bluuebunny/crossref_metadata_2025 huggingface dataset:
Freeze‐substitution without aldehyde or osmium fixatives: ultrastructure and implications for immunocytochemistry
Authors: Monaghan Paul , Robertson David
Published: August 2011

Cryo‐fixation followed by freeze‐substitution without aldehyde or osmium fixation has been investigated as a method for preparing biological specimens with a view to minimizing antigenic alteration. Samples of both solid tissues (mouse small intestine and human kidney) and a human tumour cell line grown in vitro were rapidly frozen by impact (slammed) onto a copper block cooled with liquid nitrogen. They were freeze‐substituted at −80°C in methanol, and embedded at low temperature in Lowicryl K4M or HM20. Resin blocks were polymerized by ultraviolet light. Well‐preserved ultrastructure was observed in the outer 10–15 μm of all samples. Positive immunocytochemical localization of fixation‐resistant and fixation‐labile antigens was obtained on sections of human kidney and the human breast tumour cell line ZR‐75‐1 at both light and electron microscope levels.
Primary considerations for image enhancement in high‐pressure scanning electron microscopy
Authors: Farley A. N. , Shah J. S.
Published: August 2011

The mechanisms of electron beam scattering are examined to evaluate its effect on contrast and resolution in high‐pressure scanning electron microscopy (SEM) techniques reported in the literature, such as moist‐environment ambient‐temperature SEM (MEATSEM) or environmental SEM (ESEM). The elastic and inelastic scattering cross‐sections for nitrogen are calculated in the energy range 5–25 keV. The results for nitrogen are verified by measuring the ionization efficiency, and measurements are also made for water vapour. The effect of the scattered beam on the image contrast was assessed and checked experimentally for a step contrast function at 20 kV beam voltage. A considerable degree of beam scattering can be tolerated in high‐pressure SEM operation without a significant degradation in resolution. The image formation and detection techniques in high‐pressure SEM are considered in detail in the accompanying paper.
TEM analysis of near‐coincidence grain boundaries in silicon
Authors: Bary A. , Nouet G. , Delavignette P.
Published: August 2011

High‐precision characterization of grain boundaries with a deviation from the ideal coincidence is presented. The analysis is based on the shift of Kikuchi lines at grain boundaries. The accuracy was controlled using Frank's relationship applied to intrinsic secondary dislocations which accommodate the orientation deviation. An application is presented to coincidences with Σ = 25 and 3 observed in polycrystalline silicon.
Reflection contrast microscopy performed on epi‐illumination microscope stands: Comparison of reflection contrast‐ and epi‐polarization microscopy
Authors: Velde I. Cornelese‐ten , Bonnet J. , Tanke H. J. , Ploem J. S.
Published: August 2011

In reflection contrast microscopy (RCM) special optics such as a quarter‐wave plate and a central stop are used, the latter being unique for Leitz microscope stands and contributing much to contrast enhancement of the image. In this paper we demonstrate that a cap or prism, linked by oil immersion to the glass slide, can be used instead of this central stop. Thus, microscope stands with incident illumination may be used for RCM.

We systematically investigated RCM and epi‐polarization microscopy (EPM), both incident light microscopical methods, for visualization of immunogold‐silver‐stained cells, autoradiographic stained cells and immunoperoxidase‐diaminobenzidine stained chromosomes. RCM was found to be superior (1) in terms of the intensity of reflected light from reflecting objects, (2) in the visualization of unstained structures and background and (3) with respect to the spatial resolution that is achieved, especially in the tiny stained structures in the image.
The influence of electron beam direction and atomic weight on the ionization cross‐section of metals by high‐energy electrons
Authors: Chen Z. , Bozdogan R. , Loretto M. H. , Smallman R. E. , Long S. J.
Published: August 2011

The intensities of X‐rays generated from samples of Ni3Al, Ni15 at. % A1 and (TiNb)3Al were examined over a range of accelerating voltages as a function of diffracting conditions. It was found that the intensities of the X‐rays from the heavier element in the alloys may be enhanced, with respect to the signal from the lighter element, when the sample is orientated at symmetry. This occurs for Ni and Ti with respect to the Al intensity in Ni3Al and (TiNb)3Al, even when the systematic row which is excited does not contain a superlattice reflection, and in Nil5 at. % A1 which is effectively a disordered alloy. The situation in (TiNb)3Al is more complex, since although the Ti signal is enhanced with respect to the Al signal at symmetry for all reflections, the NbK intensity is not increased relative to the Ti signal but is increased relative to the Al intensity; the magnitude of this increase is about twice as great for reflections which lie in rows which contain superlattice reflections than for rows which do not. In contrast, the NbL intensity is decreased with respect to the TiK intensity when the crystal is set at symmetry for all types of systematic rows. These observations are interpreted in terms of the influence of atomic number on the sizes of electron shells and hence on their ionization cross‐sections. The results are briefly discussed in terms of the technique of alchemi, which is used to determine site occupancy in alloys, and in terms of the quantification of energy dispersive X‐ray data from thin samples.
Contact microscopy with a soft X‐ray laser
Authors: Skinner C. H. , DiCicco D. S. , Kim D. , Rosser R. J. , Suckewer S. , Gupta A. P. , Hirschberg J.G.
Published: August 2011

