bluuebunny/crossref_metadata_2025 huggingface dataset:
On crystal size and cooling rate
Authors: Bald W. B.
Published: August 2011

A theoretical model is proposed which is used to derive a quantitative relationship between the critical cooling rate and average crystal size at any location within a biological specimen of given shape subject to rapid freezing. The model is applicable to the slamming, plunging or spraying methods of cryofixation provided the ice crystal size is at least 5 times greater than the size of the critical nucleus.

Complete vitrification of pure water or aqueous solutions is shown to take place at cooling rates in excess of about 3 × 106 K/s.
Specimen preparation technique for high resolution transmission electron microscopy studies on model supported metal catalysts
Authors: Jacobs J. W. M. , Verhoeven J. F. C. M.
Published: August 2011

A new technique is described which can be used for preparing transmission electron microscopy (TEM) specimens suitable for high resolution studies on supported metal catalysts. By conventional silicon processing techniques 200 × 200 μm2 Si3N4 membranes on Si wafers are produced. These membranes are extremely flat and have a uniform thickness of 13 nm. They can be used as a support in various kinds of thin film deposition. A TiO2 film, optimally structured with respect to the requirements for high resolution TEM work in TiO2–metal cluster systems, is deposited on the Si3N4 layer. It consists of one monolayer of 10–25 nm TiO2 crystallites. TiO2 lattice images show that a line resolution down to 0.19 nm is possible. Examples of TiO2–Pd and TiO2–Rh are given using respectively photodeposition and impregnation reduction to produce l.5–4 nm metal clusters.
Application of cryoultramicrotomy to immunocytochemistry
Authors: Tokuyasu K. T.
Published: August 2011

This paper reviews the most recent status of immuno‐cryoultramicrotomy. The technical aspects of each step of the method are also analysed in detail with the intention of providing a useful source of information for investigators using this method.
Immunogold‐silver staining of lymphocyte surface antigens on cells in suspension and in lymph node cryostat sections
Authors: De Waele M. , De Mey J. , Renmans W. , Labeur C. , Reynaert Ph. , Van Camp B.
Published: August 2011

An immunogold–silver staining (IGSS) technique for the light microscopical detection of leucocyte cell surface antigens in cell suspensions and cryostat sections is described. The specimens were first incubated with monoclonal mouse antibodies and then with colloidal gold‐labelled goat anti‐mouse antibodies. They were then immersed in a physical developer, counterstained and mounted. In light microscopy, the tissue architecture and the cellular morphology were well preserved. Positive cells showed dark granules on their surface membranes. Optimal labelling conditions were determined. This method proved to be a reliable tool for the enumeration of T‐cells and their subsets in peripheral blood. The dense labelling permitted the use of panoptic counterstains like May‐Grünwald‐Giemsa or Wright's stain. This IGSS technique was used to determine the distribution of the T‐ and B‐cell subsets in cryostat sections of reactive lymph nodes. The sensitivity of the method was comparable with that of immunofluorescence microscopy for cell suspensions and that of the biotin–avidin–peroxidase technique for tissue sections. Immunogold–silver staining was combined with enzyme cytochemistry. In dark‐field or epipolarization microscopy the labelling appeared as bright granules on a dark background. With its dense granular membrane labelling and its good morphology IGSS is an ideal method for the study of particular cell types in mixed cell suspensions. In addition, it could be a general method for the detection of cell surface antigens in all kinds of cells and tissues.
Problems in the production and use of 5 nm avidin‐gold colloids
Authors: Morris Randal E. , Saelinger Catharine B.
Published: August 2011

Over the past 5 years we have encountered several problems in the production and use of 5 nm avidin‐gold colloids for markers in electron microscopy. These problems include flocculation of colloids during reduction of chloroauric acid, insoluble gold pellets following ultracentrifugation, and non‐specific binding of avidin‐gold colloids to biological membranes. We are able to avoid these problems by: (1) avoiding the use of crystalline chloroauric acid; (2) succinoylating egg white avidin prior to adsorption on the gold sols; (3) resuspending the pellets following ultracentrifugation in 5 mM phosphate buffer, pH 7.5; and (4) using the avidin‐gold colloids within 4 weeks of production.
Simultaneous immunoenzyme double labelling using two different enzymes linked directly to monoclonal antibodies or with biotin‐avidin
Authors: Boorsma D. M. , VanBommel J. , VanderRaaij‐Helmer E. M. H.
Published: August 2011

A novel and rapid method for immunoenzyme double staining with monoclonal antibodies (McAb) is described. The principles of this method are the simultaneous application of primary antibodies and the unrelatedness of the two detection systems. One McAb is directly labelled with horseradish peroxidase and the other McAb is labelled with biotin. This second McAb is thereafter detected using an avidin‐alkaline phosphatase conjugate. This conjugate was prepared by a new method using a heterobifunctional reagent. Double staining of cell surface membranes of human tonsil was studied in cryosections using various combinations of McAbs to lymphoid cell markers. As expected, suppressor T cells were found to be contained within the pan T cell population on the basis of the distinguishable intermediate colour produced. Similarly, it was shown that most of the suppressor T cells were not HLA‐DR activated in this tonsil. In cryostat sections of human skin T6 positive Langerhans cells in the epidermis were shown to carry the HLA‐DR antigen.
Immuno‐ and enzyme‐histo/cytochemical analysis of resin sections and cell suspensions: a comprehensive diagnosis of bone marrow on a single aspiration sample
Authors: van de Sandt M. M. , Herman C. J. , Lindeman J.
Published: August 2011

