oscar W. , Mathews Steven M. , Shaffer Steven M.
Published: August 2011

The distance from the observer's eye at which the image appears to be focused was measured with monocular, convergent and parallel eyepiece bodies, interchangeable on a Spencer microscope, with a camera lucida prism and a calibrated optometer. Five optometric tests for binocular vision and one for stereopsis made on thirty‐four observers revealed no significant correlation with the positions of the images. The apparent image position from the eye using the parallel binocular eyepieces averaged 671 mm, with the convergent eyepiece body at 587 mm, and 469 mm with monocular observation. The relations of these image positions with possible rest equilibrium positions of the accommodative mechanism of the eye and optical design of binocular instruments are discussed.
The use of polystyrene microcarriers to prepare cell monolayers for transmission electron microscopy
Authors: Sargent G. F. , Sims T. A. , McNeish A. S.
Published: August 2011

A simple method is described, using polystyrene microcarrier beads, that allows cell monolayers to be prepared without distortion for transmission electron microscopy.

Cells cultured on polystyrene beads (Biosilon R, Nunc, Kamstrup, Denmark) are held in an agar matrix. Before embedding, the beads are dissolved in 1, 2‐epoxypropane.

The technique increases the number of observable cells, when compared to preparations of planar monolayers, and there is good preservation of morphological detail.
A technique for obtaining ribbons of epoxy and other plastic sections for light microscope histology
Authors: Richards Peter R.
Published: August 2011

A method is described by which ribbons of thick, large area sections of material embedded in epoxy resins prepared from standard recipes for electron microscopy, can be cut using conventional microtomes. The epoxy blocks are double embedded in an epoxy/polyethylene glycol mixture and ribbons are cut dry with fluorocarbon coated long‐edged (‘Ralph’) glass knives. The method can also be applied to other plastic embedding media such as glycol methacrylate.
Strong evidence against section thawing whilst cutting on the cryo‐ultratome
Authors: Frederik P. M. , Busing W. M.
Published: August 2011

The distribution of ice crystal sizes in frozen thin sections correlates with the size distribution in freeze fracture replicas. In the hypothetical case of melting (and refreezing) during cyro‐sectioning this correlation should be lost as was found after intentional melting or frozen thin sections. These observations demonstrate that there is neither a through section melting nor a growth of ice crystals during cryo‐ultramicrotomy.
Photographic defocusing: a means to render recognizable line traced models of negatively stained biological specimens
Authors: Rodrigues F. Carvalho , Nunes J. F. Moura , Chagas J. Pinto , Soares J. O.
Published: August 2011

A simple method of obtaining analogue images from traced models of biological specimens is presented. It consists of the photographic defocusing of traced models and it is illustrated with negatively stained cylindrical forms of the ASFV; the black lines of the trace in the model correspond to the negative stain surrounding the viral morphological subunits as seen in the electron micrograph. The photographic defocusing is the means by which the traced model is filtered and is used to introduce grey levels on an otherwise black and white image. The right amount of defocusing is attained when the width of the trace of the model equals the width of the rim of the negative stain appearing between the morphological subunits in the electron micrograph.
Polychromatic Giemsa staining for Epon semi‐thin sections
Authors: Cañete M. , Stockert J. C.
Published: August 2011

A polychromatic staining procedure which uses the Giemsa stain and differentiates between several cell and tissue structures with distinct colours has been developed for semi‐thin sections of glutaraldehyde‐fixed, Epon‐embedded animal and plant tissues.

The method does not require removal of the plastic and can be applied in a simple and rapid way for routine staining of semi‐thin sections with good results.
X‐ray microanalysis with Si(Li) detectors
Authors: Statham P. J.
Published: August 2011

The Si(Li) detector, with its relatively high collection solid angle, has brought X‐ray microanalysis within the operating current limits of both SEMs and transmission microscopes. At present, the main deficiencies arise in the low‐energy region of the spectrum, below about 2 keV. Here, detection efficiency falls off because of absorption in the entrance window, peaks invariably overlap and pile‐up rejection is less effective. High‐energy photons generated in transmission microscopes are responsible for new spectral artefacts and problems of overload. While computer processing of spectra can correct for most artefacts, background and peak overlaps, accuracy is fundamentally limited by counting statistics and the overall stability of the spectrometer. The technique will therefore benefit from improved design of both electronic circuitry and components. For example, advances in FET design may bring a substantial reduction in electronic noise which will not only improve resolution at low energies, but will also improve discrimination between small pulses and noise so that pile‐up rejection can be more effective. This will be most important when a very thin entrance window is used to permit observation down to the C Kα energy (282 eV) although the hazards involved in such ‘windowless' operation still limit its application. The stability of detection efficiency with time has been exploited in methods which attempt to remove the need for repetitive standardization of the spectrometer and new techniques have been developed for the analysis of thin films, particles and rough surfaces which make use of information contained in the bremsstrahlung continuum.
An X‐ray microanalytical examination of precipitation methods for the ultrastructural localization of potassium in plant tissue: II. Tetraphenyl boron
Authors: van Steveninck Margaret E. , van Steveninck R. F. M.
Published: August 2011