A soft X‐ray laser of output energy 1–3 mJ at 18–2 nm was used to record high resolution images of biological specimens. The contact images were recorded on photoresist which was later viewed in a scanning electron microscope. A composite optical X‐ray laser microscope ‘COXRALM’ of novel design is also described.
Efficient estimation of cell volume and number using the nucleator and the disector
Authors: Møller A. , Strange P. , Gundersen H. J. G.
Published: August 2011

The nucleator allows the unbiased estimation of absolute structural quantities of suitably sampled, arbitrarily shaped structures from observations made from arbitrary points using isotropic probes. A number of time‐saving modifications using the nucleator and the consequences of the modifications are studied in terms of their bias and efficiency. Using rat neocortex as an example, a description is given of how to estimate mean neuronal volume and total neuron number efficiently from only a few pairs of plastic sections with a thickness of about 3 μm.
Sources of error in the estimation of Leydig cell numbers in control and atrophied mammalian testes
Authors: Mendis‐Handagama S. M. L. C. , Ewing L. L.
Published: August 2011

The effects of assuming (i) that testicular tissue shrinks equally regardless of species or treatment at fixing and processing, (ii) that all Leydig cells in a given testis have spherical nuclei of identical size, and (iii) that testicular volume (i.e. the reference volume) is constant regardless of species or treatment, on the estimation of Leydig cell numbers in mammalian testes were investigated. This was accomplished by comparing the results of stereological analyses of Leydig cell numerical density and Leydig cell number in control testes of hamster, guinea‐pig, and rat and in atrophied testes of hamster, and rat, obtained via the disector method which is unbiased with respect to the particle shape under study, and the Floderus equation which assumes that the particles under study are identical spheres. In control hamster, and also in guinea‐pig, the effects of the three assumptions on the estimates of Leydig cell number per testis were negligible, because in these two treatment groups, the total shrinkage of testis tissue at fixing and processing (ST%) was low, Leydig cell nuclear profiles were circular in section, and the average volume of a testis was close to unity (i.e. 1 cm3). By contrast, in hamsters, and rats with atrophied testes, these assumptions produced incorrect estimates in Leydig cell number per testis, because the ST% was high, the majority of Leydig cell nuclear profiles were pleomorphic, and the average volume of a testis was lower than control.

In summary, this study documents that the assumptions of equal shrinkage in testis tissue at fixing and processing, a constant testicular reference volume, and spheroidal shape of Leydig cell nuclei may contribute significant errors in estimates of Leydig cell number in mammalian testes. The magnitude of the errors introduced by these assumptions depends upon the species and the experimental treatment.
Efficiency of grain‐density autoradiographs with 125I for the light microscope
Authors: Semmler M. , Junger E.
Published: August 2011

Efficiencies of grain‐density autoradiographs with 125I for the light microscope were determined using Ilford L4 emulsion and Microdol X development. Calibrated layers of 125I‐bovine serum albumin and polymer sections of autoradiographic [125I] micro‐scales were used as reference sources. Efficiency was determined as a function of the radiation dose, source thickness and thickness of the absorber foils (e.g. non‐radioactive material between the source and the emulsion), respectively. By describing the dependencies of these factors quantitatively this study provides a means for planning autoradiographic experiments.
A simple procedure for demonstrating the overall morphology of fibroblasts in routine histological preparations of adult tissues, using silver impregnation
Authors: Novotny G. E. K. , Gommert‐Novotny E.
Published: August 2011

A silver impregnation procedure for the total visualization of fibroblasts, with all their processes, is described. The method is applicable to routine formalin‐fixed paraffin sections, or to formalin‐fixed or native cryostat sections. The results are illustrated with examples from loose and dense connective tissue. Fibroblasts are visualized as cells with long, branching dendritic processes. The fibroblasts maintain contact with each other via these processes, thus forming a reticulum of cells, even in adult connective tissue.
Cytochemical observations on the centriolar adjunct of grasshopper spermatids in relation to its silver stainability
Authors: de la Torre J. , Gosálvez J. , Stockert J. C.
Published: August 2011

The silver stainability of the centriolar adjunct from grasshopper spermatids was analysed after blocking ‐SH groups with heavy cations (lead, mercury), or following the conversion of ‐SH and ‐S‐S groups into sulphonates (permanganate oxidation). These treatments produced the loss of silver affinity in the centriolar adjunct. Likewise, the selective fluorescence of this structure by mercuridibromfluorescein was abolished after these treatments. Our results indicate that ‐SH groups are involved in staining reactions of the centriolar adjunct by silver and mercuridibromfluorescein.
Fluorescence quenching; a practical problem in flow cytometry
Authors: Chapple M. R. , Johnson G. D. , Davidson R. S.
Published: August 2011

