bluuebunny/crossref_metadata_2025_split huggingface dataset:
Platinum/iridium/carbon: a high‐resolution shadowing material for TEM, STM and SEM of biological macromolecular structures
Authors: Wepf Roger , Amrein Matthias , Bürkli Urs , Gross Heinz 
Published: August 2011

SUMMARYThin Pt/Ir/C coating films (1.5 nm) show a fine granularity and provide a high structural resolution in the transmission electron microscope (TEM) when applied to freeze‐dried biological macromolecules. They keep their structure when exposed to atmospheric conditions, without the need of an additional stabilizing carbon layer, in contrast to conventional high‐resolution shadowing materials such as Ta/W and Pt/C. However, the correct ratio of the components has turned out to be crucial. When evaporating Pt/Ir/C from the source electrode in an electron‐beam‐heated evaporator, the ratio of the three elements changes progressively, and, consequently, the properties of such films depend strongly on the mass that has been pre‐evaporated.In this paper we present a quantitative analysis of the composition of Pt/Ir/C films by wavelength‐dispersive X‐ray analysis (WDX) undertaken in association with TEM experiments. We applied Pt/Ir/C shadowing to two regular biological test specimens, the phage T4 type III polyhead and the HPI‐layer of Deinococcus radiodurans. It turns out that Pt/Ir/C films containing at least 25% C are three‐dimensionally stable on the freeze‐dried macromolecular samples.By the dramatically improved resolution power of the latest scanning electron microscopes (SEM) and the invention of the scanning tunnelling microscope (STM), two new surface‐sensitive tools for the investigation of biological macromolecular structures became available. The Pt/Ir/C coating has proved to be well suited for STM and SEM imaging of freeze‐dried biological structures because of its good electrical conductivity and its direct three‐dimensional stability. We compare STM, SEM and TEM images of freeze‐dried and Pt/Ir/C‐coated polyheads.
Gap‐junction quantification in biological tissues: freeze‐fracture replicas versus thin sections
Authors: Ryerse J. S. , Nagel B. A. 
Published: August 2011

SUMMARYThe relative efficiency of freeze‐fracture replicas versus thin sections for the visualization and quantification of gap junctions in biological tissues has been evaluated. Both methods may underestimate gap‐junction number—thin sections for reasons of tissue resolution and freeze‐fracture replicas due to the mechanics of the fracturing process. Freeze‐fracture misses gap junctions in regions of plasma membrane which are highly contoured, such as the overlapping basal cell processes of Drosophila imaginal wing discs and the interdigitating lateral membrane plications of intercalated discs in cardiac tissue. If the missed gap junctions are relatively large, as they are in both of these examples, freeze‐fracture significantly underestimates the total gap‐junctional area. Thin sections may miss small gap junctions, but in tissues which contain a range of gap‐junction sizes the lost junctions constitute a relatively small fraction of the total junctional area. In neoplastic imaginal wing discs, thin sections were as efficient as freeze‐fracture replicas in identifying even the smallest gap junctions. Although freeze‐fracture may be the better technique for the qualitative and quantitative documentation of small gap junctions in tissues with relatively flat to gently contoured plasma membranes, and thin sections may be the superior method for gap‐junction quantification in tissues containing a range of gap‐junctional sizes and highly contoured cellular processes, the data suggest that a combination of the two approaches should be utilized whenever possible.
Cell surfaces and fractal dimensions
Authors: Keough K. M. W. , Hyam P. , Pink D. A. , Quinn B. 
Published: August 2011

SUMMARYThe perimeters of the surface membranes of some different cell types have been digitized from electron micrographs and the data analysed in order to discover whether the perimeter can be described by a fractal dimension, df. Micrographs obtained at various magnifications and subsequently enlarged by different amounts have been used. Values of df ranging from 1.02 to 1.34 were manifested over a scale length of about one order of magnitude. Values of df were independent of the magnification, and were the same for cells of the same type. Possible implications of these results are discussed.
Optical sectioning strength of the direct‐view microscope employing finite‐sized pin‐hole arrays
Authors: Wilson T. , Hewlett S. J. 
Published: August 2011

SUMMARYWe discuss how the strength of the optical sectioning property of the direct‐view reflection microscope varies with the size and distribution of the pin‐holes in the source and detector arrays. Finite‐sized circular pin‐holes are considered in both bright‐field and fluorescence imaging and the results are compared with those of the corresponding scanning optical microscope. We discuss the effect of using both incoherent and coherent illumination.
A confocal beam scanning white‐light microscope
Authors: Hell S. , Witting S. , v. Schickfus M. , van Resandt R. W. Wijnaendts , Hunklinger S. , Smolka E. , Neiger M. 
Published: August 2011