A protocol is described in which a single bone marrow aspiration specimen is used to prepare resin sections and cell suspensions. Using this protocol, a full battery of morphological, enzyme‐histochemical, immuno‐histochemical and cyto‐pathological techniques can be applied. This allows a definitive bone marrow diagnosis to be established without resorting to bone marrow biopsy in the majority of patients.
Inhomogeneity in epoxy resin standards for quantitative biological X‐ray microanalysis
Authors: Condron R. J. , Marshall A. T.
Published: August 2011

summary

It is shown by X‐ray point analyses and line scans that the concentrations of sodium and potassium, as crown ether complexes, in epoxy resin may not be of uniform distribution. The concentrations may be substantially higher in a thin layer at the base of the block. It is recommended that chemical analysis of a selected central region of a block, not the intact block, be carried out to establish the true concentration. This may be substantially lower than the nominal concentration. This problem appears to be less acute with cryptate complexes of sodium and potassium but a similar trend is nevertheless apparent.
Automatic description of microstructures
Authors: Gallerneault C. E. , Kulick J. , Powley G.
Published: August 2011

This is the third in a series of three papers concerned with developing automatic analysis and classification techniques for metallographic images (specifically binary eutectic alloys of aluminium). The goal of the developmental work was to capture and describe image structure, in the descriptive terminology of a metallographer, and to permit quantification of formerly descriptive parameters. The first paper dealt with the preprocessing stage of image analysis and entailed a study of two segmentation techniques. The second paper reported on work demonstrating the use of texture analysis to characterize structural aspects in images of metallic microstructures. In this paper the textural primatives, or phase particles, are subjected to shape analysis and a novel mechanism for within class characterization. The study of the distribution of these shapes may provide a new means of following the effects of mechanical processing.
Elemental concentrations in isolated rat thymocytes prepared for cryofixation in the presence of different media
Authors: Warley Alice
Published: August 2011

Rat thymocytes were isolated in suspension and the effect of preparing the cells for cryofixation in the presence of different media on the elemental content was investigated using the technique of X‐ray microanalysis. Cells prepared in the different media showed variation in the concentrations of Na, K and Cl. The isolated cells were incubated at 310 K for 1 h to allow recovery from isolation. There was a decrease in Na and Cl content after incubation. The thymocyte population was disturbed by suspension in medium containing dextran, and this resulted in a number of cells with high concentrations of Na and low concentrations of K. These cells did not take up vital dye. Thymocytes were also prepared for freezing by using high‐speed centrifugation to concentrate the cells. Thymocytes prepared by this method showed values for concentrations of Na, K and Cl similar to published values for these cells using other methods of estimation. There were, however, consistent differences in Na content between the cells prepared in Hanks' balanced salt solution and those prepared in serum. Factors which affect the apparent concentration of Na and Cl in isolated cells are discussed.
A rapid method of preparing perforated supporting foils for the thin carbon films used in high resolution transmission electron microscopy
Authors: Lünsdorf H. , Spiess E.
Published: August 2011

A simple and highly reproducible working technique is described for preparing holey foils as supports for the carbon films used in high resolution electron microscopy. By this method perforation of the Collodion film is accomplished resulting in a net. The diameters of single holes range from 1·8 to 7·9 μm with an average of 4·5 μm.
The application of X‐ray microscopy in materials science
Authors: Cunningham T. G. , Davies R. Ll. , Graham S. C.
Published: August 2011

X‐ray microscopy is a form of high resolution radiography that uses low‐energy X‐rays (≤10 keV) to enhance the contrast between light elements such as hydrogen, carbon, nitrogen and oxygen. As performed on compact laboratory equipment the technique can achieve spatial resolutions of roughly 1 μm in virtually any material given that the specimen is sufficiently thin (typically 0·2–2 mm) to be adequately transparent to low‐energy X‐rays. Notwithstanding that the technique has lately found favour in biomedical radiology, its considerable potential in other fields, notably the materials sciences, remains largely unexploited.

The scope and potential of laboratory X‐ray microscopy in the materials sciences is demonstrated here by its application to ceramics, elastomers, coal‐chars and reinforced composites. In all cases the technique provided valuable microstructural characterization often unobtainable by any other non‐destructive method. These examples demonstrate that laboratory X‐ray microscopy offers much to the materials sciences and deserves a wider application than is current.
Quantitative image analysis of finite porous media
Authors: Macdonald I. F. , Kaufmann P. , Dullien F. A. L.
Published: August 2011

Reconstruction, from serial sections, of a branch‐node chart containing the connection pattern of the pore channels in small sandstone samples, and computation of genus are described.