The possibility of using sodium tetraphenyl boron (TPB) as a precipitant for the ultrastructural localization of K+ was examined using both in vitro and in vivo studies. Because TPB salts are soluble in the usual dehydrating agents, glutaraldehyde‐urea was used for embedding. When Na TPB was introduced to the tissue together with OsO4, good cytoplasmic preservation was achieved, and deposits containing K were located within the cytoplasm and vacuoles. However, these deposits cannot be distinguished visually, so that X‐ray microanalysis is necessary to confirm the presence of K.
The development of squamous cell metaplasia in human bronchial epithelium by light microscopic morphometry
Authors: Bertram J. F. , Rogers A. W.
Published: August 2011

Bronchial biopsies from ten subjects, including five smokers, have been examined using light microscopic morphometry. The biopsies were free from identifiable disease. Using manual point counting and a Quantimet 720 image analysing computer, a number of parameters were measured. Computer‐based cluster analysis of seven of these parameters associated the subjects into three groups: visual inspection of the sections achieved the same separation. Four subjects (non‐smokers) had normal epithelia: four subjects (one non‐smoker, three smokers) showed mucous cell hyperplasia: two subjects (smokers) had squamous cell metaplasia. Three parameters in conjunction contained sufficient information to characterize accurately the histological appearance of the epithelia: the epithelial thickness, the volume density of intracellular mucus and the number of nuclear profiles per unit area of sectioned epithelium. Reduction of these three parameters to a linear plot closely approximated a similar reduction of the original seven parameters. These three parameters can be measured rapidly, either manually or by the Quantimet. The linear representation of these parameters provides a reproducible and objective basis for comparing specimens of bronchial epithelium.
Glycol methacrylate in light microscopy: nucleic acid cytochemistry
Authors: Cole Madison B. , Ellinger Joan
Published: August 2011

Techniques utilizing Feulgen, azure B bromide, methyl green‐pyronin, gallocyanin chromalum and cresyl violet stains have been modified and adapted for visualizing nucleic acids in 0.5–2.0 μm sections of tissues embedded in glycol methacrylate (GMA). Methods for evaluating the stain specificity for DNA and RNA using deoxyribonuclease and ribonuclease digestions, aldehyde blocking, and acid extractions are also described. The specificity of the stains in GMA embedded tissues is comparable to that reported for paraffin‐embedded tissues.
Scanning X‐ray microradiography of a section of a carious lesion in dental enamel
Authors: Elliott J. C. , Dowker S. E. P. , Knight R. D.
Published: August 2011

A scanning X‐ray microradiographic system is described with which it is possible to measure the X‐ray absorption in a 1 mm × 1 mm area of a section in about 27 h at a resolution of 20 μm with a reproducibility of about ± 1.5%. This system has advantages over photographic methods for certain quantitative investigations. The results can be presented in various forms including contour maps and half‐tone pictures and this is illustrated with a radiograph of a section of a carious lesion in dental enamel.
Multinucleate giant enterocytes in small intestinal villi after irradiation
Authors: Carr K. E. , Hamlet R. , Nias A. H. W. , Watt C.
Published: August 2011

Scanning electron microscopy of the small intestine of the mouse 5 days after X‐ or neutron irradiation has revealed the formation of giant cells on the villus surface.

Correlative light microscopy and transmission electron microscopy have shown that these giant cells are syncytial in nature.

Characteristic features of lipid inclusions and apical microvilli suggest that these syncytia are giant enterocytes. It has also been shown that these giant cells are in contact with the connective tissue core of the villus and have a close contact with the normal enterocytes, thus maintaining mucosal integrity. It is postulated that radiation damage has caused incomplete separation during mitosis and that attempted division occurs outside the crypts of Lieberkuhn.
The contribution of scanning electron microscopy in haematology: its role in defining leucocyte and erythrocyte disorders
Authors: Polliack Aaron
Published: August 2011

This report reviews the contribution of scanning electron microscopy (SEM) in haematology. Important observations regarding red cell shape and deformities are referred to and contributions in the definition and classification erythrocyte disorders are stressed. In this field there is no doubt that SEM has contributed much to the three‐dimensional visualization of RBC disorders.

