bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative study of spurious X‐ray production in thin film microanalysis
Authors: Glas Frank
Published: August 2011

When X‐ray microanalysis is performed in a TEM on a thin area of a specimen, some a priori indistinguishable spurious photons produced in other zones of this specimen are always recorded. Several mechanisms contribute to this production. For instance, some Bremsstrahlung and characteristic photons are generated by secondary and Auger electrons; a conservative upper bound to this particular contribution is calculated for several materials, and the present approach is compared to the Monte Carlo simulation. It is then shown that, in special test‐specimens, the total extraneous contribution of the thick parts of the specimen to the spectra recorded in a thin zone can be measured; different instruments may now be compared in this respect. In the HB5 STEM, this total contribution remains low; its main cause is beam scattering in the specimen, not before it. Finally, an experimental procedure for estimating this bulk contribution in any specimen of interest is proposed. Calculations and experiments are illustrated for the case of gallium arsenide.
Processing of secondary ion microscope images: an example of application to the thyroid
Authors: Cavellier J. F. , Berry J. P. , Escaig F. , Boumati P. , Gaume P.
Published: August 2011

Ion microscopy is a microanalytical method by which one can obtain distribution images of any chemical element with isotope discrimination even at very low local concentrations, in successive slices of the specimen. These images are obtained at the price of progressive erosion of the specimen, so that the analysis may not be replayed and it is necessary to record the maximum amount of information during specimen erosion.

We present an improvement of this method using a highly sensitive camera connected to a video analog‐digital converter. The images are acquired and digitized on line and may be processed by an image computer.

We illustrate the technique described with an application of ion microscopy that is made possible by digital recording and processing of images. This application concerns the precise comparison of iodine isotopes and phosphorus distributions in sections of the thyroid gland of rats which were submitted to an iodine‐deficient diet followed by an injection of 129 I.
Digital image analysis of two‐dimensional Na,K‐ATPase crystals: dissimilarity between pump units
Authors: Beall Harry C. , Hastings David F. , Ting‐Beall H. P.
Published: August 2011

Two‐dimensional crystals of purified Na,K‐ATPase were induced by treatment with phospholipase‐A2 and vanadate. The negatively stained crystals were imaged by electron microscopy and analysed by digital image processing. Two‐dimensional averaged projections of the crystals were calculated by the technique of correlation analysis, utilizing SPIDER (System for Processing of Image Data in Electron microscopy and Related fields) image processing software. The calculated dimensions of the unit cell were found to be 13.3 × 4.59 nm with included angle of 98°, comparable to those reported by others. However, the two protomers of the unit cell were found always to be dissimilar in shape and in orientation. All protomers of one side of the dimer ribbon had a triangular outline, and all protomers of the opposing side had a comma shape. This dissimilarity could be explained by two orientations of identical protomers: one orientation for one side of the dimer ribbon, and another orientation for the protomers of the opposing side of the ribbon. An alternative explanation is that the protomers of one side of the dimer ribbon are actually in a conformation different from that of the protomers of the opposing side of the ribbon.
Morphometric evaluation of human lens opacification
Authors: Babizhayev Mark A. , Deyev Anatoly I. , Derevyagin Vladimir I.
Published: August 2011

A method of classifying cataractous changes in excised human lenses, based on separate and independent photographic documentation, with consequent quantitative evaluation of lens opacification is proposed. The degree of lens clouding was evaluated quantitatively by measuring the optical density index and the area of the zones of clouding with certain optical density parameters (equidensities) by use of an image analyser. This technique produced an image in which contrast represented different zones of lens clouding in the frontal projection, and allowed their contribution to the total lens area to be determined. The precise topography of the opacities was established. The classification data obtained for different clinical types of cataracts can be simplified according to the needs of the scientist in his effort to measure clinically or scientifically significant quantitative associations between laboratory measures and cataractous changes.
Theoretical study of the ultimate resolution of SEM
Authors: Ze‐Jun Ding , Shimizu Ryuichi
Published: August 2011

This work aims to clarify the problem of why the ultimate resolution assessed experimentally from the observation of 0.8 nm separation of Au‐Pd fine particles is beyond the theoretical resolution limit of scanning electron microscopy (SEM). The correlation between the spatial distribution of secondary electrons on a sample surface and the resolution estimated by edge‐to‐edge separation in SEM was studied by a Monte Carlo simulation with secondary electron generation included. The result clearly indicates that the edge‐to‐edge separation can extend beyond the theoretical ultimate resolution, particularly by image processing for contrast expansion and by improving the signal to noise ratio (S/N).
Materials analysis with a position‐sensitive atom probe
Authors: Cerezo A. , Godfrey T. J. , Grovenor C. R. M. , Hetherington M. G. , Hoyley R. M. , Jakubovics J. P. , Liddle J. A. , Smith G. D. W. , Worrall G. M.
Published: August 2011