Seventy‐eight serial sections were obtained by serially grinding off layers of about 10 μm thickness from a 1 × 1 mm sample, whose pores had been filled with Wood's metal, and taking photomicrographs of the polished sections. Edge effects and rock enclosed by pore space were accounted for in the construction of the branch‐node chart. The importance of boundary effects was reflected by the difference between the minimum and maximum estimates of genus, Gmin and Gmax, respectively.

The Berea sample (with porosity of 23% and average grain size of about 200 μm) was found to have essentially one main network containing over 87% of all pore space features, plus a number of very much smaller boundary networks representing ‘dents' on the sample sides. The number of anomalies from missed connections was found to be small and unlikely to cause significant error in the genus calculations. Both Gmin and Gmax for the sample were found to be essentially those of the one continuous main network.
Analog enhancement of videomicroscope images
Authors: Burton J. L. , Bank H. L.
Published: August 2011

A simple, inexpensive technique for enhancing the contrast and resolution of videomicroscope images has been developed. The system has manual controls for gain and pedestal (black level) which permit expansion of low contrast images to the full white‐to‐black video range. Analog delay‐line based circuits are used to sharpen the edges and enhance fine details in the image. These circuits also produce an effective increase in the information content of the image by selectively amplifying low amplitude, high frequency components of the video signal. When live, unstained cells were examined at high magnifications, cytoplasmic structures which were only faintly visible in the unenhanced image became clear. The images of fluorescent objects appear in pseudo‐relief, which improves visibility even in the presence of background fluorescence. The system enhances images by performing signal processing functions that otherwise require expensive digital image processing equipment.
Why low temperature embedding for X‐ray microanalytical investigations?
Authors: Wróblewski Joanna , Wróblewski Romuald
Published: August 2011

Freeze‐drying followed by infiltration with resin and polymerization by UV light at low temperatures and under constant vacuum conditions is an alternative tissue preparation technique for microprobe analysis. Embedding is carried out with the nonpolar low‐temperature embedding resin (Lowicryl HM20) which allows infiltration and polymerization at temperatures down to −50°C. Sections of low temperature embedded material can be cut dry at −60°C or at room temperature. Sectioning at low temperatures is an alternative for preparations that are difficult to cut at room temperature.

The morphological preservation is adequate for the identification of structures such as mitochondria, lysosomes and different types of endoplasmic reticulum in liver cells. Some physical properties of Lowicryl resins, such as mass loss under the electron beam and high contrast, are positive characteristics for the analysis of semi‐thick sections. No significant differences in the elemental composition could be detected between tissue which was freeze‐dried or freeze‐substituted prior to embedding. Freeze‐drying is less time consuming. By avoiding contact with organic solvents the risks of ion loss and redistribution are diminished. In contrast to freeze‐dried thin cryosections, low temperature embedded material can be sectioned for light microscopy and areas of interest chosen for further thin sectioning. This is of great importance in work with tissues with complicated morphology and heterogeneous cell populations. The initial preparative step—the cryofixation— determines to a high degree the morphological preservation of freeze‐dried and embedded tissue.
Water content from analysis of freeze‐dried thin sections
Authors: Warner Ronald R.
Published: August 2011

The analytical electron microscope and related electron beam instruments are uniquely able to measure the water content of biological tissue at a subcellular level. An analysis of the Rick procedure for quantifying this water content (Dörge et al., 1978; Rick et al., 1979) reveals a hidden assumption; namely, that the sample and standard densities, in the hydrated state, must be equal. In general this will not be valid. Equations are derived that do not invoke this hidden assumption. These equations show that errors in using the Rick technique for measuring water content should be small, a few per cent, unless the sample contains a very heterogeneous distribution of mass or the water content of the standard is a poor representation of the sample. For freeze‐dried samples with extensive mass heterogeneity, such as skin, the equations for calculating water content presented in this paper should be used.
Quantitative electron microscope autoradiography: application of multiple linear regression analysis
Authors: Markov Dimiter V.
Published: August 2011

A new method for the analysis of high resolution EM autoradiographs is described. It identifies labelled cell organelle profiles in sections on a strictly statistical basis and provides accurate estimates for their radioactivity without the need to make any assumptions about their size, shape and spatial arrangement. The radioactivity in interfacial membranes and transitional junctional regions between pairs of adjoining cell structures can be determined without any additional measurements. The uniformity of internal labelling of large cell organelles can be also assessed. Correcting for cross‐fire does not need any predictive information from a frequency distribution function describing the image spread about a radioactive point source. Instead, the section area is subdivided into regions in such a way that each region with cross‐fired silver grains over it includes also the sites of the radioactive disintegrations that have produced them. The method is based on the quantitative regression relationship between the number of developed silver grains overlying any given region and the size of the cell organelle profiles included. A transparent overlay screen bearing a regular array of circles and a point‐counting procedure are applied to divide the section area into regions and to measure their area and the size of the cell organelle profiles. The least squares method is used for the simple and rapid calculation of specific activity estimates and exact standard errors to be attached to them. A modest computing facility and a standard library program are required. Simulation modelling and analysis of residuals were used to lend support to the validity of the method.
Direct imaging of paracrystalline phospholipid structure in the electron microscope
Authors: Fryer J. R. , Dorset D. L.
Published: August 2011

A long chain amphiphilic molecule—the phospholipid 1,2‐dihexadecyl sn glycerophosphoethanolamine—has been crystallized epitaxially so that the interlamellar molecular periodicity is parallel to the substrate and hence normal to the electron beam in the electron microscope. This has permitted the direct resolution of the 55·6 Å lamellae in unstained crystals at room temperature.