As far as leucocyte pathology is concerned the situation is less clear. SEM has contributed much to current knowledge and understanding of the surface properties of white blood cells. Normal leucocytes have different surface features and can be distinguished under the SEM. However, some overlap does occur, making individual distinction on the basis of surface architecture alone extremely difficult. The difficulties in this regard are discussed in this review and factors influencing the variability of surface microprojections are reviewed briefly.

Leukaemic cells of different origins may also be distinguished under the SEM. However, while ‘hairy’ cells have typical surface features and non‐lymphoid leukaemic cells, in particular monocytes, are readily distinguished from lymphoid leukaemic cells, there is much overlap of surface topography. Undifferentiated cells and early myeloblasts and lymphoblasts have similar surface features and cannot be distinguished under the SEM. While SEM adds a valuable third dimension to morphology and ultrastructure, it cannot be used alone in the definition of difficult cases of acute leukaemia.
Colloidal gold markers and probes for routine application in microscopy
Authors: Goodman S. L. , Hodges G. M. , Trejdosiewicz L. K. , Livingston D. C.
Published: August 2011

Colloidal gold can be used as an electron‐dense cytochemical probe for direct or indirect labelling techniques. Gold markers can be prepared in a size range of 5–150 nm, and show size‐dependent shape characteristics and absorption spectra. Size and shape distribution increases with mean particle diameter, with appreciable overlapping between populations of different mean size. Using radio isotope‐binding assays, spectrophotometric analysis and an innovative rapid microtitration technique, the effect of pH, ionic strength and protein concentration on gold‐protein interaction has been studied. Efficient adsorption of protein to gold occurs at, or near, the pI of the protein. The amount of protein needed to effect stabilization is both a function of pH and of ionic strength, but does not reflect the amount of protein binding for all proteins. There is evidence for multilamellar adsorption of proteins to gold, which is discussed in context of the bioactivity, and stability of the probe. A working protocol for the routine reproducible manufacture of protein‐gold probes is given, making use of the microtitration assay.
Study of supported ruthenium catalysts by STEM
Authors: Pennycook S. J.
Published: August 2011

A VG Microscopes' HB5 STEM has been used to study supported ruthenium catalysts provided by ICI Ltd. They consist of small ruthenium particles 3–10 nm in diameter and have been imaged using a variety of detectors. The signal combining strongest contrast from the particles with minimum contrast variations due to diffraction effects is from a high angle annular detector. The detector is particularly useful with crystalline supports. For microanalysis purposes, X‐ray spectra, electron energy loss spectra, and microdiffraction patterns have been recorded and together show conclusively that even the smallest particles are hexagonal ruthenium metal.
The measurement of atomic number and composition in an SEM using backscattered detectors
Authors: Ball M. D. , McCartney D. G.
Published: August 2011

The atomic number dependence of electron backscattering can be used as the basis of a microanalysis technique. The operating procedures and condition for quantitative measurements of specimen atomic number are outlined and an expression relating the accuracy of composition to the atomic number sensitivity has been derived. Some measurements of the spatial resolution of backscattered electron microanalysis are also presented and compared with the resolution of X‐ray microanalysis.

Although the range of application of this technique is limited, where it can be applied it has the following advantages: (i) higher spatial resolution than X‐ray microanalysis for bulk specimens; (ii) very rapid measurement; (iii) can be applied to compounds of low atomic number elements, (e.g. borides, carbides, nitrides, etc.); (iv) specimen preparation is often relatively straightforward.
A surveying optical diffractometer
Authors: Salmon E. D. , DeRosier David
Published: August 2011

A surveying optical diffractometer is described which provides an efficient means of obtaining high quality optical diffraction patterns of particularly suitable specimens from electron micrographs.
Quantitative studies of the autofluorescence derived from neuronal lipofuscin
Authors: Dowson J. H. , Harris S. J.
Published: August 2011

The degree of accumulation of neuronal lipofuscin in the human brain can indicate the age of some neurones, may be correlated with certain disease processes and can be affected by drugs. The estimation of the volume of neuronal lipofuscin has usually employed relatively unreliable methods such as counting techniques or visual ratings, while the present paper describes and illustrates a method for measuring the intensity of the autofluorescence derived from neuronal lipofuscin. The relationship between autofluorescence intensity and amount of lipofuscin is discussed.
Size determination of transparent spheres in an opaque specimen from a slice
Authors: Coleman Rodney
Published: August 2011

A practical procedure is described for estimating the size distribution of transparent spherical particles embedded in an opaque specimen from the sizes of the profiles in a thin slice of the specimen. This complements Rose (1980), the case of opaque spheres in a transparent medium.
The etching behaviour of silicon carbide compacts
Authors: Jepps N. W. , Page T. F.
Published: August 2011