Dual fluorescence analysis with a single‐laser fluorescence‐activated cell sorter is dependent on the use of two fluorochromes with similar excitation wavelengths but different emission wavelengths. The dye pair fluorescein and R‐phycoerythrin (RPE) have been widely employed for this purpose and interaction between the two dyes has not been observed. Here evidence is presented to show that at high concentrations RPE can completely quench the fluorescein signal in dual fluorescence analysis of human tonsil lymphocytes labelled with pairs of monoclonal antibodies. Reduction in the fluorescein signal correlated with the intensity of red (RPE) staining. This phenomenon can seriously compromise interpretation of dual immunofluorescence carried out on a single laser instrument and can best be avoided by careful analysis of single colour controls.
Flow cytometric evaluation of DNA digestion with micrococcal nuclease on isolated HeLa nuclei
Authors: Prosperi E. , Giangaré M. C. , Supino R. , Bottiroli G.
Published: August 2011

Flow cytometric assessment of DNA digestion with micrococcal nuclease has been performed on isolated HeLa nuclei by determining the relative reduction in stainability with the DNA‐specific fluorochrome, propidium iodide. At the nuclease concentrations used, DNA histograms of digested nuclei showed the typical bimodal pattern, when the enzymatic reaction was performed in a medium maintaining chromatin in its native (i.e. condensed) or partially decondensed form. In contrast, when nuclei were digested in a buffer lacking both the mono‐ and divalent cations K+ and Mg2+, an extensive decrease in fluorescence intensity, with loss of the histogram shape, was observed. In nuclei with native chromatin, DNA stainability decreased as a function of time and enzyme concentration, to reach a lower limit of about 46%, as compared with undigested control samples. Removal of the histone H1 induced a significant increase (approximately by a factor of 2) in the extent of digestion, although only in nuclei with partially decondensed chromatin. These results suggest that the sensitivity of DNA to digestion with micrococcal nuclease can be quantitatively monitored with flow cytometry when appropriate reaction conditions are chosen.
Flow cytometric analysis of the functional ability of neutrophils from patients with autoimmune neutropenia
Authors: Macey M. G. , Sangster J. , Veys P. A. , Newland A. C.
Published: August 2011

Flow cytometry has been used to evaluate the functional ability of neutrophils and the expression of IgG Fc receptors (FcRII and FcRIII) in autoimmune neutropenia. Quantification of the neutrophil oxidative burst was made by assaying the production of 2′7′‐dichlorofluorescein (DCF) from non‐fluorescent 2′7′‐dichlorofluorescin trapped within the cell, by flow cytometric analysis of cellular fluorescence. In the present study the DCF assay was used to examine the response of neutrophils to stimulation by opsonized and non‐opsonized Staphylococcus aureus. In addition, the rate of uptake of S. aureus labelled with the red nuclear dye propidium iodide was determined.

The presence of surface‐bound immunoglobulin, which may affect the phagocytic capacity of the neutrophil, was also measured. No correlation between the neutrophil count and level of membrane‐bound IgG or the rate of bacterial uptake was found. The studies were performed on twenty patients with autoimmune neutropenia, twelve with other autoimmune disorders and fourteen normal controls. The rate of uptake of bacteria was considered in relation to the expression of FcRII and FcRIII. Good correlation was found with the level of expression of FcRII, the major receptor for neutrophil activation, and the rate of uptake of bacteria (r = 0.64).
x‐y‐Recording in transmission electron microscopy. A versatile and inexpensive interface to personal computers with application to stereology
Authors: Rickmann M. , Siklós L. , Joó F. , Wolff J. R.
Published: August 2011

An interface for IBM XT/AT‐compatible computers is described which has been designed to read the actual specimen stage position of electron microscopes. The complete system consists of (i) optical incremental encoders attached to the x‐ and y‐stage drivers of the microscope, (ii) two keypads for operator input, (iii) an interface card fitted to the bus of the personal computer, (iv) a standard configuration IBM XT (or compatible) personal computer optionally equipped with a (v) HP Graphic Language controllable colour plotter.

The small size of the encoders and their connection to the stage drivers by simple ribbed belts allows an easy adaptation of the system to most electron microscopes. Operation of the interface card itself is supported by any high‐level language available for personal computers. By the modular concept of these languages, the system can be customized to various applications, and no computer expertise is needed for actual operation.

The present configuration offers an inexpensive attachment, which covers a wide range of applications from a simple notebook to high‐resolution (200‐nm) mapping of tissue. Since section coordinates can be processed in real‐time, stereological estimations can be derived directly “on microscope”. This is exemplified by an application in which particle numbers were determined by the disector method.
Estimation of surface area and length with the orientator
Authors: Mattfeldt Torsten , Mall Gerhard , Gharehbaghi Hassan , Möller Peter
Published: August 2011

The orientator is a new technique for the estimation of length and surface density and other stereological parameters using isotropic sections. It is an unbiased, design‐based approach to the quantitative study of anisotropic structures such as muscle, myocardium, bone and cartilage. A simple method for the practical generation of such isotropic planes in biological specimens is described. No special technical equipment is necessary. Knowledge of an axis of anisotropy can be exploited to optimize the efficiency. To randomize directions in space, points are selected with uniform probability in a square using various combinations of simple random, stratified random, and systematic random sampling. The point patterns thus produced are mapped onto the surface of a hemisphere. The mapped points define directions of sectional planes in space. The mapping algorithm ensures that these planes arc isotropic, hence unbiased estimates of surface and length density can be obtained via the classical stereological formulae. Various implementations of the orientator are outlined: the prototype version, the orientator‐gencrated ortrip, two systematic versions, and the smooth version. Orientator sections can be generated without difficulty in large specimens; we investigated human skeletal muscle, myocardium, placenta, and gut tissue. Slight practical modifications extend the applicability of the method to smaller organs like rat hearts. At the ultrastructural level, a correction procedure for the loss of anisotropic mitochondrial membranes due to oblique orientation relative to the electron beam is suggested. Other potential applications of the orientator in anisotropic structures include the estimation of individual particle surface area with isotropic nucleators, the determination of the connectivity of branching networks with isotropic disectors, and generation of isotropic sections for second‐order stereology (three‐dimensional pattern analysis).
A direct method for fast three‐dimensional serial reconstruction
Authors: Odgaard Anders , Andersen Kurt , Melsen Flemming , Gundersen Hans Jørgen G.
Published: August 2011

