bluuebunny/crossref_metadata_2025_split huggingface dataset:
Confocal microscopy and image processing in the study of plant nuclear structure
Authors: Shaw Peter , Highett Martin , Rawlins David 
Published: August 2011

SUMMARYWe describe measurements of the point spread function (PSF) for a confocal microscope and compare them with the PSF for a conventional (wide‐field) fluorescence microscope. In situ hybridization with probes to telomere and ribosomal rDNA sequences, combined with three‐dimensional (3‐D) microscopy, has been used to study interphase nuclei in root tissue of Pisum sativum and Vicia faba. Nearly all the telomeres in both species are located at the nuclear envelope, and are highly clustered in the Vicia tissues, suggesting specific binding interactions. rDNA labelling in P. sativum shows four brightly staining knobs, corresponding to condensed regions of the rDNA genes from the two pairs of nucleolar organizer genes in this species, arranged approximately tetrahedrally around each nucleolus. Deconvolution using the measured PSFs can be used to improve these images, revealing a fibrous substructure in the perinucleolar knobs, and a large amount of interconnecting internal structure, which we suggest represents rDNA both in the fibrillar centres and also more diffuse, widely dispersed rDNA. Finally we show that accurate conventional data coupled with deconvolution can produce 3‐D reconstructions comparable to those obtainable with confocal microscopy, but that the clearest images are obtained by applying deconvolution to the confocal data.
Co‐localization of the cellulose framework and cell‐wall matrix in helicoidal constructions
Authors: Vian B. , Temsah M. , Reis D. , Roland J. C. 
Published: August 2011

SUMMARYAffinity methods (enzymes, lectins and antibodies used as specific probes) were applied in order to target cellulose and the major matrix components in cell walls of dicotyledons built up as helicoids. The probes were the enzyme cellobiohydrolase, CBH1, for cellulose, polyclonal antibodies and the lectin RCA (Ricinus communis agglutinin) for xyloglucans, and monoclonal antibodies JIM5 or JIM7 for homogalac‐turonan sequences (according to their degree of esterification). Observations were performed: (i) in muro and/or on heteromolecular fractions following controlled cell‐wall dissociation experiments; and (ii) at the light microscope level and/or at the electron miscroscope level by means of various visualization markers. Affinity labelling was complemented by subtractive cytochemistry and by the labelling of available anionic groups by means of cationic gold particles. Data confirm the importance of using a variety of probes, the combination of which allows the acquisition of convergent and complementary results. Concerning the particular case of helicoidal walls of elongating cells, it was shown that cellulose was always co‐localized with xyloglucans and homogalacturonan polymers in zones where the cholesteric order was well defined. Cellulose was always associated with compatible hemicellulose polymers capable of binding tightly. Moreover, residual charges were always present along the microfibrils, forming an anionic coat able to repel the adjacent cellulose microfibrils. A possible role of the heteromolecular association of xyloglucan and pectate as a surfactant allowing the cholesteric assembly is hypothesized.
Contrast enhancement and depth perception in three‐dimensional representations of differential interference contrast and confocal scanning laser microscope images
Authors: Schormann Thorsten , Jovin Thomas M. 
Published: August 2011

SUMMARYA commonly used three‐dimensional reconstruction method in confocal scanning laser microscopy (CSLM) involves the generation of stereo views by stacking optical sections. Under certain conditions the resulting stereo pairs exhibit inferior quality with respect to contrast and depth perception. A method based on digital image processing is described in which the individual images are enhanced prior to reconstruction, thereby increasing the number of usable optical sections by a factor of up to five. Furthermore, we introduce a new contrast enhancement transformation based upon local statistics and a grey‐level probability density function that provides improved depth perception. Digital image processing methods map a discrete grey scale onto a continuous, unbounded interval. Inasmuch as a closed, discrete grey scale is required for computer display purposes, we present an appropriate mapping function derived from an entropy criterion.
Resolution in light microscopy studied by computer simulation
Authors: Goldstein D. J. 
Published: August 2011