A position‐sensitive detector has been combined with time‐of‐flight mass spectrometry in the atom probe field‐ion microscope to yield a system in which both chemical identity and spatial information are obtained for individual ions field‐evaporated from the specimen surface. This allows the variations in composition originally present in the sample to be reconstructed in 3‐D with sub‐nanometre resolution. The prototype position‐sensitive atom probe is being used to study phase chemistry in a number of metallurgical alloys, including accurate composition determination of 1–2 nm Cu‐rich precipitates formed in Fe–1.3% Cu–1.4%Ni aged to peak hardness. Other applications of the position‐sensitive atom probe (POSAP) include the analysis of surface layers on superconductors and atom probe studies of semiconductor multiple quantum wells. These initial applications of the instrument are reported, and the limitations and intended improvements to the instrument are discussed.
Axial resolution of confocal fluorescence microscopy
Authors: Sheppard C. J. R.
Published: August 2011

A simple analytic expression is given for the axial resolution of a confocal fluorescence microscope. The expression, which is based on the spatial frequency cut‐off criterion of resolution, is valid for high aperture optics and arbitrary fluorescence wavelength.
Tomographic reconstruction from energy‐filtered images of thick biological sections
Authors: Olins Ada L. , Olins Donald E. , Levy Henri A. , Margle Stephen M. , Tinnel Ed P. , Durfee Richard C.
Published: August 2011

Energy filtration makes it possible to image a ∼0.5 μm biological section at 80 kV in the electron microscope. Based on spectra taken at different tilt angles, we chose the most probable energy loss, ΔEp ± 10 eV for each tilt angle, as the imaging energy window. A complete tilt series from + 60° to −60° at 2.5° intervals was collected on the Zeiss EM902 and used in a tomographic reconstruction of transcriptionally active chromatin in the Balbiani ring of Chironomus tentans.
STEM imaging of monatomic surface steps and emergent dislocations
Authors: Milne R. H.
Published: August 2011

Surface features such as dislocations and monatomic steps have previously been imaged using reflection microscopy with conventional transmission electron microscopes. Here it is shown that similar features can be observed with a scanning transmission electron microscope by using phase contrast. Bragg contrast is very small unless a displaced aperture technique is employed.
Identification of an electron‐beam‐induced reaction product in the praseodymium oxide system
Authors: Eick H. A. , Eyring L. , Smith David J.
Published: August 2011

Under the influence of the electron beam of a 400‐keV electron microscope, Pr7O12 decomposes and exhibits a projected unit cell in the (211)F zone which contains four cations. By calculating images of postulated structures which contained either four cations or multiples thereof, and comparing them to the observed images, an attempt was made to develop a model of the structure. The most reasonable model is one in which vacancies are aligned parallel to the electron beam.
A hot stage SEM for gas‐solid reaction studies
Authors: Brown A. M. , Hill M. P.
Published: August 2011

An ultra‐high vacuum, field emission source, scanning electron microscope (SEM) equipped with a gas cell to permit the in situ study of high temperature gas‐solid reactions is described.

High resolution SEM (50 Å) and scanning Auger microscopy (300 Å resolution) can be carried out at specimen temperatures up to 1000°C. Reaction products and processes can be imaged continuously in the SEM at pressures up to ∼0.2 Torr. Gas pressures up to 50 Torr are possible but with some limitations.

Methods of specimen mounting, problems of temperature measurement, and the microscope performance at high specimen temperatures and gas pressures are discussed. Examples of metal surface preparation and impurity segregation under high temperature annealing and gas exposure are reported.
A dynamic SEM study of solution‐precipitation processes in carbon deposition
Authors: Brown A. M. , Hill M. P.
Published: August 2011

In situ high resolution scanning electron microscopy (SEM) has been used to study carbon deposition processes occurring in the Fe/CH4 reaction at temperatures of 620–850°C.

The morphology of deposits formed by carbon dissolution and precipitation processes has been investigated. The development of particulate‐type deposits has been examined. These are associated with the enhancement of catalytic activity of transition metals and the break‐up of the metal structure as the deposition reaction progresses. A mobile Fe‐C phase has been observed that can explain how complex carbon morphologies develop.
Unbiased estimation of particle number using sections—an historical perspective with special reference to the stereology of glomeruli
Authors: Bendtsen T. F. , Nyengaard J. R.
Published: August 2011

Methods for estimating particle number may be based on at least three different principles. Two of them, isolation of particles and indirect counting, have been predominant until recently. The new methods based on section pairs (disectors) are unbiased and far more efficient. The three principles and their historical developments are described, emphasizing that the newly developed principles have old roots, which were forgotten until recently. It is concluded, that ‘learned societies’ combined with individual enthusiasm are material for the spread and development of new ideas.
Reconstruction of the complex reflectance function in acoustic microscopy
Authors: Fright W. R. , Bates R. H. T. , Rowe J. M. , Spencer D. S. , Somekh M. G. , Briggs G. A. D.
Published: August 2011

We describe the theory and practical implementation of an iterative computer algorithm (an extension of the Gerchberg Saxton algorithm, originally developed for electron microscopy) for enabling an estimate of the complex reflectance function of a material to be reconstructed from measured values of only the magnitude of the response of an acoustic microscope (i.e. without the phase of the transducer voltage). Results are presented for eight materials measured with a spherical lens (at 320 MHz) and five with a cylindrical line‐focus lens (at 210 and 228 MHz).
The accuracy of one‐dimensional systematic sampling
Authors: Mattfeldt T.
Published: August 2011