The lattice images have shown the presence of line dislocations and lenticular cracks in the crystals. Of significance to their biological properties is that the lattice is undulating with a periodicity of 0·1–0·5 μm. This would also account for the difficulties encountered by X‐ray and electron diffraction techniques when examining these crystals.
Scanning acoustic microscopy visualizes cytomechanical responses to cytochalasin D
Authors: Bereiter‐Hahn Jürgen
Published: August 2011

The reactions of XTH‐2 cells (line derived from Xenopus laevis tadpole hearts) to cytochalasin D (CD) were followed using scanning acoustic microscopy (SAM) at 0.9 GHz, fluorescence and electron microscopy. The first reaction to CD which can be detected by SAM is a loss of image contrast, indicating a decrease in acoustic impedance of about 30%. Based on structural changes revealed by staining of actin with TRITC‐phalloidin, and taking theoretical considerations into account, a relationship between impedance decrease and tension in the actin fibrillar system is deduced.
Microcomputer‐aided reconstruction: a system designed for the study of 3‐D microstructure in histology and histopathology
Authors: Yaegashi Hiroshi , Takahashi Tohru , Kawasaki Masami
Published: August 2011

We have designed a microcomputer system to reproduce 3‐D tissue structures graphically from serial microscopic sections. The system, based on a Hewlett‐Packard model 310 desktop computer, comprises a floppy disc drive, a hard‐disc that extends the available user memory by adding 40 Mbytes, a colour memory‐mapped graphics display, a graphics tablet and a graphics printer. A set of serial 2‐D images, manually extracted from serial sections using a microprojector, are digitized on the tablet by tracing the contours of the structure of interest; up to ten different structures can be inputted and reproduced using a ‘hidden line’ effect. The software, written mainly in HP‐BASIC 4.0, produces 3‐D pictures of a tissue structure either as a ‘stack of slices' with or without being ‘tiled’ with triangular patches, or in ‘wire framing’. Any angle of rotation around the x, y and z axes is assigned for the image to be reconstructed, allowing the operator to obtain the best perspective. The system also discloses the internal connectivity of an object by reducing the structure to a network diagram; this is especially useful in analysing the topological properties of tissue structure. It is shown how, in some examples, the system contributes to a better understanding of tissue microstructures and their morbid changes and how, as an effective tool in morphology, it will aid future studies of histology and histopathology.
Estimation of organelle water fractions from frozen‐dried cryosections
Authors: Zglinicki Thomas von , Bimmler Martina , Krause Werner
Published: August 2011

Local dry mass or water fractions can be measured on frozen‐dried cryosections assuming constant section thickness in the hydrated state and no net water movements and no differential shrinkage during freezing and drying. These assumptions have been tested on a model consisting of isolated rat liver mitochondria in an albumin matrix with a concentration similar to the dry mass concentration of the cytoplasm.

The dry mass concentrations of mitochondria before freezing as measured by interference microscopy and after freezing and freeze‐drying of the sections as measured by X‐ray microanalysis and scanning microdensitometry are shown to be equal as long as the ice crystals in the medium are smaller than about 100 nm. It is concluded, therefore, that the above‐mentioned assumptions could also hold for the cryopreparation of cells and tissues.
An evaluation, by Fourier analysis, of some of the errors introduced in reflectance measurements
Authors: Marcos C. , Virgos J. M. , Nicieza C. G.
Published: August 2011

In order to evaluate the errors which can be introduced in the measurement of reflectance, we have made a study of data obtained for several minerals and in two immersion media on turning the microscope stage to a succession of positions relatively orientated by 45°. Fourier analysis of the results allows an estimate of the different errors as a function of the periodicity with which they appear on a full rotation of the stage.