SUMMARYMicroscopic resolution can be characterized by K = x NA/δ, where x is the distance between two objects, or the interval of a grating, just resolved with light of wavelength λ and an objective of aperture NA. Using a computer simulation of imaging the following K values were obtained on the Sparrow resolution criterion for line and grating objects and various imaging methods (figures for the Rayleigh criterion, which assumes a finite contrast‐sensitivity of the light detector, are in parentheses). Several results appear to be novel. Due to limitations discussed in the text some data are only approximate.With a grating object K is 1.0 (1.0) for axial coherent illumination, 0.5 (0.5) for coherent illumination at an obliquity NAobi which just enters the objective aperture, 0.5 (about 0.53) for incoherent illumination, 0.5 (about 0.52) for illumination with a condenser aperture NAc equal to NA, 0.5 (about 0.515) for transmitted‐light confocal scanning, and 0.25 (about 0.38) for fluorescent confocal scanning. If the object consists of two parallel lines K is about 0.68 (0.71) for axial coherent illumination, about 0.44 (0.5) for incoherent illumination, 0.375 (about 0.48) for optimal partially coherent illumination in which NAc may exceed NA, 0.44 (0.48) for transmitted‐light confocal scanning, and 0.32 (0.41) for fluorescent confocal scanning.For inter‐object distances of 1, 1.5 and 2 wavelengths, respectively, NAc values of about 0.69, 0.5 and 0.375 gave optimal contrast and resolution irrespective of NA. The practice of setting NAc to about two‐thirds of the NA of a high‐power objective is supported by the fact that a condenser aperture of about 0.69 gives excellent or optimum resolution and contrast for most inter‐object distances and objective apertures tested, although with some distances and apertures reducing NAc improved contrast slightly.The rule (sometimes attributed to Abbe) that resolving power is proportional to the mean of NA and NAc is correct for oblique coherent illumination in the case of a grating object, provided NAobl does not exceed NA. In the case of two isolated objects the rule is only approximately correct, but applies even if NAobl is greater than NA. Coherent light at an obliquity of 0.5λ/x introduces a half‐wavelength phase difference between two objects and permits their resolution (with perhaps an incorrect apparent inter‐object distance) even with objective apertures approaching zero.In confocal scanning the width of the scanning spots has only a moderate effect on resolution, and two objects can sometimes be resolved with scanning spots wider than the inter‐object distance provided the lens apertures are neither too small nor too large.
An improved cryo‐jet freezing method
Authors: Greene William B. , Walsh Lyle G. 
Published: August 2011

SUMMARYA new cryo‐jet freezing apparatus is described that is easy to use and gives good results using a propane‐butene mixture (3: 1). Our use of the freezer in the study of mouse spinal cord explant cultures is discussed. At the tissue surface, the quality of tissue preservation from freezing, followed by freeze substitution, rivals that of conventional electron microscopic methods. Certain intracellular structures are better visualized using our methods. There is no evidence of the tissue being distorted by the cryogen jet when the freezer is operated correctly. A new freeze substitution device is also discussed.
Investigation and use of plasmon losses in energy‐filtering transmission electron microscopy
Authors: Fromm I. , Reimer L. , Rennekamp R. 
Published: August 2011

SUMMARYThe electron spectroscopic imaging (ESI), diffraction (ESD) and different types of electron energy‐loss spectroscopy (EELS) modes in an energy‐filtering transmission electron microscope can all be used for the investigation and analytical use of plasmon losses. Shifts of plasmon losses caused by differences in composition can be detected with an accuracy of 0.1 eV by parallel‐recorded EELS (PEELS). The dispersion of plasmon losses and the cut‐off angle θc can be observed by angle‐dispersive EELS and by recording spectra at different scattering angles θ. ESD patterns with a selected energy window width of 1 eV enable the dispersion and its anisotropy to be imaged by characteristic intensity distributions between the primary beam and the first Bragg diffracted beams. The ESI mode can be used for the selective imaging of precipitates and for the investigation of the excitation volume of plasmons in small particles.
Quantification procedures for electron energy‐loss spectroscopy and imaging: the use of correspondence analysis for element determination
Authors: Gelsema E. S. , Beckers A. L. D. , Sorber C. W. J. , De Bruijn W. C. 
Published: August 2011