A method for accurate three‐dimensional reconstruction of openly connected porous structures is described. The method is based on embedding of a specimen in a contrast coloured epoxy resin and serial sectioning in a standard hard tissue microtome. A PC‐based image processing system is used for direct digitization of the cut surface, and by thresholding two‐phase images are obtained. The process is fully automated, and about 170 sections can be produced, digitized, dichotomized, and stored per hour. As an example of its applications, the method is used on trabecular bone, which is an anisotropic porous structure.
Compton‐scattering X‐ray artefacts observed in an STEM with high take‐off angle EDS detector
Authors: Wilson A. R. , Lambrianidis L. T.
Published: August 2011

A new spectral artefact has been identified in a high take‐off angle energy‐dispersive X‐ray detector used in a JEOL 2000FX STEM. This artefact has been verified to be due to Compton scattering of Mo K X‐rays which are produced in the standard JEOL condenser lens Mo apertures. These Mo X‐rays are Compton scattered by a thick carbon insert in the upper objective lens pole‐piece of the microscope and are detected as two broad X‐ray peaks centred on 16.44 and 18.3 keV. The presence of this artefact severely limited attempts to quantify small concentrations of Nb in a nickel alloy. Possible ways to avoid or minimize the artefact include the use of large apertures, apertures constructed from different materials or thick apertures.
Submicrometre resolution measurement of the composition profile through E‐glass fibres subjected to stress‐corrosion
Authors: Caddock B. D. , Devenish R. W. , Evans K. E.
Published: August 2011

A replication method has been developed to obtain high‐resolution composition profiles through E‐glass fibres subjected to corrosion by aqueous acids. STEM/EDX analysis of fragments of glass fibres has provided compositional information at a spatial resolution better than 0.1 μm. It has been shown that the core‐sheath profile observed in long‐time exposures of glass fibres to aqueous acids is indeed an extremely abrupt change, with calcium and aluminium ions fully leached from the sheath region. A surface region less than 0.2 μm thick of severe demineralization has also been detected in fibres undergoing a combination of aqueous acid corrosion and tensile load, which fail at short times and in which no core‐sheath morphology is detectable by SEM examination.
On the empirical variance of a fractionator estimate
Authors: Cruz‐Orive Luis M.
Published: August 2011

The fractionator consists of several sampling stages with systematic sampling at each stage; data are collected only at the last stage. Therefore, predicting the error variance of a fractionator estimator is a non‐trivial problem, because the observations are correlated in a complicated, unknown way. Gundersen proposed to split the material sampled at the first stage into two subsets, and to compute the variance of the pooled estimate empirically using the corresponding pair of observations made in these two subsets. The idea is very effective, but the estimator thus proposed needed some corrections. The purpose of this paper is to present an improved estimator of the coefficient of error of a fractionator estimator using Gundersen's design.
Characterization of low‐light‐level cameras for digitized video microscopy
Authors: Tsay Tsong‐Tseh , Inman Richard , Wray Barnaby , Herman Brian , Jacobson Ken
Published: August 2011

The use of low‐light‐level video cameras and solid‐state image detectors in conjunction with image digitizers for optical microscopy is increasing dramatically as more people learn about such systems, and as they become more powerful, less expensive, and easier to use. However, there is currently little information available allowing comparison between cameras, or for determining camera suitability for a given experiment. In this paper, we describe a series of tests designed to characterize the performance of low‐light‐level cameras. The results of these tests should assist in the selection of appropriate cameras for given video microscopy applications.
Experimental ionization cross‐sections of phosphorus and calcium by electron spectroscopic imaging
Authors: Heng Y. M. , Simon G. T. , Boublik M. , Ottensmeyer F. P.
Published: August 2011

The absolute partial electron scattering cross‐section for the phosphorus L2,3‐shell ionization was measured by electron spectroscopic imaging using poliovirus as a primary standard. The equivalent calcium cross‐section was obtained in relation to phosphorus using the stoichiometric ratio for these two elements in hydroxyapatite, Ca10(PO4)6(OH)2. At 80kcV, the partial cross‐section of phosphorus was 2.26 times 10−20 and 2.68 × 10−20cm2/atom for poliovirus and hydroxyapatite, respectively, at 150eV loss for a 15‐cV energy window and an acceptance angle of 15 mrad. Under the same conditions the calcium cross‐section was 0.49 × 10−20 cm2/atom at 360 eV loss. The experimental values are slightly higher than the theoretical cross‐sections calculated either by hydrogenic or Hartree—Slater approaches.
Bordered pits in spruce from old Italian violins
Authors: Barlow C. Y. , Woodhouse J.
Published: August 2011