Sampling designs with equally spaced measurements along a temporal or spatial axis (one‐dimensional systematic sampling) are widely used in stereology (volume estimation from serial sections, linear integration), for Monte Carlo integration, in forest research (line sampling), in time‐series analysis and in demographic studies. An unbiased estimate of the integral of a function f(x) in an interval [0, h] is obtained from a discrete number of values of f(x) at arguments separated by a constant distance d, provided that one of the arguments is selected with uniform probability within an interval of length d. In this paper the accuracy of one‐dimensional random systematic sampling is explored. The relative mean square error of the estimate, CE2(Â), is determined for six geometrical models of f(x) directly by integration (which leads to explicit results) or indirectly via a Fourier transformation of f(x) (which leads to results in form of series expansions). Some CE2s thus obtained depend only on n=[m] and p=m‐[m], the integer and decimal part of the real number m=h/d. These non‐elementary expressions have recently been denoted as np‐functions (Mattfeldt, 1987). In practical studies lack of independent replications in systematic sampling often precludes the unbiased estimation of CE(Â). Short‐cut estimators were based on treatment of the systematic sample as a simple random sample, on the empirical autocorrelation, on various differences between the elements, on interpenetrating subsamples, and on the theory of regionalized variables. More involved methods which usually require a computer rely on Monte Carlo simulation, regression of log CE on log m, Fourier analysis, the Euler‐MacLaurin theorem, and methods of time‐series analysis. In an empirical study the absolute volumes of ten rat hearts were estimated from sets of systematic slices. Acceptable estimates of CE(Â) were obtained with the short‐cut estimators based on the theory of regionalized variables, by Monte Carlo methods, and with regression of log CE on log m.
On the precision of systematic sampling: a review of Matheron's transitive methods
Authors: Cruz‐Orive Luis M.
Published: August 2011

G. Matheron's theory of regionalized variables provides a suitable basis for obtaining variance approximations for estimators of integrals from systematically sampled observations, with applications in geostatistics, image analysis, stereology and numerical quadrature techniques in general. The approximations are often fairly accurate for practical purposes. The methods are, however, not sufficiently widespread outside the field of geostatistics. The purpose of this paper is to present in an informal way the transitive part of the methods (relevant to the design‐based approach) and a number of stereological applications.
Contrast in the electron spectroscopic imaging mode of a TEM. I. Influence of zero‐loss filtering on scattering contrast
Authors: Reimer L. , Ross‐Messemer M.
Published: August 2011

Measured values of the transmission of amorphous films as a function of the objective aperture and film thickness can be described by a single‐scattering theory for unfiltered and zero‐loss filtered images in the electron spectroscopic imaging mode of a transmission electron microscope. The theory can be applied to estimate the gain of contrast by zero‐loss filtering for specimen structures larger and smaller than the chromatic aberration disc.
Defocus modelling correction for wavelength dispersive digital compositional mapping with the electron microprobe
Authors: Marinenko R. B. , Myklebust R. L. , Bright D. S. , Newbury D. E.
Published: August 2011

A new, simplified procedure for correcting the defocusing observed in low‐magnification digital maps obtained with the electron microprobe using wavelength spectrometers is described. This procedure uses a wavelength scan of the analysed element and the geometric relationship between the specimen and the diffracting crystal to calculate a model of a standard map, which is subsequently used in the quantification of each pixel of the unknown map. The results of this new procedure are compared with the earlier method of using an experimentally obtained standard map.
Patronage and publishing: Journals of microscopy 1839–1989
Authors: Brock W. H.
Published: August 2011

The development of microscopical journals in the United Kingdom, America and in France and Germany since the 1840s is reviewed. Particular attention is paid to the periodicals issued by the Royal Microscopical Society, the Quekett Club and the Postal Microscopical Club. Conclusions are drawn concerning the roles of societies and commercial publishers as patrons of scientific research.
The social history of the microscope
Authors: Bennett J. A.
Published: August 2011

The motivation for elevating the microscope from an optical toy to an optical instrument derived from the mid‐seventeenth‐century belief in the subliminal, mechanical organization of the natural world. The microscope failed to reveal this corpuscular realm, but its success in popular instruction and entertainment secured its place in the growing amateur pursuit of natural philosophy in the eighteenth century. In the early nineteenth century the development of the achromatic objective gave the microscope a new technical potential, a potential exploited to its full extent by the members of the Microscopical Society of London. The British makers were very successfully engaged with advanced technical development in the mid‐century, but saw their dominance of the market decline with the growing demand for large numbers of instruments aimed at professional applications.
Landmarks in biological light microscopy
Authors: Bradbury S.
Published: August 2011

The pioneering seventeenth‐century work of Hooke (using the compound microscope) and Leeuwenhoek (using the simple microscope) was followed in the eighteenth century by a period of comparative stagnation until the early nineteenth‐century work of Lister (who evolved the principle of aplanatic focal points in lens design). The founding of the Microscopical Society of London in 1839 reflected the increasing importance of the microscope as a scientific instrument and its involvement in specimen preparation techniques, cell theory and bacteriology is described.