SUMMARYA systematic investigation of the application of correspondence analysis to the determination of element responses in electron spectroscopic imaging is presented. The formalism of correspondence analysis has been applied to a set of artificially generated images, in which the effects of non‐homogeneous specimen thickness and noise were incorporated. The simulation experiments indicate that the results may be used for both qualitative (element distribution) and quantitative (element concentration) purposes.
A new background subtraction for low‐energy EELS core edges
Authors: Tenailleau H. , Martin J. M. 
Published: August 2011

SUMMARYA new three‐parameter function is proposed that improves the ‘classical’ power‐law function for background subtraction processing at low energy losses in electron energy‐loss spectroscopy. The variation of the edge integrals with the width of the pre‐edge window Γ is reduced by a factor of at least five (of interest for accurate quantitative analysis), as is the variation in the height of energy‐loss near‐edge structure. The lower energy limit used for previous background functions has been cut by a factor of two or more. However, the principal advantage of this background function is that the difficulty in the choice of the pre‐edge window Γ is removed since the widest possible need only be taken.
Analysis of the calcium distribution in predentine by EELS and of the early crystal formation in dentine by ESI and ESD
Authors: Plate U. , Höhling H. J. , Reimer L. , Barckhaus R. H. , Wienecke R. , Wiesmann H. P. , Boyde A. 
Published: August 2011

SUMMARYPredentine is a collagen‐rich extracellular matrix between the odontoblasts and the dentine with a width of about 15–20 μm. Electron energy‐loss spectroscopy of rat incisors shows a significantly higher calcium content in the predentine at the predentine‐dentine border than in the middle region of the predentine.At the predentine‐dentine border in the dentine, the calcium and the phosphate groups combine to form apatite crystallites. Electron spectroscopic diffraction with zero‐loss filtering revealed that the earliest crystallites contain only Debye‐Scherrer rings of apatite, which are fewer in number and more diffuse than the diffraction rings from the mature crystallites. We therefore conclude that the early crystallites still contain lattice defects, which are annealed out to some degree with crystal growth. Electron spectroscopic imaging with zero‐loss filtering also showed that the earliest crystallites are chains of dots (or small islands); they build up strands composed of islands, which rapidly acquire a needle‐like character and coalesce laterally to form ribbon‐ or plate‐like crystallites. The parallel strands sometimes appear to reinforce the macroperiod of the collagen microfibrils (67 nm) by tiny holes without any crystal‐substance lined up perpendicular to the parallel strands of the crystallites.
The electron energy‐loss spectroscopic analysis of inhaled smoke particles
Authors: Mitchelson Brian P. 
Published: August 2011

SUMMARYThis paper details the use of electron spectroscopic imaging in the elemental analysis of smoke particles inhaled by smoke‐death victims. The results show that these particles have a varied structure and composition. Because of this, these particles may play a far more significant role in smoke inhalation injuries than has previous been recognized.
Zero‐loss energy‐filtered imaging of frozen‐hydrated proteins: model calculations and implications for future developments
Authors: Schröder R. R. 
Published: August 2011

SUMMARYEnergy‐filtered transmission electron microscopes operating in zero‐loss mode are used increasingly to study biological material in frozen‐hydrated conditions. The contrast enhancement and improved structural resolution obtainable by this method have been studied using Monte‐Carlo model calculations for the scattering processes occurring in such samples. Three models representing typical situations have been analysed, each normalized to minimal beam damage. It is shown that for proteins in thin layers of ice an optimal signal‐to‐noise ratio is achieved in the 80–120‐keV electron energy range. For proteins which have to be embedded in thicker ice layers, a considerably higher acceleration voltage is required. In particular, electron energies above 200 keV would be desirable for electron diffraction work on microcrystals.
Electron spectroscopic study (ESI, EELS) of Nanoplast‐embedded mammalian lung
Authors: Fehrenbach H. , Richter J. , Schnabel Ph. A. 
Published: August 2011