A series of microstructural investigations has been undertaken in order to explore the reliability of particular etches in revealing microstructural detail in silicon carbide compacts. A series of specimens has been etched and examined following complete prior microstructural characterization by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and X‐ray diffractometry techniques. In particular, the sensitivity of both a molten salt (KOH/KNO3) etch and a commonly‐used oxidizing electrolytic ‘colour’ etch to crystal purity, crystallographic orientation and polytypic structure has been established. The molten salt etch was found to be sensitive to grain boundaries and stacking disorder while the electrolytic etch was found to be primarily sensitive to local purity and crystallographic orientation. Neither etch appeared intrinsically polytype sensitive. Specifically, for the ‘colour’ etch, the p‐ or n‐type character of impure regions appears critical in controlling etching behaviour; p‐type impurities inhibiting, and n‐type impurities enhancing, oxidation. The need to interpret etching behaviour in a manner consistent with the results obtained by a variety of other microstructural techniques will be emphasized.
Factors affecting the ion‐beam thinning characteristics of ceramics
Authors: Salehpoor B. , Marquis P. M.
Published: August 2011

A study of the factors that affect thinning rates and damage processes during the ion‐beam thinning of ceramics has been undertaken. The results of these studies are reported and related to the conditions necessary for the preparation of uniform damage free specimens. Particular emphasis is placed on the variations in specimen stoichiometry that can occur during ion‐beam thinning. It is shown that under certain conditions the positively charged ion‐beam can induce charged interstitial migration into the bulk of the sample. The extent to which this effect, atomic bonding, crystal orientation and beam conditions can produce non‐uniform samples is discussed.
Microstructure dependence of the creep and creep‐fracture behaviour of ceramic materials
Authors: Wilshire B.
Published: August 2011

The microstructure dependence of the creep and creep‐fracture properties of ceramics is illustrated with particular reference to the behaviour of magnesia, lime and silicon nitride materials. From a consideration of the various models proposed to account for deformation and fracture under high temperature creep conditions, a consistent interpretation can be provided for the diverse behaviour recorded for different ceramics as a result of variations in crystal structure, composition and phase distribution.
The crystallization of calcium aluminate glasses
Authors: Marquis P. M.
Published: August 2011

The crystallization characteristics of two commercial calcium aluminate glasses have been studied using scanning electron microscopy. In particular it is demonstrated how atomic number contrast can be used to identify rapidly crystalline phases without the need for etching and the related loss of compositional and morphological information. From these studies and concurrent X‐ray analysis it is shown that the presence of barium oxide and silica has a marked effect on the crystallization behaviour of calcium aluminate glasses.
The characterization of surfaces of technical ceramics used in the synthetic fibres industry by microscopy
Authors: Gallant P. E. , Merigold R. J.
Published: August 2011

In the manufacture of synthetic fibres such as nylon and Terylene, ceramic surfaces are used to guide and control the threadlines. Successful operation is critically dependent on correct friction between the threadline and the surface it contacts; this is achieved by control of surface topography. Microscopy, particularly the SEM, has played an important role in understanding the characteristics of different surfaces, control of manufacture of the surfaces, development of new surfaces and processes, and the elucidation of plant problems. Examples of these applications are illustrated and discussed.
Energy dispersive X‐ray microanalysis of air dried casein micelles from bovine milk
Authors: O'Brien Timothy L. , Baumrucker Craig R.
Published: August 2011

Secretory proteins (casein micelles) were air dried prior to elemental analysis either without treatment, following glutaraldehyde fixation and dialysis, or following dialysis against distilled water. Samples were then examined by energy dispersive X‐ray microanalysis in a scanning transmission electron microscope (STEM). Untreated samples and samples dialysed against distilled water retain colloidal (bound) elements as compared to glutaraldehyde treated samples. Contamination of air dried casein micelles by ionic constituents in the non‐protein aqueous phase was observed in the untreated sample and in the glutaraldehyde treated sample. Dialysis of casein micelles against distilled water reduced contamination from the aqueous phase without diminishing the apparent concentrations of colloidal elements.
A simple device for freeze‐drying electron microscope specimens
Authors: Roberts I. M. , Duncan G. H.
Published: August 2011

A simple, inexpensive and easily made device is described in which specimens can be freeze‐dried on electron microscope grids before shadowing. The device can be used with almost any type of vacuum coating unit, and can take up to twenty grids from four different samples under identical conditions. Freeze‐drying with this device, as compared to air‐drying, considerably improved preservation of particles of plant viruses, and it was also suitable for other types of electron microscope specimens.
Measuring line length with the Quantimet 720 image analysing computer
Authors: Gaskell S. A. , Pearse A. D.
Published: August 2011