The mean magnitude of two of the Fourier coefficients is shown to provide a measure of the quality of the measurements further allowing evaluation of the equipment used, the orthogonality of the sample in relation to the incident light, and other errors of different periodicity.
Quantitative digital X‐ray imaging using frozen hydrated and frozen dried tissue sections
Authors: Saubermann Albert J. , Heyman Robert V.
Published: August 2011

Application of quantitative X‐ray imaging to frozen hydrated tissue sections has presented a number of major problems including lack of a suitable algorithm which could deal effectively with mass loss due to radiation damage, problems of low characteristic X‐ray signal to background ratios, and provide a means of analysis of the same location in both hydrated and dried states. This paper presents details of the application of our algorithm for analysis of frozen hydrated, then dried cryosections applied to quantitative X‐ray imaging, which provides relatively high precision quantitative measurement of elemental content (related to both wet and dry weight) and water content of each pixel. This algorithm largely circumvents many of the problems of analysis of frozen hydrated tissue sections. Our algorithm for X‐ray imaging obtains reasonably precise quantitative measurements coupled with morphological information by trading speed and image resolution.
What has the Penrose tiling to do with the icosahedral phases? Geometrical aspects of the icosahedral quasicrystal problem
Authors: Mackay A. L.
Published: August 2011

The Penrose tiling is indeed a geometrical model appropriate for the description of the phenomena of icosahedral quasicrystals, although it is probably a related example, rather than the central theory itself, but many aspects of the theory remain to be developed. The transition from X‐ray crystal structure analysis (XRCSA) to high resolution electron microscopy (HREM) as the paradigmatic method for the investigation of crystal structures has facilitated this development and promises other advances from classical crystallography.
High‐resolution electron microscopy of Al‐Mn‐Si icosahedral and Al‐Mn decagonal quasicrystals
Authors: Hiraga K. , Hirabayashi M. , Inoue A. , Masumoto T.
Published: August 2011

Al‐Mn‐Si icosahedral and Al‐Mn decagonal quasicrystals have been studied by high‐resolution electron microscopy. High‐resolution micrographs of the Al‐Mn‐Si icosahedral quasicrystal taken with the incident beam along the five‐, three‐ and two‐fold symmetry axes are well interpreted on a three‐dimensional Penrose tiling model. An image of the Al‐Mn decagonal quasicrystal with the incident beam parallel to the ten‐fold symmetry axis shows a pattern of an aperiodic tiling with two types of rhombs which can be distinguished from the Penrose tiling using matching and inflation rules. The Al‐Mn decagonal quasicrystal examined coexists coherently with that of icosahedral symmetry exhibiting a definite crystallographic orientation relationship.
Quasicrystalline grain boundary precipitates in aluminium alloys through solid‐solid transformations
Authors: Cassada W. A. , Shiflet G. J. , Poon S. J.
Published: August 2011

We have observed the formation of icosahedral quasicrystals of Al‐Mg‐Zn, Al‐Mg‐Cu, Al‐Li‐Zn and Al‐Li‐Cu by low temperature annealing of solid solution alloys. The icosahedral phase particles form at the grain boundaries of the matrix alloys. The orientational relationships between the icosahedral crystallites, their corresponding Frank‐Kaspar phase particles and the aluminium matrix are reported. The stability of the icosahedral phases is also discussed. Finally, the importance of grain boundary icosahedral phase particles to the physical properties of commercial aluminium alloys is pointed out.
Techniques for combining light microscopy and scanning electron microscopy: a survey of the literature
Authors: Wouters C. H.
Published: August 2011

A survey of methods combining light microscopy and scanning electron microscopy is presented. A simple correlation is made when two preparations from adjacent parts of one specimen are investigated in two different microscopes. A more sophisticated method is the consecutive investigation of one specimen with two microscopes. A major problem in this method is the relocation of the area of interest. Several authors have presented solutions for this problem. It is preferable when one preparation is investigated in only one instrument, combining the two microscopical (LM and SEM) techniques, thus making relocation redundant.
Experimental conditions affecting coherent bremsstrahlung in X‐ray microanalysis
Authors: Vecchio Kenneth S. , Williams David B.
Published: August 2011

Coherent bremsstrahlung (CB) peaks from several metallic crystals have been observed in X‐ray spectra obtained in the analytical electron microscope. The dependence of CB peaks on certain microscope and specimen parameters was studied with the aim of being able to distinguish CB peaks from small elemental peaks in the X‐ray spectra. It was found that although CB generation is unavoidable in thin crystals, careful choice of experimental conditions allows the detection of CB to be controlled and minimized.
Influence of some practical factors on background extrapolation in EELS quantification
Authors: Liu D‐R. , Brown L. M.
Published: August 2011

The detectability of a minor element with EELS in a STEM is of great interest. This paper first describes errors resulting from a conventional background extrapolation scheme and other faults in the collected spectra which in effect greatly reduce the seemingly good signal to noise ratio of a spectrum and therefore impose much more stringent statistical counting requirement on spectra if a small amount of element is to be quantified with good accuracy. Secondly, the systematic variation of the index r of the power law background fitting curve of an ionizatoin edge tail is discussed. Its effect on the detectability of a minor element is analysed. It puts a lower limit on the detectability of an element, however many counts are accumulated. Also a scheme is proposed of some non‐conventional linear least squares fits by which one might suffer from less quantification error due to background extrapolation and achieve lower detectability of a minor element.
Electron beam modifications of InP surfaces
Authors: Milne R. H. , Fan T. W.
Published: August 2011