Spruce from a variety of old Italian musical instruments has been studied in the scanning electron microscope, to examine the membranes of bordered pits for evidence of bacterial degradation. Such degradation would be evidence in favour of a suggestion which has been made recently that the makers of such instruments may have used wood which had been immersed in water for a significant period before use. No such evidence was found—no undamaged bordered pits could be found with membranes absent or damaged in this way. Some possible sources of confusion arising from the wide range of appearances of bordered pits are illustrated and discussed.
Image formation in scanned heterodyne microscope systems
Authors: Somekh Michael
Published: August 2011

Several new scanning microscopic techniques have recently been developed which rely on modulation of the optical beam to enhance the imaging performance. These systems have the common feature that the image is formed by detection or demodulation of an a.c. signal. Techniques have been developed which are sensitive to both intensity and phase information in the sample. We refer to such microscopes as heterodyne imaging systems (and by analogy single‐frequency microscopes, operating at d.c. only, are referred to as homodyne) although as we will point out this term is only strictly applicable to the interferometric‐based methods. Although the theory for the conventional homodyne scanning optical microscope is well developed there has been no systematic study of heterodyne microscopic techniques, which is the purpose of this paper. Several techniques are discussed and compared and the different ways of extracting amplitude and phase information are considered in detail with reference to experimental systems which have been demonstrated to have good imaging performance.
Generalizing the confocal microscope via heterodyne interferometry and digital filtering
Authors: Hobbs Philip C. D. , Kino Gordon S.
Published: August 2011

We describe a generalized confocal optical microscope which measures both phase and amplitude, separately and simultaneously. The system is based on heterodyne inferometry, and is extremely fast and accurate. Together with the associated signal processing and data acquisition system, it can take data at the rate of 50,000 points/s, to an accuracy of 12 bits in amplitude and about the same in phase. The 1s̀ phase error is 01°, corresponding to a height uncertainty of 0.1 nm. Unlike most phase‐sensitive systems of this accuracy, the phase data are not differential, and we are not restricted to small phase changes. Having phase and amplitude enables use of digital filters to modify the coherent transfer function of the microscope in almost any way we desire (within the bandwidth available), and in particular to double the spatial resolution of the system without resorting to ultraviolet light or ad hoc image processing procedures such as non‐linear transformations or the introduction of additional assumptions about the sample. In contrast to ordinary apodizing schemes involving annular pupils and graded neutral‐density filters, the digital filters used here are very easily changed to optimize the trade‐off of resolution versus ringing. Computationally efficient expressions for the line‐spread function and step response of a confocal microscope are developed for use in comparisons.
Parameters affecting imaging of the horseradish‐peroxidase‐diaminobenzidine reaction product in the confocal scanning laser microscope
Authors: Deitch Jeffrey S. , Smith Karen L. , Swann John W. , Turner James N.
Published: August 2011

Neurons intracellularly filled with biocytin and labelled with nickel‐intensified diaminobenzidine (DAB/Ni) can be imaged on the confocal scanning laser microscope in order to obtain three‐dimensional and optical section images of neurons. Intensification of the DAB reaction product with nickel was found to be crucial for obtaining a workable signal level. On the other hand, the high absorption of light by the reaction product severely attenuated the detection of structures lying directly underneath, and the intensity of the unattenuated laser used for imaging faded or damaged the DAB/Ni reaction product. We have determined that reduction of the laser intensity combined with the use of proper objective lenses and non‐laser‐based imaging for preliminary adjustments of the specimen all work to reduce or eliminate damage, and also improve the image. These items must be kept in mind when imaging and analysing DAB‐labelled structures in the laser‐based confocal microscope.
Staining sections of water‐miscible resins
Authors: Gerrits Peter O. , Horobin Richard W. , Wright David J.
Published: August 2011

Penetration of hydrophilic acid and basic dyes into sections cut from glycol methacrylate (GMA)‐embedded tissues was studied; as were the effects on such staining of superficial coatings of thin layers of GMA.

Dye size was a major factor in controlling penetration of resin and staining of tissues. ‘Large’ dyes (>1000 Da) entered GMA very slowly, and only stained those tissue components poorly infiltrated by resin. ‘Small’ dyes (< 550 Da) penetrated GMA readily, and stained tissue components whether or not they were resin‐infiltrated. Dyes of intermediate size penetrated the resin, but the staining of resin‐infiltrated tissue elements was slow. Background staining of resin also varied with dye size. Large dyes gave no staining of GMA. Small dyes did, but were readily removed by water washing. Dyes of intermediate size penetrated resin slowly, and once inside were lost slowly. This gave background staining which required use of the plasticizing solvent ethanol for its removal. Increases in resin cross‐linking also reduced staining rates.