The pursuit of increased resolving power—in particular the development of apochromatic lenses by Abbe and Zeiss and enhanced contrast techniques—polarized fight, u.v. microscopy, u.v. absorption and fluorescence, phase contrast and the differential interference contrast techniques, etc., are discussed. The story of microscopy in the twentieth century is one of interlocking discoveries and advances and the techniques of epi‐illumination in scanning fluorescence microscopy, video acquisition and analyses of images in digital form and confocal scanning microscopy are surveyed. It is concluded that these techniques have opened up whole new areas for the future development of light microscopy.
SEM—past, present and future
Authors: McMullan D.
Published: August 2011

The development of the technology of the scanning electron microscope (SEM) is traced from the early pioneering work in the 1930s to the present day. Recent trends are described and, based on these, some speculative forecasts are made of possible future developments. It is suggested that these will include better vacuum conditions in conventional SEM to permit more accurate surface characterization, and improved electron optics for higher resolution, particularly with low‐voltage operation.
Catalyst particle sizes from Rutherford scattered intensities
Authors: Treacy M. M. J. , Rice S. B.
Published: August 2011

We show that the number of atoms in a small supported catalyst cluster can be estimated from the strength of electron scattering into a high angle annular detector in the STEM. The technique is related to the Z contrast methods developed by Crewe, Wall, Langmore and Isaacson. It works best for high atomic number catalyst particles when supported on low atomic number supports, such as Pt on γ‐aluminium oxide. The method is particularly useful for detecting and measuring particles in the sub‐nanometre size range where bright field images are unreliable. Unlike the Z contrast methods, a high angle annular detector is used, which avoids intensity modulations arising from Bragg reflections. The signal is mostly high angle diffuse scattering, which is predominantly Rutherford scattering, and is proportional to the number of atoms probed by the beam, weighted by their individual scattering cross‐sections.

Scattering strengths of individual clusters are computed from digitized high angle annular detector images. Data for Pt on γ‐aluminium oxide, when plotted as imaged area1/2 against intensity1/3, define a straight line. Such plots provide calibration of the intensity increment per atom without the need of external calibration, although assumptions about particle morphology must be made. Reliable results require high signal‐to‐noise and optimum sampling of the specimen. For an STEM probe size of 0.35 nm, Pt clusters containing as few as three atoms can be detected when supported on typical, 20 nm thick, γ‐aluminium oxide supports.
Microspectroscopy on single domains of phase‐separated monolayers
Authors: Meller P.
Published: August 2011

A versatile and inexpensive, but fully equipped apparatus is presented, which enables detailed optical studies on amphiphilic molecules at the liquid‐gas interface. Structural and spectroscopic information, particularly in small areas of single domains of phase‐separated monolayers, can be achieved by combination of this miniaturized Langmuir trough and spectralphotometer microscope. The potential of this apparatus is demonstrated with some measurements on a diyne‐substituted polymerizable lipid analogue at the air‐water interface.
Scanning tunnelling microscopy measurements of carbon deposited on to graphite
Authors: Nakagiri Nobuyuki , Kaizuka Hiroshi
Published: August 2011

Topographic STM images were taken of carbon deposited onto cleaved Kishgraphite. Hillocks were seen at places in the STM images for 1‐ and 2‐nm depositions. The hillocks covered the entire surface with depositions in the range 3–8 nm and the r.m.s. roughness did not change with deposition in this region. The Fourier spectra for 3–8‐nm depositions show peaks at around 5 and 8 nm. The peak‐to‐valley distance was proportional to roughness with a coefficient of 7.8.
A rotary multiple‐specimen holder for scanning electron microscopy
Authors: Watt Peter Ridgway , Warrack John
Published: August 2011

For many applications it is a considerable benefit to be able to compare readily several samples within a set while avoiding the inconvenience of opening the microscope chamber to exchange individual specimens. For complex specimens full specimen manipulation is also required. This paper describes the design and construction of a specimen holder which carries six specimens, is rapidly exchangeable with alternative holders, and retains the full range of movements of the original eucentric single‐specimen holder of a Philips PSEM 501.
EELS quantification of the elements Ba to Tm by means of N45 edges
Authors: Hofer Ferdinand
Published: August 2011

The quantification of electron energy‐loss spectra requires determination of the partial scattering cross‐sections. For N45 edges these data are unknown. Therefore, we have determined a set of cross‐section ratios (k‐factors) of N45 and oxygen K energy‐loss edges for a number of elements between the atomic numbers 56 and 70 using thin‐film standards (oxide compounds), which will enable quantification of these rare earth elements.
Quantification of axonally transported material using cytofluorimetric scanning
Authors: Dahlström A. , Kling‐Petersen T. , Bööj S. , Lundmark K. , Larsson P. A.
Published: August 2011