InP surfaces have been modified by using the intense electron beam of a V.G. HB501 STEM and subsequently examined in the same instrument using the reflection microscopy geometry. The surface changes can be divided into two parts; first, a fairly local removal of material, and second, more widespread effects including faceting and the formation of an oxide layer which extends for several microns around the point of incidence of the electron beam.
Preparation of the mammalian organ of Corti for scanning electron microscopy*
Authors: Davies Stephen , Forge Andrew
Published: August 2011

The effect of various standard tissue preparation procedures for scanning microscopy on the morphology of the mammalian organ of Corti has been examined. Comparison was made of (i) different fixatives: glutaraldehyde (GA) alone, osmium (Os) alone, GA and Os together and GA followed by Os; (ii) freeze‐drying (FD) versus critical point‐drying (CPD); (iii) ligand binding of osmium with thiocarbohydrazide (TOTO procedure) versus sputter coating to provide a conductive coat. Results were assessed in terms of known structural parameters in fresh tissue. It is shown: (i) that double fixation, either GA, Os together or GA followed by Os, consistently produced less tissue distortion than either fixative alone, (ii) that FD produced less shrinkage of the tissue than CPD, and (iii) that the TOTO procedure appeared to rigidify the tissue and enhanced preservation of the morphology.
Time lapse video microscopy using an animation control unit
Authors: Allen T. D.
Published: August 2011

A system for time lapse video microscopy incorporating an edit recorder and animation control unit is described, allowing both colour recording, forward and reverse playback without synchrony bars, and the identification of each individual frame recorded. This is achieved by using the edit recorder, controlled by the animation unit to produce continuous series of insert edits using the preroll facility of the recorder. A feature of this system is the preview of the last hour' s recording which is constantly played as the preroll of the edit recorder. Tissue culture flasks modified for stable focus are also described.
Scanning low energy electron loss microscopy (SLEELM)
Authors: Gomati M. M. El , Matthew J. A. D.
Published: August 2011

High resolution scanning low energy electron loss imaging of a well characterized system of W on Si is demonstrated in reflection geometry at a primary electron energy Ep of 740 eV and spatial resolution of 3 μm. Characteristic losses below 60 eV are chosen to examine contrast between two types of regions (tungsten and silicon dominated, respectively). By differencing between a maximum in loss intensity and an adjacent trough, high quality chemical images are obtained with contrast far superior to Auger electron images produced in comparable times. It should be noted that, in contrast to scanning Auger microscopy, the spatial resolution of SLEELM is limited only by the profile of the incident beam. In addition, the technique is readily capable of chemical analysis at the surface of bulk specimens. A further advantage of low electron loss imaging is that the depth resolution may be varied by altering the primary electron energy, with the possibility of bridging the gap between Auger electron microscopy and X‐ray microprobe analyser. Possible disadvantages of the technique lie in the weakness of the elastic backscattering which is essential to observe the losses in reflection geometry, and the possibility of overlapping low energy characteristic loss peaks, an effect which is, however, less bad than for low energy Auger peaks. It is shown that electron probes with characteristics rather different from those currently used in Auger microscopes are required to optimize the technique, but that SLEELM has the potential to be a very useful form of surface microscopy which will complement other surface imaging techniques.
Scatter diagrams in energy analysed digital imaging: application to scanning Auger microscopy
Authors: Gomati M. M. El , Peacock D. C. , Prutton M. , Walker C. G.
Published: August 2011

In order to improve methods for the systematic characterization of inhomogeneous materials the procedures of multi‐spectral imaging and scatter diagram construction have been deployed. Although the techniques described are relevant to all instruments which detect signals on several different information channels (e.g. wavelength or energy analysed optical, X‐ray or electron imaging), they are illustrated with scanning Auger microscopy (SAM). The construction and use of bivariate correlation diagrams is described by reference to simple samples consisting of patterns of Al film upon Si substrates. The method is then applied to LaNi5 and GaInAs samples each with different signal/noise ratios and chemical characteristics. The software windowing of scatter diagrams by computer combined with presentation of false colour images is demonstrated. The multi‐spectral Auger mapping (MUSLAM) procedure thus evolved is demonstrated to be a powerful, general analytical technique for characterizing the number, abundance and chemistry of each type of region in the surface of an inhomogeneous solid.
A simplified ‘sandwich’ technique for in situ embedding and perpendicular sectioning of monolayer cultures of human skin fibroblasts
Authors: Govaerts P. J. , Bernaer I. I. , du Caju M. V. L. , Jacob W. A.
Published: August 2011

In the processing of cell cultures, grown as a monolayer in tissue culture dishes for electron microscopy, the sectioning of the monolayer is an essential step. The monolayer can be sectioned either parallel or perpendicular to the plane of growth. Several methods for the perpendicular way of sectioning have already been described. We propose a simplified method in which the monolayer is sandwiched between two layers of resin, one of which is a pre‐polymerized block, the other being a layer of resin, applied at a second stage. Sectioning of this ‘flat embedded’ specimen yields thin sections perpendicular to the plane of growth of the monolayer without elaborate orientating procedures. The advantage of this procedure is that it can be done using only routine embedding techniques, avoiding special materials or complex manipulations. This sandwich technique provides an excellent mechanical fixation of the monolayer and protects it against external damage.
Horizons for new semiconducting materials*
Authors: Goodman C. H. L.
Published: August 2011