As a consequence, it is possible to predict the probable suitability, or otherwise, of various staining reagents proposed for use with GMA sections; and also the probable influences of histoprocessing on stain penetration. In particular it is suggested that penetration of colloidal metals and macromolecular reagents (e.g. labelled antibodies and lectins) will be limited to resin‐free structures, and to the surface of resin sections. The use of superficial GMA coatings as convenient semipermeable membranes for enzyme histochemistry is also noted.
Acoustic imaging of high‐temperature superconducting materials
Authors: Bukhny M. A. , Chernosatonskii L. A. , Maev R. G.
Published: August 2011

A new method of microstructure examination, acoustic microscopy, was used to study high‐temperature superconducting materials. The high‐resolution images obtained (up to 0.4 μm resolution) visualize the specimen topography, variations in local acoustic properties, in particular surface and subsurface defects such as cracks, twins, phase and structural discontinuities and film peeling. For bulk specimens a quantitative characteristic was obtained: a local Rayleigh wave velocity (within an area of 10 × 10 μm), which makes it possible, in particular, to characterize individual crystallites in ceramics.
Complementary TEM characterization of a squeeze‐cast metal‐matrix composite by dissolution of the sample in butanol
Authors: Henriksen Bjorn Rune
Published: August 2011

Transmission electron microscopy observations of ion‐beam thinned samples and samples extracted using a butanol dissolution technique gave information regarding the interface microstructure which could not be obtained from the ion‐beam thinned samples, and that therefore both these sample preparation routes should be considered when observing aluminium metal‐matrix composites in the transmission electron microscope.
Light budgets, light and heavy losses: One‐ or two‐sided tandem scanning (real‐time, direct‐view, confocal) microscopy
Authors: Boyde Alan , Petran Mojmir
Published: August 2011

A practical arrangement was constructed which permitted the demonstration of the equivalence of the autocollimation variant of the rotating‐aperture‐disc, real‐time, direct‐view, confocal microscope (the one‐sided tandem scanning microscope—1sTSM) and the original, centrally symmetric, two‐sided TSM (2sTSM). It permitted the direct examination and disproof of claims that 1sTSM has an advantageous ‘light budget’. It is shown that more efficient use of available light is made in 2sTSM and that contrast may be further improved by the (future) more efficient rejection of waste light.
Contrast in the electron spectroscopic imaging mode of a TEM
Authors: Reimer L. , Ross‐Messemer M.
Published: August 2011

The ratio of inelastic‐to‐elastic total cross‐sections has been measured in an energy‐filtering electron microscope for different elements. Formulae for the transmission of elastically and inelastically scattered electrons in part I were used to calculate the optimum conditions for a Z‐ratio contrast in the electron spectroscopic imaging mode. Structure‐sensitive contrast can be observed for all non‐carbon atoms in biological sections when filtering with an energy loss at ΔE ∼ 250 eV below the carbon K edge. Model experiments with evaporated layers of different elements on a carbon film allow measurement of the contrast increase. Filtering with the carbon plasmon loss shows a lower phase contrast than with zero‐loss filtering. This can be explained by calculating contrast transfer functions for inelastically scattered electrons.
Contrast in the electron spectroscopic imaging mode of a TEM
Authors: Bakenfelder A. , Fromm I. , Reimer L. , Rennekamp R.
Published: August 2011

An energy‐filtering microscope working at 80 keV is used for the investigation of the effect of inelastic scattering on Bragg contrast. Inelastic scattering results in a preservation of Bragg contrast but edge and bend contours are blurred by a spectrum of excitation errors due to the angular distribution of inelastic scattering. This blurring and the chromatic aberration results in a decrease of contrast and resolution for thick specimens. Therefore, contrast and resolution can be increased by zero‐loss filtering as shown by evaporated films of increasing thickness below 150 μg/cm2. Up to ∼300 μg/cm2 an energy‐filtered image at the most probable energy shows the best results. The results obtained are extrapolated to energy‐filtered high‐voltage electron microscopy.
Three‐dimensional image formation in confocal microscopy
Authors: Sheppard C. J. R. , Cogswell C. J.
Published: August 2011

Three‐dimensional (3‐D) imaging in confocal microscopes is considered in terms of 3‐D transfer functions. This leads to an explanation of axial imaging properties. The axial response was observed in both object‐scanning and beam‐scanning microscopes and the influence of off‐axis examination investigated. By simple processing of multi‐detector signals, imaging in both the axial and transverse directions can be improved.
Concentration of solutes during preparation of aqueous suspensions for cryo‐electron microscopy
Authors: Trinick J. , Cooper J.
Published: August 2011

It is demonstrated that solutes are likely to be significantly concentrated under conditions commonly used to prepare vitrified aqueous suspensions for transmission electron microscopy. Muscle thick filaments in such suspensions were largely dissolved, probably due to an increase in salt concentration caused by evaporation of water immediately prior to freezing. The extent of solubilization indicated that salts had been concentrated by at least 50%. Simple tests showed that, under the conditions used, the rate of reduction in the height of a thin water layer was ≅ 1 μm/s. Apparatus is described which reduces evaporation, by clamping the hydrated grid in a filter paper sandwich until just before it enters the ethane coolant. High‐speed cine photography showed that, using this device, exposure of the thinned specimen to the atmosphere was approximately 200‐fold less. Frozen‐hydrated thick filaments prepared using the device had the expected length of about 1.6μm.
Contrast matching in a sequence of images by histogram normalization
Authors: Breen Edmond J. , Williams Keith L.
Published: August 2011