SUMMARYThe potential of Nanoplast melamine resin embedding for the study of mammalian lung parenchyma was examined by means of electron spectroscopic imaging (ESI) and electron energy‐loss spectroscopy (EELS). Samples were either fixed with glutaralde‐hyde‐paraformaldehyde or glutaraldehyde‐tannic acid, or were directly transferred to the embedding medium without prior fixation. Organic dehydrants, as well as fixatives containing heavy metals and stains, were omitted.A very high level of ultrastructural detail of chromatin, ribosomes, mitochondria and plasma membranes was achieved by ESI from the Nanoplast‐embedded samples. The most prominent gain in ultrastructural detail was achieved when moving from an energy loss just below the L2,3 edge of phosphorus at 132 eV to an energy loss just beyond this edge. This reflects the prominent P L2,3 edge observed by EELS of Nanoplast‐embedded samples in comparison with conventionally processed samples. Thus, taking into account possible sectioning artefacts, excellent heterochromatin images which rely on the phosphorus distribution can be obtained from Nanoplast‐embedded samples by computer‐assisted analysis of electron spectroscopic images. In this respect glutaraldehyde‐paraformaldehyde fixation is preferable to glutaraldehyde‐tannic acid fixation because the presence of silicon, revealed by EELS, in tannic‐acid‐fixed samples may introduce artefacts in phosphorus distribution images obtained by the three‐window method because of the close proximity of the L2,3 edges of silicon and phosphorus.
A 360° single‐axis tilt stage for the high‐voltage electron microscope
Authors: Barnard David P. , Turner James N. , Frank Joachim , McEwen Bruce F. 
Published: August 2011

SUMMARYA new type of specimen stage that permits more than 180° of tilting about the axis of a side‐entry rod has been developed for a high‐voltage electron microscope (HVEM). Roughly cylindrical specimens, with radial dimensions of less than a few micrometres, that can be mounted on the tip of a microneedle or micropipette are applicable. For glass micropipettes, the energy of the 1‐MeV beam of the HVEM is sufficient to image specimens through both walls. The stage employs a spindle mechanism that holds these needles or micropipettes coaxial with the tilt axis, allowing the specimen to be rotated without restriction. This arrangement, along with the cylindrical form of the specimen, is an important development for single‐axis tomography, because it permits a complete 180° set of projections to be recorded. The angular accuracy of the stage was demonstrated to be within ±0.20°, with a cumulative error of less than 1.0° over a 180° span.The new stage was tested using puffball spores mounted on a micropipette. A 180° tilt series was recorded and processed to yield a tomographic three‐dimensional reconstruction which was displayed both as a cross‐sectional view perpendicular to the tilt axis, and as a shaded surface viewed from different directions. The same computations were repeated using subsets of the tilt series to assess the effect of various amounts of missing information. Visual inspection of a selected cross‐section from these reconstructions indicated that limiting the angular range to 160° produced results nearly as good as the full data set. Limiting the range to 140°, however, produced a noticeable geometric distortion, which became increasingly severe with ranges of 120° and 100°.
Evanescent‐wave microscopy: A simple optical configuration
Authors: Murray John M. , Eshel Dan 
Published: August 2011