This paper describes in detail a cytofluorimetric scanning technique used for studying amounts of material axonally transported in antero‐ and retrograde direction in peripheral nerves. Operating procedures, preparation of tissues and instrumental set‐up are described. The basis for quantification of material in a nerve section treated for immunofluorescence is discussed. The reliability of the method has been tested by comparing results with biochemical data. There are several advantages of the technique. (1) Many different substances can be studied in one single nerve segment, thus reducing biological variation and costs. (2) Both morphological data and quantitative figures can be obtained; following scanning the section can be photographed. (3) The method can also be used on studies in the central nervous system and on tissue cultures, since it is possible to scan on single axons or bundles of fibres.
Scanning and detection techniques used in a confocal scanning laser microscope
Authors: Carlsson K.
Published: August 2011

A two‐mirror scanning mechanism for confocal microscopy is described. No optical components, in addition to the scanning mirrors, are used. Design criteria and performance of the scanner are discussed. The photometric linearity of a detector unit incorporating a photomultiplier tube is reported, and a dual detector unit with tunable split wavelength is described.
A confocal video‐rate laser‐beam scanning reflected‐light microscope with no moving parts
Authors: Goldstein Seth R. , Hubin Thomas , Rosenthal Scott , Washburn Clayton
Published: August 2011

A no‐moving‐parts, 30 frames/s, laser‐beam scanning confocal reflected‐light microscope has been developed. In principle, the technique can be extended to fluorescence and transmission light microscopy. Acousto‐optic beam deflectors controlled by digital electronics move a laser beam in a 512‐line interlaced 8·5 times 8·5‐mm raster. The light passes through a beam splitter, enters an inverted microscope through the side camera port, and is imaged at the object by the microscope objective. Reflected light returns through the objective, exits the camera port, is reflected off the beam splitter, and is imaged on to the photocathode of an image dissector tube (IDT). Confocality is provided by raster scanning the IDT aperture coincident with the congruent image of the laser beam incident on the object. Real‐time jitter‐free reflected light images of a variety of biological objects have been produced. Computer‐controlled alignment of the laser scan and IDT is performed in several seconds.
Positron emission tomography—a new technique for studies of the central nervous system
Authors: Eriksson Lars , Dahlbom Magnus , Widén Lennart
Published: August 2011

Positron emission tomography (PET) has become an important tool to study the central nervous system. Examples of such studies are cerebral blood flow and metabolism and determination of receptor characteristics of the brain. In the following the basic principles and the physics behind PET are given. Different aspects are discussed such as detector design, image reconstructions and data analyses. Since quantification is essential in PET, data have to be corrected for absorption, scatter and random coincidences. These corrections and their influence on image data are discussed.

A review of state‐of‐the‐art PET research of the brain is given.
A quantitative approach to cytoarchitectonics: Analysis of structural inhomogeneities in nervous tissue using an image analyser
Authors: Schleicher Axel , Zilles Karl
Published: August 2011

Cytoarchitectonic investigations are based on the analysis of structural inhomogeneities in the neuronal composition of nervous tissue. Boundaries of brain regions are established at locations where local structural properties, such as density, shape, orientation or arrangement of the nerve cells, change. A quantitative approach requires a complete scanning of histological sections and the measurement of at least one of these properties using an automatic device. In contrast to recently developed stereological methods which result in unbiased estimates of parameters and which are based on efficient sampling procedures, measurements with automatic devices are biased due to section thickness and problems in the segmentation of nerve cell bodies. Automatic measurements are necessary, however, if a complete scanning of histological sections is required in order to localize areal or laminar boundaries. In this approach, the grey level index (GLI) is measured with a TV‐based image analysing system from routine histological sections. Using Nissl‐staining, this parameter is a biased estimate of the local volume density of Nissl‐positive structures (cell bodies). The histological section is digitized into a GLI image by a scanning procedure. The GLI image is processed by image enhancement procedures in order to visualize the laminar pattern. Areal boundaries are localized at positions where this laminar pattern changes. GLI statistics of single brain regions can easily be evaluated by delineating these regions with a cursor in the GLI images of the sections. Information from a series of sections is compiled by specific application programs.
A rapid preparation technique for leucocytes
Authors: McCarthy D. A. , Bernhagen J. , Liu Y‐C. , Perry J. D.
Published: August 2011

A procedure is described for making leucocyte preparations from blood samples fixed ex vivo. Briefly, blood is treated with buffered formaldehyde and the erythrocytes and platelets removed subsequently by lysis and differential centrifugation. The fixed leucocyte preparations can then be processed or fixed further for various types of microscopy as required. In stained preparations examined by light microscopy, the different leucocytes, i.e. lymphocytes, polymorphonuclear leucocytes (PMN) and monocytes, can readily be identified by their characteristic nuclear morphology, and polarized PMN can easily be recognized by their non‐spherical shape. Experiments with chemotaxins in vitro indicated that the procedure could be used to determine whether polarized PMN circulate in the blood of normal individuals and patients with inflammatory disease. A study of the blood from eleven normal subjects showed that 84.0 ± 10.8% (mean ± 2 SEM) of circulating PMN were spherical, while the majority of the remainder (14.3 ± 10.5%) were only ‘slightly’ polarized.
Carbon‐based immunocytochemistry
Authors: Baigent C. L. , Müller G.
Published: August 2011