SUMMARYWe report on a confocal beam scanning microscope utilizing a continuous Xe short‐arc lamp operating in the visible spectrum with unprecedented radiance. Measurements of lateral and vertical resolution will be presented and compared with those of an equivalent scanning laser microscope. Resolution of the white‐light microscope is equivalent to that of the scanning laser microscope. White‐light microscope images positively stand out from those of the scanning laser microscope by their lack of artefacts caused by interference.
Topological description of the densification of a granular medium
Authors: Prasad P. Bhanu , Jernot J. P. 
Published: August 2011

SUMMARYThe densification of a granular medium is described in terms of topological parameters. Two successive cases are quantitatively analysed starting from regular three‐dimensional (3‐D) packings (in continuous and digitized space) and simulated random 3‐D packings of monosized spheres.
Electron microscopic study of the atomic structure of OMVPE‐grown GaInAs/InP quantum wells
Authors: Hsu Tung , Wang T. Y. , Stringfellow G. B. 
Published: August 2011

SUMMARYThe as‐grown (001) surfaces of InP single crystals and GaInAs/InP multilayers grown under different conditions by organometallic vapour phase epitaxy (OMVPE) have been studied using reflection electron microscopy (REM). It was found that atomically smooth surfaces can be grown by OMVPE using the proper growth conditions. A small density of elliptical defects also found on these surfaces may be attributed to abnormal growth due to defects formed during the growth process.
In defence of aldehyde—osmium fixation and critical‐point drying for characterization of seed‐storage fungi by scanning electron microscopy
Authors: Mycock D. J. , Berjak Patricia 
Published: August 2011

SUMMARYLow‐temperature scanning electron microscopy cannot be used as a general approach when resolution of definitive surface features is required for fungal characterization, because of the production of a pervading exudate by some of the species, strains or varieties. As the resolution of the light microscope is obviously limiting, scanning electron microscopy remains the best microscopical mode. Results are presented for four species within the Aspergillus flavus group which indicate that minimal (if any) distortion or collapse can be achieved with concomitant preservation of surface morphology, when particular precautions are taken during fixation and critical‐point drying. Details of the steps pertaining to preparation for scanning electron microscopy following critical‐point drying that are considered collectively to contribute to the success of the approach are discussed.
A strategy for the reconstruction of structures possessing axial symmetry: sectioned axonemes in sperm flagella
Authors: Lanzavecchia Salvatore , Bellon Pier Luigi , Afzelius Björn A. 
Published: August 2011

SUMMARYThe images of complex biological structures seen in the electron microscope, possessing an n‐fold rotational symmetry, can be enhanced by averaging the axially repeating motif in order to improve their signal‐to‐noise ratio; this requires that the slices with n‐fold symmetry do not exhibit distortions relative to one another.A strategy is proposed to detect the relative distortions and to remove them in order to obtain reliable results from the averaging process. Extensive use is made of cross‐correlation analysis and of interpolation; because the procedure involves iterated resampling of the image, it is essential to adopt interpolation algorithms which preserve the spectral power.The procedure is illustrated by the analysis of transverse sections of the sperm flagellum axoneme of a stick insect; it might be used for the reconstruction of microtubules, nuclear pore complexes, virus capsids, and other supramolecular aggregates. The reconstructed images of axoneme sections reveal new information about the interactions between adjacent doublets; in particular, a double conjunction connecting the inner dynein arm with the nearest B‐tubule has been consistently observed.
A method for rapid fixation and dehydration of nematode tissue for transmission electron microscopy
Authors: Jones John T. , Gwynn Iolo Ap 
Published: August 2011

SUMMARYA method is described using a microwave oven and acidified 2,2‐dimethoxypropane to fix and dehydrate nematode tissue rapidly in preparation for transmission electron microscopy. Nematode tissue prepared using this method is well preserved compared with tissue fixed by conventional methods. The technique also enabled fixation of unhatched nematodes in the egg, a process that is almost impossible to achieve using conventional methods of fixation. Using the method described, differences were found between the cuticles of different nematode stages.
Suggestions for the quantitative X‐ray microanalysis of thin sections of frozen‐dried and embedded biological tissues
Authors: Hall T. A. 
Published: August 2011