The properties of existing semiconductors limit performance in a whole range of devices and leave many interesting potential applications (e.g. for lighting, power generation or air‐conditioning) possible in principle but technologically impracticable. Better properties, then, could be valuable. They could be obtained in two ways, by discovering new materials (a long‐range option apt for academic investigation) or by modifying existing materials either chemically (e.g. by forming solid solutions, perhaps of highly unconventional type) and/or physically (e.g. by low dimensional confinement effects). These options are reviewed and some of their implications explored.
Illumination and detection systems for quantitative fluorescence microscopy
Authors: Spring Kenneth R. , Smith Paul D.
Published: August 2011

Systems are described for the illumination and detection of light microscope images of fluorescence in living cells at low levels of excitation. The illumination system is based on the use of an acousto‐optic modulator to control the wavelength and power of the excitation beam. The detection system utilizes an image intensifier coupled with a television camera to acquire images at extremely low intensities. Both the illumination and detection systems have superior performance compared to existing devices.
Dark‐field electron microscopy of unstained biological materials embedded in Nanoplast
Authors: Frösch Dieter , Westphal Christel , Bauer Richard
Published: August 2011

Extremely thin sections of unstained materials (beef liver catalase, double‐stranded calf thymus DNA, horse spleen ferritin and mammalian skeletal muscle), embedded in the water‐soluble melamine resin Nanoplast FB101, were studied by dark‐field electron microscopy and electron spectroscopic imaging.

While ferritin molecules so recorded show 04 and 09 nm lattice fringes within the crystalline iron core, double‐stranded DNA shows a helical repeat with a spacing of 34 nm.

The gain in resolution of structural detail reported here is probably due mainly to the reduced section thickness as compared to traditional thin‐sectioning techniques. As we reported earlier (Frösch & Westphal, 1984), melamine resins can be sectioned extremely thinly (<10 nm) and observed without a supporting film, making them especially suitable for dark‐field electron microscopy.
Upside‐down etching; a simple method for reducing unetchable sediment during deep‐etching of fresh untreated tissues
Authors: Shibata Y. , Izumi T. , Yamamoto T.
Published: August 2011

A simple new method of deep‐etching, ‘upside‐down etching’, has been developed in an attempt to obtain clearer images or replicas, especially of fresh, untreated biological tissues. During etching, specimens were kept facing downwards and shaken periodically so that non‐volatile materials in the subliming ice could fall out of the frozen tissue. This improvement leads to a considerable reduction of sedimented debris of unetchable materials on ‘true’ membrane surfaces, as well as in the cytoplasm exposed by deep‐etching.
A simple method of measuring the thickness of semi‐thin and ultra‐thin sections
Authors: Bedi K. S.
Published: August 2011

The exact measurement of the thickness of semi‐thin and ultra‐thin sections is of importance for some stereological methods. Although section thickness can be measured with great precision and accuracy using suitable microinterferometers, such instruments are expensive and not easily available to many laboratories. This paper describes a simple method of measuring the thickness of sections actually used for stereological counting procedures. It involves the division of any given section into two portions, one of which is used for the counting procedures. The other portion is re‐embedded and re‐sectioned through its thickness making it a simple task to measure its thickness.
Failure analysis of plastic encapsulated components—the advantages of IR microscopy*
Authors: Brown J. , Footner P. K. , Richards B. P.
Published: August 2011

This paper demonstrates that the use of an IR microscope in non‐destructive failure analysis of plastic encapsulated devices overcomes many of the problems previously associated with conventional failure analysis techniques. Moreover, the devices are amenable to subsequent examination and testing. Its advantages are highlighted in a number of case histories, concerning bond quality, corrosion and features of poor processing. The consequential modification to failure analysis methodology and its applicability to VLSI devices are discussed.
The projection of a negatively‐stained filamentous object down its central axis as revealed by image reconstruction from tilt series
Authors: Seymour J. , DeRosier D. J.
Published: August 2011

Helical reconstructions of negatively stained biological objects contain distortions arising from filament flattening and the non‐cylindrical profile of the stain envelope. Current methods of estimating flattening do not make full use of the information available in electron micrographs. We have applied the more rigorous approach of reconstructing the density profile from tilt series using digital image processing techniques.

Tilt series were collected for tobacco mosaic viruses (TMV) and seven independent reconstructions were calculated using equatorial data out as far as ∼1/9·3 nm—1. They indicated that the filaments were flattened with an axial ratio of about 2·4:1, which was probably closer to 3:1 in the original specimen, because the limited resolution caused flattening to be underestimated. The stain envelope around TMV and some indication of the underlying carbon substrate were also observed.