Variations in contrast between digital light microscopy images of a video sequence can be filtered out by matching the contrast of each individual image in the sequence to a reference image using the principle of histogram matching. This ensures the reliability of grey‐scale threshold values in each frame within an image sequence. Here we describe examples of digital image‐to‐image contrast matching as applied to video light microscopy.
Interfacial energies and surface‐tension forces involved in the preparation of thin, flat crystals of biological macromolecules for high‐resolution electron microscopy
Authors: Glaeser R. M. , Zilker A. , Radermacher M. , Gaub H. E. , Hartmann T. , Baumeister W.
Published: August 2011

It is generally agreed that surface‐tension forces and the direct interaction between the specimen and either the air‐water interface or the water‐substrate interface can influence significantly the preparation of biological materials for electron microscopy.

Even so, there is relatively little systematic information available that would make it possible to control surface‐tension forces and interfacial energies in a quantitative fashion. The main objective in undertaking the present work has been to understand somewhat better the factors that influence the degree of specimen flatness of large, monolayer crystals of biological macromolecules. However, the data obtained in our work should be useful in understanding the preparation of specimens of biological macromolecules in general. Data collection by electron diffraction and electron microscopy at high resolution and high tilt angles requires thin crystals of biological macromolecules that are flat to at least 1°, and perhaps less than 0·2°, over areas as large as 1 μm2 or more. In addition to determining empirically by electron diffraction experiments whether sufficiently flat specimens can be prepared on various types of modified or unmodified carbon support films, we have begun to use other techniques to characterize both the surfaces involved and the interaction of our specimen with these surfaces. In the specific case of large, monolayer crystals of bacteriorhodopsin prepared as glucose‐embedded specimens on hydrophobic carbon films, it was concluded that the initial interfacial interaction involves adsorption of the specimen to the air‐water interface rather than adsorption of the specimen to the substrate. Surface‐tension forces at the air‐water interface and an apparently repulsive interaction between the specimen and the hydrophobic carbon seem to be major factors influencing the specimen flatness in this case. In the more general case it seems likely that interfacial interactions with either the substrate or the air‐water interface can be variously manipulated in the search to find desirable conditions of specimen preparation.
Immunocytochemical localization of apolipoprotein A‐I using polyclonal antibodies raised against the formaldehyde‐modified antigen
Authors: Harrach Bärbel , Robenek Horst
Published: August 2011

The preparation of cells for electron microscopy, in particular the fixation and embedding routine, influences the antigenicity, often resulting in a markedly reduced labelling intensity. To overcome the difficulties associated with fixation‐induced changes in antigenicity, we produced antibodies against pre‐fixed human apolipoprotein (apo) A‐I. Purified apo A‐I was fixed with 4% formaldehyde and was used to raise polyclonal antibodies in rabbits. The antiserum was purified by protein‐A‐Sepharose followed by affinity chromatography with the fixed antigen coupled to vinylsulphone‐activated agarose. The specificity of the antibodies was ascertained by enzyme‐linked immunosorbent assay (ELISA) and Western blot analysis against different fixed and unfixed lipoproteins. In ELISA, the reaction of the antibodies was markedly enhanced with the fixed antigen, indicating that the antibodies were directed against epitopes characteristically modified by the fixation. The efficacy of the antibodies for light and electron microscopy was tested on HepG2 cells and on human liver cells. When HepG2 cells were exposed to anti‐apo A‐I antibodies followed by a secondary FITC‐labelled antibody, fluorescence was found intracellularly in distinct regions. Electron microscopy revealed that the endoplasmic reticulum, and in particular the trans elements of the Golgi complexes, were the main compartments stained for apo A‐I both in HepG2 cells, as shown by the immunoperoxidase technique, and in human hepatocytes, as shown by the protein‐A‐gold technique on ultrathin cryosections. The findings demonstrate the potential of using antibodies to fixed antigens as a strategy to overcome impaired localization due to fixation in cytochemistry at the light microscopic and electron microscopic levels.
Immunogold labelling in combination with cryoultramicrotomy, freeze‐etching, and label‐fracture
Authors: Boonstra J. , van Bergen en Henegouwen P. M. P. , van Belzen N. , Rijken P. J. , Verkleij A. J.
Published: August 2011

During the past years, the methods of ultrastructural visualization of intracellular and cell‐surface proteins have been improved considerably, mainly as the result of the development of low‐temperature preservation in combination with immunocyto‐chemical labelling procedures using poly‐ or monoclonal antibodies. In this contribution we will discuss the combination of immunogold labelling with cryoultramicrotomy and two replica methods, i.e. freeze‐etching and label‐fracture. The main advantage of cryoultramicrotomy is that it enables post‐sectioning labelling, thus providing complete accessibility of all cellular antigens, located both intracellularly and on the cell surface. Important parameters that influence the labelling (i.e. label‐efficiency), including penetration of the label and antibodies in the section, effects of fixatives on antigenicity, and steric hindrance, will be discussed in detail. The replica methods have the advantage of enabling an analysis of the lateral distribution of antigens located at the cell surface. The label efficiency is of particular importance in these studies and in this context several parameters will be discussed, including accessibility and effect of fixatives.
The measurement of water distribution in frozen specimens
Authors: von Zglinicki Thomas
Published: August 2011