A carbon‐based immunocytochemistry (CarBIC) method, whereby the epoxy resin is completely removed from a section prior to immunocytochemistry (IC), is described. The resulting absence of embedding medium allows for an optimal access of both primary antibodies and marker systems to structures at various levels throughout the depth of the section. A carbon film evaporated onto the surface of the section before extraction of the resin forms a base to which the section adheres and maintains structural integrity during subsequent processing. The conventional and accepted electron microscopical (EM) appearance for epoxy‐resin‐embedded material is regained by re‐embedding in Epon before contrasting in the normal manner. The technique is demonstrated using human cytomegalovirus (HCMV)‐infected human fibroblasts. HCMV+ serum is the source of primary antibodies and 10‐nm protein A‐gold (pA‐G10) together with rabbit anti‐human IgG + IgM is the marker for IC.
Preparation of the nematode‐trapping fungus, Arthrobotrys oligospora, for scanning electron microscopy by freeze substitution
Authors: Wharton D. A. , Murray D. S.
Published: August 2011

A freeze‐substitution technique for preparing fungal specimens for scanning electron microscopy is described. This involves cryofixation in liquid nitrogen, freeze substitution in methanol at — 20°C and critical‐point drying. The trapping complexes and conidiophores of the nematophagous fungus Arthrobotrys oligospora are well preserved and retain their normal three‐dimensional arrangement.
Three‐dimensional reconstruction of a plant dictyosome from series of ultrathin sections using computer image processing
Authors: Lockhausen Jürgen , Kristen Udo , Menhardt Wido , Dallas William J.
Published: August 2011

A single dictyosome from an actively secreting ovary gland cell of Aptenia cordifolia has been reconstructed in 3‐D from a series of twenty‐nine electron micrographs by computer image processing. The reconstruction is presented under different viewing angles in the form of shaded perspective displays. From these displays the entire dictyosome, surrounded by numerous vesicles, appears to be more a spherical than a flat body. The plate‐like region of the dictyósome is demonstrated when only a portion of the electron micrographs is used for the image processing, leading to ‘cut‐off’ displays. Since some upper planes were removed, such ‘cut‐off’ displays revealed both tubular connections between cisternae of the dictyosome and the neighbouring endoplasmic reticulum as well as tubular continuities between adjacent Golgi cisternae within the same stack. Possible consequences of both types of interconnections on transport and processing of proteins and glycoproteins are discussed.
Confocal microscopy as a tool for the study of the intranuclear topography of chromosomes
Authors: van Dekken H. , van Rotterdam A. , Jonker R. , van der Voort H. T. M. , Brakenhoff G. J. , Bauman J. G. J.
Published: August 2011

A scanning confocal microscope was used to investigate the spatial positions of specific regions within blood cell nuclei. These centromeric regions were fluorescently labelled by in‐situ hybridization to suspended nuclei with a centromere‐1‐specific DNA probe. The 3‐D image data sets, obtained by optical sectioning of the cells, were used to determine the spatial position of the centromeric regions in the nuclei by means of specially developed software. The centromeres were found to be localized near the nuclear boundary. This spatial pattern was tested against a random distribution model by means of the Kolmogorov—Smirnov test. The difference between the two patterns was at a P < 0ċ01 significance level.
Confocal fluorescence microscopy: some applications in bone cell biology
Authors: Jones S. J. , Taylor M. L.
Published: August 2011

Three‐dimensional information is necessary for the proper investigation of the interrelationships of bone cells, and of the complex interface between these cells and the bone matrix they form and destroy. The use of fluorescence confocal microscopy was explored in the determination of the distribution of immunolabelled actin and vinculin—cytoskeletal and attachment proteins—in isolated chick bone cells cultured on dentine, and in neonate rat and rabbit calvaria. Confocal imaging, compared with conventional fluorescence imaging, greatly enhanced the interpretation possible.
In vitro confocal imaging of the rabbit cornea
Authors: Masters Barry R. , Paddock Stephen
Published: August 2011

We were able to observe in vitro the fine structure of the rabbit cornea using a laser scanning confocal microscope, especially in the regions between Descemet's membrane and the epithelial basal lamina. We observed submicrometre filaments throughout the stroma with high concentrations adjacent to Descemet's membrane, and found extensive interconnecting processes between stromal keratocytes. There are numerous regions containing nerve plexuses in the stroma. We found a deeply convoluted basal lamina adjacent to the epithelium, and observed regions containing junctions between endothelial cells in fluorescent images of rabbit corneas stained with the actin‐specific compound fluorescein phalloidin.
Microwaves for microscopy
Authors: Kok L. P. , Boon M. E.
Published: August 2011