The line mode of the Quantimet 720 image editor used in conjunction with the MS3 computer to measure lengths of sloping or curved lines gives considerably inaccurate results. Depending on the degree of accuracy required, two methods are proposed for improving these results.
Cellular sieving by a natural, high‐flux membrane
Authors: Skaer R. J.
Published: August 2011

Human platelets can be sieved from plasma by means of insect peritrophic membrane.
Point‐projection imaging of macromolecular contours
Authors: Panitz J. A.
Published: August 2011

A point‐projection microscope is described which uses an electric field of several volts per nanometre to generate ions from a layer of benzene condensed in high vacuum on to the apex of a cryogenically cooled field‐emitter tip. Clusters of ferritin (a spherical protein molecule) embedded in the layer, have been imaged by exposing their contours during controlled field‐desorption. Image contrast is high. A magnification of 2 times 105 has been achieved at a spatial resolution estimated to be better than 3 nm. Three‐dimensional reconstructions of cluster morphology from a series of molecular contours are in qualitative agreement with transmission‐electron micrographs of the ferritin covered emitter apex. Reproducibility of the cluster contours, and TEM images of the apex before and after imaging suggest that the imaging procedure is nondestructive.
A new fixation procedure for preserving the ultrastructure of marine invertebrate tissues
Authors: Eisenman Edward A. , Alfert Max
Published: August 2011

Addition of 0·05% OsO4 to a conventional glutaraldehyde fixative for the first 10 min of fixation was found to improve greatly the preservation of ultrastructure in the eggs of Urechis caupo. Several workers have since confirmed this result in other marine invertebrate tissues. Specific protocols and techniques are given. We believe that the OsO4 rapidly renders the plasma membranes of cells freely permeable to glutaraldehyde, allowing faster penetration of this fixative. This method should be applicable to a wide variety of tissues that are difficult to fix.
Freeze‐substitution and isothermal freeze‐fixation studies to elucidate the pattern of ice formation in smooth muscle at 252 K (‐21°C)
Authors: Hunt C. J. , Taylor M. J. , Pegg D. E.
Published: August 2011

Taenia coli muscle was cooled to 252 K in the presence of the cryoprotectant dimethyl‐sulphoxide, at cooling rates known to reduce viability by significantly different amounts. The reduction in viability was known to be related to ice formation. Freeze‐substitution and isothermal freeze‐fixation studies were carried out to determine the distribution of ice within the muscle at this temperature. Freeze‐substitution using ethylene glycol was unsuccessful but a new method, using high concentrations of the cryoprotectant as the substituting solvent, was able to maintain ice configurations at this relatively high substitution temperature.

The results of freeze‐substitution in dimethylsulphoxide were confirmed by isothermal freeze‐fixation when both techniques were conducted under identical cooling conditions. The results indicated that the functional differences produced by cooling muscle at either 0·3 K min−1 or 2 K min−1 were related to the distribution of the ice phase within the tissue.
Low temperature scanning electron microscopy of fungal material
Authors: Beckett A. , Porter R. , Read N. D.
Published: August 2011

A preliminary evaluation has been made of different preparatory techniques used for the scanning electron microscope with regard to their application to a number of fungal specimens. A brief description of the use of the Emscope sputter cryo apparatus for the preparation of frozen‐hydrated specimens is given and the results obtained with this equipment are compared with those obtained by critical point‐drying and freeze‐drying. Perithecia from 11‐day‐old cultures of Sordaria humana, germinating conidia of Erysiphe graminis f.sp. hordei and uredosori and intercellular hyphae of Uromyces fabae were examined. In all cases the frozen‐hydrated specimens consistently showed the best preservation; particularly with regard to their external morphology, internal morphology, cell exudates and in relation to dimensional changes.
Freeze‐fracture electron microscopic analysis of solutions of biological molecules
Authors: Gulik A. , Aggerbeck L. P. , Dedieu J. C. , Gulik‐Krzywicki T.
Published: August 2011

Freeze‐fracture electron microscopy was used to study the morphology of proteins in solution. The sizes of the particles appearing on the fractured surfaces, replicated with tungsten‐tantalum, were measured in a direction perpendicular to the shadowing angle. The distributions of the measured particle sizes could be correlated with the known shape and dimensions for each protein. It is concluded that freeze‐fracture electron microscopy is a useful technique to study the morphology of biological molecules in solution, particularly hydrophobic proteins which may be difficult to study by other microscopic techniques.
Some experiences in the use of a polymeric cryoprotectant in the freezing of plant tissue
Authors: Wilson A. J. , Robards A. W.
Published: August 2011

Recently it has been suggested that polymeric cryoprotectants might be usefully employed for reducing ice crystal size during ultrastructural and analytical studies of frozen biological tissues. Furthermore, it was reported that they have little physiological effect and cause negligible structural changes in the tissue.