There are three techniques to measure local water fractions in the cryomicroscope. First, water content may be measured by a direct analysis of oxygen in bulk samples using a windowless detector. Secondly, mass thickness may be estimated in frozen‐hydrated, then frozen‐dried sections. This technique offers unrivalled spatial resolution, especially if the radiation dose in the frozen‐hydrated state is kept low by the use of electron scattering techniques instead of an X‐ray microanalytical background determination. External water content standards can be used instead of frozen‐hydrated sections and the whole analysis can even be performed exclusively on frozen‐dried sections at room temperature. Thirdly, local water fractions can be evaluated from X‐ray microanalytical measurements of element concentrations per mass in the frozen‐hydrated and frozen‐dried state. Corrections necessary for the other techniques cancel out. However, the high radiation dose required for a fully quantitative analysis excludes the use of these methods in thin or ultrathin sections.
Cryofixation and ultra‐low‐temperature freeze‐drying as a preparative technique for TEM
Authors: Livesey S. A. , del Campo A. A. , McDowall A. W. , Stasny J. T.
Published: August 2011

We have developed cryofixation and ultra‐low‐temperature molecular distillation drying as a method for preparing biological samples for electron microscopic analysis. To validate this approach, we have investigated the relationship between the drying characteristics and ice phases present within frozen samples.

Two sample types were investigated. In the first, pure deuterium oxide (D2O), or heavy water, was vapour condensed under vacuum conditions onto a gold‐coated copper sample holder held at −175 or −110°C. Additionally, D2O was slow‐rate cooled from room temperature under an ultra‐pure dry nitrogen gas atmosphere. The second sample type was rat liver biopsies from animals after 5 days of feeding with D2O loaded water and ultra‐rapid cooling by metal‐mirror cryofixation. Ice forms present in the latter samples, determined by electron diffraction of frozen‐hydrated cryosections, were amorphous, cubic, and hexagonal.

Drying of samples was achieved using a molecular distillation configuration with continuous, microprocessor‐controlled sample heating. The vacuum contents of the drying column were monitored by residual gas analysis (RGA) throughout the drying cycle.

D2O vapour in the vacuum chamber, as analysed by RGA, was found to increase in a phasic manner across a broad temperature range. These phases had characteristic onset temperatures and could be removed sequentially. For condensed D2O samples, these onset temperatures were — 160, — 148, — 125 and — 90°C. Rat liver samples also demonstrated phasic drying patterns which were more complex than those detected with pure D2O samples.

Ultrastructural analysis of samples cryofixed and dried in this manner demonstrated a morphology consistent with the ice phases demonstrated in the frozen‐hydrated cryosections. This, together with the RGA results, suggests the absence of devitrification or ice crystal growth during the drying procedure.
Perspective and limitations of cryo‐electron microscopy
Authors: Frederik P. M. , Stuart M. C. A. , Bomans P. H. H. , Busing W. M. , Burger K. N. J. , Verkleij A. J.
Published: August 2011

We investigated the possibility of vitrifying temperature‐sensitive lipid phases as well as (small) biological specimens. From a suspension of unilamellar vesicles, prepared from dipalmitoyl‐phosphatidylcholine (DPPC), thin aqueous films were formed at various temperatures. With cryo‐electron microscopy vesicles were found to be smooth, rippled and faceted or faceted only, depending on the temperature of thin‐film formation (318, 312 and 296 K respectively). The morphology and the electron diffraction patterns indicate that membranes can by physically fixed by vitrification in their high‐temperature configuration and studied at low temperature by cryo‐electron microscopy. This finding suggests that it may also be possible to preserve, in their original state, the more complex membrane systems found in living organisms by initiating rapid‐cooling at a physiological temperature. This was explored by vitrification of thin films formed on specimen grids with (human) blood platelets adhering to collagen fibres. Low‐temperature observation with an acceleration voltage of 120 kV revealed subcellular details. More details were observed when using higher accelerating voltages (200 and 300 kV) of the electron beam. The results presented in this paper illustrate the great potential of cryo‐electron microscopy in the study of membrane dynamics, both in relatively simple model membrane systems and in more complex biological membrane systems.
Low‐temperature SEM for detection of fungicide activity
Authors: Guggenheim R. , Düggelin M. , Mathys D. , Grabski C.
Published: August 2011

Low‐temperature scanning electron microscopy (LTSEM) combined with cryopreparation methods provided images of well‐preserved biological surfaces and structures on a routine basis. Fractures of wheat leaves revealed epidermal and parenchymatous cells and masses of fungal hyphae growing in intercellular spaces. Freeze‐fractured plant cells contained haustoria of the brown rust fungus Puccinia triticina. Extrahaustorial matrices were clearly distinguishable and at infection sites granular material was found. Activity of the triazole fungicide cyproconazole was mainly directed towards fungal hyphae and sporogenic tissue, resulting in a stronger branching and swelling of hyphal tips and collapse of fungal cells.

Cryofixation methods combined with the use of a cryopreparation unit (Balzers SCU 020) were more reliable in interpreting the observed biological events through easier discrimination between evidence and artefacts. More advantages in the use of the cryopreparation unit are described in detail.