Sample preparation for microscopy is based on physical and chemical processes. These processes can be influenced by microwave irradiation. The prerequisite for the development of good microwave procedures is knowledge of histochemistry combined with understanding of the physics of microwave irradiation. Examples of superior results of fixation, processing, and (immuno) staining performed in the microwave oven are presented, both for light‐ and electron microscopy.
Fine structure of unstained human chromosome fibres dried with no fixative as observed by X‐ray contact microscopy
Authors: Shinohara K. , Nakano H. , Kinjo Y. , Watanabe M.
Published: August 2011

X‐ray contact microscopy was used to examine unstained human chromosomes dried without any fixative. This is the first report of the observation of single human chromosome fibres at high resolution without any modification(s) such as staining or fixation. The ‘beads‐on‐a‐string’ structure was observed in stretched portions of chromosome fibres. The diameters of the thin filaments and small particles were 7–15 nm (mean ± SD of 12·2 ± 2·1 nm) and 16–69 nm (31·4 ± 13·2 nm), respectively. These sizes correspond to those of fibres composed of nucleosomes (10–15 nm in diameter) and particle units (15–50 nm in diameter) called superbeads (unit structures composed of a multiple association of nucleosomes). These results are in good agreement with the current nucleosome and superbead model for chromosome fibre structure.
Ultrastructural localization of nucleic acids in plant tissues following the use of malachite green or neutral red in the fixative solution
Authors: Lawton June R.
Published: August 2011

Malachite green and neutral red, when added to glutaraldehyde for fixation of various tissues, yielded high‐contrast images of cell ultrastructure. Malachite green, in acid conditions, appeared to increase contrast of heterochromatin material in the nucleus whereas neutral red gave greater clarity to the nucleolus and to cytoplasmic ribosomes. Control tissue fixed under acid conditions showed little damage but there were ‘crystalline’ areas at the periphery of the nucleolus. RNase did not digest cytoplasmic ribosomes from tissue after neutral red glutaraldehyde fixation. These results suggested that neutral red became bound to RNA in the tissues. Fixation with malachite green, at a pH below 6, did not affect the digestion of RNA by RNase but did protect chromatin against the bleaching action of the chelating agent EDTA. The addition of malachite green (pH < 6) or neutral red to glutaraldehyde are useful techniques for the investigation of the ultrastructure of nuclear material and cytoplasmic ribosomes.
Cooling rate and ice‐crystal measurement in biological specimens plunged into liquid ethane, propane, and Freon 22
Authors: Ryan K. P. , Bald W. B. , Neumann K. , Simonsberger P. , Purse D. H. , Nicholson D. N.
Published: August 2011

Specimens sandwiched between copper planchettes were plunged up to a depth of 430 mm into coolants used for cryofixation. Hydrated gelatin containing a miniature thermocouple was used to mimic the behaviour of tissue during freezing. Gelatin and red blood cells were used for ice‐crystal analysis. Ethane produced the fastest cooling rates and the smallest ice‐crystal profiles, and Freon 22 produced the slowest cooling rates and the largest crystal profiles. Smaller crystal profiles were often seen in the centre of the specimens than in subsurface zones. The results show that ethane, rather than propane, should be used for freezing metal‐sandwiched freeze‐fracture specimens by the plunging method, and probably also in the jet‐cooling method. They further suggest that good cryofixation could occur at the centre of thin specimens rather than only at their surfaces. Comparison between theoretical and experimental ice‐crystal sizes was satisfactory, indicating that where the experimental parameters can be defined then realistic predictions can be made regarding cryofixation results.
The value of assessing cell proliferation in breast cancer
Authors: Lawry J. , Giri D. , Rogers K. , Duncan J. L.
Published: August 2011

Using three methods to measure cell proliferation, namely DNA cell cycle; anti‐proliferating cell monoclonal antibody (MAb) (Ki67, P145) analysis by flow cytometry; and the histological silver (argyrophilic) staining technique to visualize nuclear‐organizing regions (AgNOR), twenty‐two paired samples of primary breast tumour and axillary lymph node were analysed. The results showed variable levels of correlation between the methods used for the tumour group (r = 0.915, P <0.001 for Ki677 versus P145; r = 0.42, P <0.005 for percentage S/G2/M‐phase versus P145; r = 0.16, P <0.5 for percentage S/G2/M‐phase versus AgNOR; r = 0.400, P < 0.1 for Ki67 versus AgNOR). The lymph‐node group showed slightly poorer correlations, yet involved nodes showed a consistently higher level of proliferation than non‐involved nodes by all methods used.