Our experiences with one such polymer, polyvinyl pyrrolidone (PVP), in the cryopreservation of mature plant roots prepared for electron microscopy, have led us to conclude that preservation deep into the structure of this tissue is not improved. Even short periods of exposure of tissue to polymer cause rapid withdrawal of water from vacuolated cells of plant roots, resulting in shrinkage and collapse.

Low temperature techniques have confirmed that little if any improvement in the reduction of ice crystal size results if the root is first treated with PVP.
A simple airlock system for hoar frost free specimen exchange in freeze‐etch vacuum plants
Authors: Niedermeyer W.
Published: August 2011

This paper describes a simple airlock system which allows hoar frost free quick specimen exchange on freeze‐fracture machines. The time needed for one preparation is reduced to about 15–20 min. The described modifications improve vacuum conditions and reduce the total water content within the recipient to less than 20% of the total residual gases. These conditions also enhance the topographic resolution of the specimen under research.
Freeze‐fracturing at low temperatures: I. A device for fracturing biological specimens at 77–10 K under high vacuum
Authors: Niedermeyer W.
Published: August 2011

Standard freeze‐etching or freeze‐cleaving is performed at 173 K in a vacuum of 133 μPa or at 77 K under liquid nitrogen with subsequent transfer of the specimen into a vacuum chamber. It has been suggested that the frequent poor resolution of morphological details, the poor complementarity of innermembrane protein particles and the semi‐crystalline substructures in biomembranes are caused by structural distortion or plastic deformation due to sheer forces which occur even at 77 K during fracturing or cleaving. In addition, water contamination and radiant heat damage occurring during replication introduce artefacts to the structural record. These artefacts could be avoided or reduced by lowering the temperature at which fracturing or cleaving and shadowing is carried out, to about 10 K. Therefore, a device for cleaving biological specimens at 15–10 K under high vacuum was constructed. To allow the use of existing equipment, the device was built into a standard Balzers 301 vacuum unit, where the specimen transfer is done via an airlock system which allows hoar frost contamination free transport of the specimen holder onto the specimen table.

To reduce or prevent the condensation of water and other residual gases in the vacuum onto the freshly cleaved specimen surface at 10 K, the specimen is surrounded by two cooled surfaces of 6 and 20 K. All condensable gases outside those shielding shrouds will condense on these surfaces before reaching the specimen. This makes it possible to work at a high vacuum of 3 μPa outside the cooled shrouds, which can be reached with standard turbomolecular pumps. The actual vacuum within the cooled shrouds is estimated to be approximately 13 nPa. Residual gas analysis before and during replication reveals equal conditions to ultra high vacuum systems.

An analysis of the yeast cell paracrystalline plasmalemma structure shows that the topographic resolution of the crystalline arrays has been improved by working at 12 K. However, plastic deformation still occurs under these conditions. This observation points to the possibility that what is described as plastic deformation, for at least some membrane proteins, may be a loss of resilience at low temperatures.
Replica production for scanning electron microscopy: a test of materials suitable for use in field settings
Authors: Scott Eugenie C.
Published: August 2011

Seven dental impression materials were tested in combination with five epoxy resins for the purpose of producing positive replicas suitable for use with the scanning electron microscope. Goals of the project were that the materials and procedures could be used in vivo, on high or low relief surfaces, involve a minimum of equipment, and produce high fidelity replicas. The combination of the dental impression material Xantopren with the resin Stycast 1266 was superior to all others tested at magnifications up to x 600.

The Stycast also has the additional advantage of not requiring a heat cure, which further minimizes equipment in the field.
Age dependence of collagen fibril and subfibril diameters revealed by transverse freeze‐fracture and ‐etching technique
Authors: Itoh T. , Klein L. , Geil P. H.
Published: August 2011