This information could enable correction factors for flattening to be developed, which could be useful when calculating helical reconstructions or indexing helical diffraction patterns. This method could also be extended to non‐equatorial layer planes, which would provide three‐dimensional information on a wide range of macromolecules that possess a one‐dimensional repeat.
Mass loss rate in collodion is greatly reduced at liquid helium temperature
Authors: Lamvik M. K. , Kopf D. A. , Davilla S. D.
Published: August 2011

The mass thickness of collodion films has been monitored at several temperatures, under conditions typical of electron microscopy, through the use of an electron energy loss spectrometer. Compared to room temperature, only a five‐fold reduction in the rate of mass loss was observed through the use of a commercial liquid nitrogen cooled stage; in contrast, the rate of mass loss was reduced more than one hundred fold when these films were held at liquid helium temperature.
High spatial resolution secondary ion imaging and secondary ion mass spectrometry of aluminium‐lithium alloys
Authors: Williams D. B. , Levi‐Setti R. , Chabala J. M. , Newbury D. E.
Published: August 2011

Samples of aluminium‐lithium alloys have been observed by scanning ion microscopy and analysed by secondary ion mass spectrometry. The high signal‐to‐noise ratio of the positive secondary lithium ion opens up the possibility of both high resolution imaging and microanalysis of lithium distributions in aluminium and other materials. Some of the problems encountered due to sample preparation are discussed and ion images of both the artefacts and the true lithium distribution are shown.
A Monte Carlo calculation of secondary electrons emitted from Au, Ag and Cu
Authors: Suichu Luo , Yongsheng Zhang , Ziqin Wu
Published: August 2011

Monte Carlo calculation of slow secondary electrons (SE) has been performed with a hybrid model of the exponential decay law and cascade process. The contributions of both valence electron excitation and core electron excitation have been considered. The calculated yield, energy distribution, angular distribution and depth distribution for Au, Ag and Cu (28 keV, at normal incidence) are in agreement with the experimental results and better than those previously reported.
Single‐electron effects observed with a low‐temperature STM
Authors: van Bentum P. J. M. , van de Leemput L. E. C. , Smokers R. T. M. , van Kempen H.
Published: August 2011

We have used a low‐temperature scanning tunnelling microscope to study the effect of the Coulomb charging energy on the tunnelling behaviour of low capacitance point‐contact junctions. The tunnelling I‐V characteristics between a tungsten tip and various materials, such as stainless steel, aluminium, carbon and YBa2Cu3O7‐δ, show a quadratic behaviour at low voltages and a displaced asymptotic behaviour at high voltages. The I‐V characteristics can be quantitatively understood using the model of single‐electron tunnelling induced by the Coulomb blockade. The capacitances of this type of point‐contact tunnel junctions are in the 10−18 F range, and are adjustable by varying the distance between tip and surface. These capacitances are at least two orders of magnitude lower than can presently be achieved by electron lithography.

In a series configuration of two low‐capacitance tunnel junctions, with an isolated small particle as a common electrode, we have observed the so‐called Coulomb staircase, due to the quantization of the charge on a single small particle.

The experimental results are in good agreement with semi‐classical Monte‐Carlo simulations.

The low capacitance of an STM assembly can cause a serious complication in the interpretation of low temperature spectroscopic data. We will discuss the implications for the measurements on superconductors, and for the case of inelastic tunnelling spectroscopy.
Coherent electron emission from point sources
Authors: Serena P. A. , Escapa L. , Sáenz J. J. , García N. , Rohrer H.
Published: August 2011

We study the electron emission and propagation in ultrasharp tips field‐emission experiments. The intensity‐voltage characteristics as well as the angular spreading of the electron beam is analysed. We show that non‐coherent emission cannot explain the very narrow experimental electron beams. The properties of coherent electron sources and the beam spatial coherence width are discussed. The quantum mechanical current distribution of field‐emitted electrons from tubes seems to explain the experimental beam widths.
The importance of current density ‘focusing’ in STM image resolution
Authors: Aers G. C. , Leavens C. R.
Published: August 2011

In a previous paper we incorporated Tersoff and Hamann's model of an STM tip into Lang's transfer Hamiltonian result for the tunnelling current density and applied the resulting simple expression to Tersoff's six‐plane‐wave model of a monolayer of graphite. In this paper the results for a more realistic model of a graphite surface and a more complete selection of tip positions are presented. They support the previous conclusions that the normal component of the current density takes on both positive and negative values in a complex flow pattern and that its lateral falloff away from the (projected) tip position is much slower than expected from the extraordinary lateral resolution (∼1 Å) evident in the best STM images of graphite. This shows that sharp ‘focusing’ of the current density directly under the tip is not a necessary condition for high lateral resolution.
A combined scanning tunnelling and field ion microscope
Authors: Michely Th. , Besocke K. H. , Teske M.
Published: August 2011

The construction of a combined scanning tunnelling and field ion microscope allowing the in situ preparation and analysis of the tunnelling tip is described. The apparatus is based on the Besocke type STM and includes a fast, automated sample approach, a sample transfer manipulator and a tip‐cooling mechanism for the FIM operation mode. The design is simple and needs a mechanical feedthrough only for sample transfer. A method has been developed to produce sharp tungsten tips with small clusters on the apex plane. Test‐run results of FIM and STM operation modes are discussed.