bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bordered pits in spruce from old Italian violins
Authors: Barlow C. Y. , Woodhouse J. 
Published: August 2011

SUMMARYSpruce 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

SUMMARYSeveral 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

SUMMARYWe 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

SUMMARYNeurons 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

SUMMARYPenetration 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

SUMMARYA 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

SUMMARYTransmission 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

SUMMARYA 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

SUMMARYThe 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

SUMMARYAn 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

SUMMARYThree‐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

SUMMARYIt 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

SUMMARYVariations 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

SUMMARYIt 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

SUMMARYThe 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

SUMMARYDuring 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

SUMMARYThere 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

SUMMARYWe 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

SUMMARYWe 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.