Collagenous tissues taken from tail tendons and cornea of freshly killed rats and rabbits, respectively, were fractured transversely and longitudinally at 80 K without any chemical pretreatment. The tissues were subsequently etched at 195 K for 30 min under a vacuum in the 130 μPa range. Carbon‐platinum replicas of the tissues were examined by transmission electron microscopy. It is demonstrated that the collagen fibrils are very closely packed and are composed of subfibrils whose size may vary with each tissue. Fibrils and subfibrils grow in diameter during growth and maturation but their size remains unchanged during the ageing process. The subfibril is suggested to be the growing unit in the collagen fibril based on the observation that the ratio of the diameter of fibril to subfibril does not change through growth, maturation and ageing. The ratio is approximately 10 for rat tail tendon and 5 for rabbit cornea and ear cartilage, suggesting the tissues from the two animals contain on the order of 100 and 25 subfibrils per fibril, respectively. It is proposed that the usual chemical fixation and dehydration processes used in conventional electron microscopy cause significant shrinkage in the diameters of subfibrils and fibrils.
Monolayer freeze‐fracture autoradiography: origins and directions
Authors: Fisher Knute A.
Published: August 2011

Monolayer freeze‐fracture autoradiography (MONOFARG) is a product of two earlier methods: freeze‐fracture autoradiography (FARG) and cell monolayer freeze‐fracture. MONOFARG incorporates many of the basic principles and cytochemical goals of FARG while exploiting the technical advantages of monolayer freeze‐fracturing. The latter method offers the opportunity to process freeze‐dried ‘half’ membranes at room temperature. Although the feasibility of MONOFARG has been shown for qualitative analyses of split membranes, its quantitative feasibility for transmembrane and in‐plane analyses has just begun to be documented. An example of one aspect of that documentation is included in this report. The distribution of 125I‐FITC‐Concanavalin‐A in the plane of split plasma membranes, human erythrocyte extracellular fracture faces, was examined and found to be homogeneous. The relevance of this finding to recently described double labelled membrane splitting experiments is discussed. The future of MONOFARG appears promising, especially in the application of the technique to biologically significant questions.
Optimum conditions for cryoquenching of small tissue blocks in liquid coolants
Authors: Elder H. Y. , Gray C. C. , Jardine A. G. , Chapman J. N. , Biddlecombe W. H.
Published: August 2011

Three approaches were taken with the aim of defining the optimum conditions for rapid cryopreservation in liquid quenchants. In a theoretical approach, two mathematical models were used. The first is of value in defining the absolute maximum rates of cooling which could be achieved at various depths in the tissues. The second highlights the poor thermal properties of liquid coolants and therefore emphasizes the essential requirement for vigorous quenchant mixing and rapid specimen entry.

Experimental work with thermocouples showed that fastest cooling rates occur at the leading edge of the object entering coolant. Of five liquid quenchants investigated, cooling rates were in the order, propane> Freon 22> Freon 12> liquid nitrogen slush> liquid nitrogen. Other considerations, however, may affect the choice of quenchant. For a given quenchant, cooling rate is maximal near the equilibrium freezing point. The consequences of quenching in the presence of thermal gradients either within the coolant or in the gas layer above it are shown. Cooling rate was found to be approximately proportional to entry velocity at least up to ∼2 m s−1 in our system.

Stereological analysis of rapidly quenched, freeze‐substituted tissue samples, of geometry which imposed an approximately unidirectional heat flow, revealed four zones: (i) a narrow surface layer (∼10 μm) of low image contrast and apparent absence of ice crystals; (ii) a zone of enhanced contrast with ice crystals whose size increased rapidly with depth from the surface (the ‘slope’); (iii) a sharply defined zone (the ‘ridge’) of maximum ice crystal size beyond which there is (iv) an extensive ‘plateau’ with smaller ice crystals and no marked increase in size with depth. The ‘ridge’ of maximal ice‐crystal damage was consistently found but varied considerably in depth from the surface (∼25–120 μm) between samples. The existence of the deeper plateau region of relatively uniform ice‐crystal‐size may be of significance in X‐ray microanalytical studies of physiological processes at some depth from the sample surface.

In terms of our present understanding of the quenching process, the conditions for optimal cryofixation of small tissue samples are listed.
Resin development for electron microscopy and an analysis of embedding at low temperature*
Authors: Carlemalm E. , Garavito R. M. , Villiger W.
Published: August 2011

We have tested a wide range of acrylate and methacrylate resin formulations and have concluded that embedding resins can be developed which are usable within a broad range of environmental conditions. To demonstrate this versatility, we have designed two highly‐cross‐linked resins, one polar and the other nonpolar, that are usable to temperatures from 243 to 223 K. Both of these resins formulations, which are now commercially available, show that systematic experiments can be easily done to study the effects of environmental parameters, such as water content, temperature, or resin polarity, on biological material during embedding. Using these resins and aldehyde‐fixed protein crystals, it can be shown that low temperature minimizes the loss of molecular structure to an extent that is not often obtainable with conventional methods of dehydration and embedding. Embedded crystals of aspartate aminotransferase still retained molecular order to 0·6 nm. Embedded crystals of catalase show X‐ray diffraction maxima out to 0·8 nm. When sectioned, catalase revealed stain‐limited electron and optical diffraction patterns to 2·5 nm. Nevertheless, our work clearly demonstrates that low temperature embedding procedures are superior and that versatile, general purpose resins can be designed to take advantage of this fact.
Estimating the effect of etching agents on plastic sections
Authors: Horobin Richard W. , Proctor John
Published: August 2011