Overall, MAb binding of Ki67 or P145 was seen to be a good indicator of cycling cells, detecting G1‐phase cells in addition to S/G2/M‐phase cells indentified by the other methods used. However, no advantage was found over the usual DNA flow cytometric analysis of cells, which had clear prognostic value. AgNOR scores were found to be able to discriminate between diploid and aneuploid; and dividing and non‐dividing cells, but areas of score overlap limited the application of this technique to that of a positive discriminator only.
A variable‐thickness., multipurpose culture chamber for high‐magnification observation
Authors: Braga P. C.
Published: August 2011

A new, versatile, general‐purpose, high‐magnification perfusion chamber for ex vivo microscopy, which can be machined at low cost, and which can be assembled very rapidly, is described. An additional useful feature is the special lock system, with an annular threaded screw tap with the inside wall cut to fit against the slant of the microscope objective, to increase the field of observation.
Acoustic microscopy of rocks
Authors: Rodriguez‐Rey A. , Briggs G. A. D. , Field T. A. , Montoto M.
Published: August 2011

Acoustic images of two common rocks, granodiorite and limestone, are studied to explore the kind of geological information that they can provide. The contrast is primarily due to Rayleigh waves; in rock specimens they are mainly affected in their propagation by crystal anisotropy and surface discontinuities. Therefore, acoustic microscopy makes possible detailed identification of intragranular structures in the rock‐forming minerals (very fine cracks, grain and subgrain boundaries, etc.); these features may appear more sharply than in transmitted polarized light microscopy or in scanning electron microscopy. In particular the acoustic images are effective in microfractographic studies, they can show not only the finest cracks, even those much less than a wavelength wide, but also their position in relation to the grain and subgrain structure.
Biological cryosection preparation and practical ion yield evaluation for ion microscopic analysis
Authors: Sod Earl W. , Crooker Allen R. , Morrison George H.
Published: August 2011

A mounting method utilizing an indium substrate is described for preparing freeze‐dried cryosections of biological tissue for ion microscopic analysis. Using this procedure, a qualitative comparison between cryosection, conventional chemical, and freeze‐substitution specimen preparations is made with rat liver tissue. Practical ion yield variations in cryosections are found to be minimal (± 13% relative standard deviation) in tissue‐containing areas of rat liver and small intestine.
Measurement of boundaries using a digitizer tablet
Authors: Mercer Robert R. , Randell Scott H. , Young Stephen L.
Published: August 2011

The perimeter is the most error prone of the primary measurements (length, perimeter and area) made when using a device such as a digitizer tablet to trace profiles on micrographs. To allow for minimization of this error an expression is developed relating the error in the perimeter to the digitizer resolution plus other errors in the entry process. The predictions of this expression are shown to be in good agreement with the results of computer simulation of the tracing process, as well as results from manual tracing using a digitizer tablet. This analysis of the digitizer entry process also provides a method by which an optimal choice of the sample spacing between digitizer coordinates can be made.
Occupational risks of (meth)acrylate compounds in embedding media for electron microscopy
Authors: Tobler M. , Freiburghaus A. U.
Published: August 2011

(Meth)acrylates are the main constituents of embedding media widely used in electron microscopy research for low‐temperature embedding of biological tissue. (Meth)acrylates toxicology is still incompletely understood and therefore an estimation of health hazards involved in handling must be inaccurate. (Meth)acrylate monomers are known to be harmful to skin and other tissues and may sensitize workers. Since low‐temperature electron microscopy techniques have gained popularity in research laboratories, it is important to establish safety rules for handling the (meth)acrylate‐containing solutions.

The aim of our report is to review briefly the toxicological properties and occupational hazards of the chemicals involved, summarize our own experiences with resins and protective devices in this respect, give guidelines for safe embedding and pass on these data to all interested researchers in order that workers are not discouraged from using (meth)acrylate embedding media, but know the risks and how to minimize them.
A revisionist history of microscopical sciences
Authors: Phinney Harry K.
Published: August 2011

Indices of productivity have been constructed using the scientific literature of the seventeenth through mid‐nineteenth century for use in judging the accuracy of generalized statements citing low or no productivity in microscopical science during this time. The evidence produced refutes the expressed opinions that: (1) microscopes of that period were considered useless for scientific studies; (2) microscope images were considered unreliable by the scientific community; (3) the trivial activities of ‘amateurs’ precluded serious studies by ‘professionals’; (4) the microscope was simply unappreciated as a research tool until the middle of the nineteenth century.
Visualization and coding in three‐dimensional image processing
Authors: Forsgren Per‐Ola
Published: August 2011

A system for calculating orthographic views of three‐dimensional objects from a confocal microscope has been implemented in a high‐level language. It is used on a regular basis in a number of projects and on different computers. The system enables the user to filter the original data and make a selection of which points and parts of the objects to show in a projective view. The information to be shown is coded in a compact format that is well suited for projection calculations. Several display principles were implemented that enhance different aspects of the objects.
Measuring the forces acting during microtomy by the use of load cells
Authors: Allison Russell T. , Vincent Julian F. V.
Published: August 2011

The forces acting upon the cutting blade during microtomy can be accurately measured by using a load cell. From the information obtained, the optimal knife angles (rake, clearance and slant) can be determined. In addition, the speed of cutting and thickness of sections can also be optimized. The information obtained from the load cell additionally reveals variations in tissue composition which affect the cutting forces. This paper is a preliminary communication to illustrate the possible roles for a modified microtome in (a) the study of microtomy and (b) comparative studies of tissue density.
Relocation of minute specimens for scanning electron microscopy
Authors: Dvorachek M. , Rosenfeld A.
Published: August 2011

A relocatable stub‐holder assembly for SEM that can record the position of small specimens by x‐y‐coordinates is described. It enables the accurate pin‐pointing of objects for further study later.