SUMMARYUnder suitable conditions, the region of the aqueous phase immediately adjacent to a glass‐water interface can be selectively illuminated using the evanescent wave created when total internal reflection occurs at the interface. Objects in the aqueous phase away from the glass become effectively invisible, since the intensity of the evanescent wave decays exponentially with distance from the interface. Previous methods of generating evanescent waves for light microscopy have employed accessory light sources and optical components that directed the illumination on the specimen from one side. The asymmetric illumination creates a surprising orientation dependence of visibility for some specimens. Objects such as microtubules are totally invisible unless they are orientated nearly perpendicular to the direction of illumination. An explanation of this phenomenon is provided in terms of the geometry of diffraction of light by long thin objects.A simple method of achieving evanescent‐wave illumination is described and shown to be useful in practice for biological specimens. In contrast to previously described methods, the present arrangement has the advantage of producing circularly symmetric illumination, and of utilizing only standard optical microscope components. The system has been used for imaging specimens both by light scattering and by fluorescence. It has proved useful for following the fragmentation of flagella into isolated microtubules, observing microtubules gliding over dynein adsorbed to a surface, and also for determining the arrangement of vinculin and alpha‐actinin in cell‐substratum attachment sites and termini of growing myofibrils in cardiac cells.
The application of electron microscopy in dairy research
Authors: Schmidt D. G. , Buchheimi W. 
Published: August 2011

SUMMARYA review is presented of the application of various electron microscopical techniques in dairy research. Special attention is given to the use of cryofixation techniques, such as freeze‐fracturing, and of cryomicroscopy. These techniques are particularly well suited for the investigation of dairy products, which are mostly characterized by a high water content and by the presence of milk fat, having a low melting point. With these techniques, fat globules in milk, cheese and cream, and casein micelles in milk and milk products have been studied in an unperturbed state. By using immunogold‐labelled cryosections it could be demonstrated that in casein micelles in milk the kappa‐casein is located at the surface of the micelles, whereas the other caseins are distributed throughout the whole micelle.
Preparation of hard particle powders for examination in the transmission electron microscope
Authors: Boswell E. , Reece M. J. , Gee M. G. 
Published: August 2011

SUMMARYA new technique for preparing electron‐transparent specimens of hard particle powders is described. It consists of mechanically embedding the powder into a thin foil of soft metal followed by ion‐beam etching. This has numerous advantages over other methods of examining powders, such as their direct examination when supported by a carbon film, or by using epoxy resin as an embedding medium. The metal foil can be chosen to minimize overlap of the spectrographic peaks of the metal and the hard particles.
Fixation for biological ultrastructure. II. Cross‐linking of bovine serum albumin by nanosecond pulses of ionizing radiation
Authors: Luft John H. 
Published: August 2011

SUMMARYVery large doses of ionizing radiation were delivered quickly to concentrated albumin solutions by pulses of 500 keV in air. The electrons penetrated the aluminium foil bottom of the test cell and into the solution. A dose of 4.6 Mrad (4.6 × 104 Gy) produced a gel in the albumin solution comparable to the long‐term effect of 0.36% glutaraldehyde in the same albumin solution. The cross‐links created by the radiation leading to gel formation are probably irreversible and quite different from those leading to gelation in the glutaraldehyde‐albumin reaction. Single large pulses of ionizing radiation may be useful for fast fixation of cells and tissues for microscopy.
Confocal and differential phase imaging of thin organic films
Authors: Hudson A. J. , Wilson T. 
Published: August 2011

SUMMARYThis paper discusses the imaging of thin organic films in scanning optical microscopes using both differential phase contrast and confocal modes. The Lang‐muir‐Blodgett technique is used to deposit thin films of controllable thickness. Step structures in these films are considered and theoretical models of the imaging are compared with experimental data. The model provides a measurement of film parameters such as thickness and permittivity. The differential phase contrast mode is also proposed as a simple method of assessment of film quality.
Further modification of the LKB 7800 series KnifeMaker for improved reproducibility in breaking ‘cryo’ knives
Authors: Morewood Christopher R. , Elliott Edith , Dennison Clive , Bruto Anthony G. 
Published: August 2011

SUMMARYThis note describes further modifications to the LKB 7800 series KnifeMaker to improve the precision of scoring of glass strips and the consequent reproducibility of breaking of ‘cryo’ knives, by largely eliminating play in the mechanism, and by accurate adjustment of the cutting wheel position.