The effects on epoxy embedding media of certain etching procedures have been studied in two ways. Firstly the resin present in thick sections, before and after etching, was demonstrated by staining the plastic with Sudan Black B. Secondly the change in weight of plastic blocks after their exposure to etching procedures was measured. Methods involving halogens in acetone, alkoxides in ethanol or in methanol‐benzene, and performic acid resulted in gross removal of plastic. Additionally, in keeping with the widespread belief that etching may increase permeability of plastic sections to stains, most of the etch methods studied, including organic solvents alone, did remove some plastic from the blocks.
Investigation of fading and recovery of fluorescence intensity at 73·5 K
Authors: Tiffe H.‐W. , Hundeshagen H.
Published: August 2011

Fading and recovery of Af‐feulgen stained sperm heads are investigated at 73·5 K. The fast fluorescence signals are measured and stored by two coupled transient recorders. 100% recovery was reached after a dark time of 3 s. This shows that the photodecomposition is mainly caused by change of the probability density of energy level and not by destruction of the chromophore groups. The recovery effect allows measurement of the fluorescence intensity of the same sample more than 50 times. Therefore the unaffected spectrum can be measured directly, provided that between the short‐term measurements at constant wavelength an appropriate dark phase has been put into operation.
Low‐temperature X‐ray microanalysis of the differentiating vascular tissue in root tips of Lemna minor L
Authors: Echlin Patrick , Lai C. E. , Hayes T. L.
Published: August 2011

The fracture faces of bulk‐frozen tissue offer a number of advantages for the analysis of diffusible elements. They are easy to prepare, remain uncontaminated, and, unlike most frozen‐hydrated sections, can be shown to exist in a fully hydrated state throughout examination and analysis. Root tips of Lemna minor briefly treated with a polymeric cryoprotectant are quench frozen in melting nitrogen. Fractures are prepared using the AMRAY Biochamber, lightly etched if necessary to reveal surface detail and carbon coated while maintaining the specimen at 110 K. The frozen‐hydrated fracture faces are analysed at 110 K using the P/B ratio method which is less sensitive to changes in surface geometry and variations in beam current. The method has been used to investigate the distribution of seven elements (Na+, Mg++, P, S, Cl−, K+ and Ca++) in the developing vascular tissue of the root tip. The microprobe can measure relative elemental ratios at the cellular level and the results from this present study reveal important variations in different parts of the root. The younger, more actively dividing cells, appear to have a slightly higher concentration of diffusible ions in comparison to the somewhat older tissues which have begun to differentiate into what are presumed to be functional vascular elements.
On the size of small protein particles determined by electron microscopy of unidirectionally shadowed freeze‐etched preparations
Authors: Schmidt D. G. , Buchheim W.
Published: August 2011

The influence exerted by the thickness of the deposited metal layer and the ionic strength of the solution on the apparent size of particles of bovine serum albumin in unidirectionally shadowed freeze‐etch preparations of spray‐frozen specimens was investigated. It appeared that the size increase due to shadowing is nearly twice the thickness of the deposited metal layer. Apparent particle size was shown to increase linearly with the inverse square root of the ionic strength of the solution. At ionic strength 0·001 the particles appeared about 30% larger than at infinite ionic strength.
On‐line digital computer techniques in electron microscopy: general introduction
Authors: Smith K. C. A.
Published: August 2011

Digital computers may be interfaced to electron microscopes in a variety of ways to provide on‐line facilities for the acquisition, storage, processing and display of data. The data may be in the form of either images or spectra, the former necessitating a large amount of storage or memory space within the computer system. This has led to the development of special‐purpose digital memory systems, known as framestores, capable of holding one or more complete TV size images. Framestores can be used for both image acquisition and image display at normal TV frame rates. On‐line computer systems with, and without, framestores are being applied increasingly in scanning and conventional electron microscopy. The introduction of new computer technology, coupled with a reduction in costs, appears likely to accelerate this trend.
Reduction of noise in TV rate electron microscope images by digital filtering
Authors: Erasmus S. J.
Published: August 2011

Digital framestores can be used to reduce noise in TV rate electron microscope images. Three operating modes: averaging, recursive filtering and Kalman filtering have been investigated. It is shown that there is an optimum choice of filter parameters.