SUMMARYWhen a microregion in a thin section of frozen‐dried and embedded tissue is analysed by the conventional electron‐probe X‐ray continuum‐normalization method, the measured quantity is in mmol of element per kg of embedded specimen. As each microregion contains an unknown amount of embedding medium, this quantity generally lies indeterminately somewhere within the wide range between mmol of element per kg of hydrated tissue and mmol of element per kg of dehydrated tissue. However, if a ‘tag’ element is incorporated in the embedding medium, the contribution of the medium to the local continuum count in each probed field should be measurable, and the X‐ray data may then unambiguously yield mmol of element per kg of dehydrated tissue. This result should not be affected by shrinkage on freeze‐drying or by incomplete replacement of water by embedding medium.The same X‐ray data can additionally provide estimates of mmol of element per unit volume, mmol of element per kg of hydrated tissue and local dry‐mass fraction. However, these estimates are subject to errors due to tissue shrinkage, incomplete replacement of water and beam damage.
A comparison of some methods for measuring the size distribution of two‐dimensionally clustered latex particles in TEM images using mathematical morphology
Authors: Froehling P. E. 
Published: August 2011

SUMMARYThe particle size distribution of polymer latexes of, for example, polybutadiene can be measured on transmission electron micrographs using image analysis methods. By taking suitable measures in preparing the sample it can be ensured that the image only consists of projections of two‐dimensional clusters of adhering latex spheres. For the deagglomeration of these clusters a number of methods based on mathematical morphology are discussed. Speed and accuracy of the methods were tested on a personal‐computer‐based image analysis system and compared to manual measurements.Repeated openings or erosions applied to a binary image are relatively fast global methods with a low accuracy due to the fact that particle diameters are measured in whole numbers of pixels; only the horizontal diameters of rectangular pixels are measured.With an iterative method the individual components of the clusters are found by repeated conditional thickening of the erosion kernels. The method is accurate but very slow due to the large number of processing steps.The watershed method as applied to the original grey‐scale transmission electron micrograph makes use of the sharp local maxima which arise at the interface of the projection of two adhering particles. This method is as accurate as the iterative method, but about eight times as fast.
On the contraction and dilation of the plastic support membranes under electron‐beam irradiation in the transmission electron microscope
Authors: Dimitriu Corneliu 
Published: August 2011

SUMMARYTime relation curves are given for Formvar membrane contraction or collodion membrane dilation under electron‐beam irradiation. Also, numerical values are given for the contraction or dilation measured in different zones of the Formvar or collodion support membranes.
A method using an on‐line digital computer for the improvement of resolution of backscattered electron images
Authors: Oho Eisaku , Ogihara Akinori , Kanaya Koichi 
Published: August 2011

SUMMARYIn principle, the resolution of backscattered electron (BSE) images can be little improved, even though an infinitely small beam size is achieved by various improvements in the intrinsic instrument. In order to circumvent this problem, a method is proposed which utilizes an on‐line digital computer for the image recording and processing. The major image‐processing tools are reduction, expansion, super‐imposition with matching of the images, and high‐emphasis filtering in the Fourier domain. By using various combinations of these techniques, the resolution of BSE images has been significantly improved. The validity of these improved images has been confirmed. In the case of a BSE image with too wide a dynamic range, both the present method and digital homomorphic filtering provide successful results.
Anisotropy analysis for particle systems
Authors: Stoyan Dietrich , Beneš Victor 
Published: August 2011

SUMMARYThis paper discusses some methods for the quantitative description of anisotropy of planar structures. These methods can be applied to systems of particles randomly distributed. It is also possible to detect anisotropics in particle arrangements. Finally, a simple Monte Carlo test of an isotropy hypothesis is suggested. The methods are applied to a planar section of a Cd‐Zn eutecticum with clear anisotropy both in particle orientation and arrangement.
Increased depth of field and stereo pairs of fluorescence micrographs via inverse filtering and maximum‐likelihood estimation
Authors: Holmes Timothy J. , Liu Yi‐Hwa , Khosla Deepak , Agard David A. 
Published: August 2011

SUMMARYImage processing methods are presented for effectively increasing the depth of field and for generating stereo pairs of fluorescence micrographs from a conventional optical microscope. In developing these methods the slice theorem of computed tomography is used. In this way the image reconstruction problem is reduced to one of processing only two‐dimensional arrays rather than three‐dimensional arrays and the classical difficult problem of restoring missing Fourier components within the missing cone region is circumvented. Two different approaches to such processing are presented. One approach is based on inverse filtering. Another approach is based on previous development of iterative image restoration algorithms for quantum‐limited incoherent imagery, founded on maximum‐likelihood estimation. Limited experimentation with real micrographs shows that both approaches work well. Some preliminary comparisons are made between the different variations of the methods tested, which point out the advantages and present limitations. Both methods can be implemented on IBM‐AT‐compatible computers with relatively fast execution times. Advantages that these methods have over confocal microscopy are (i) the optical and computing equipment required is less expensive, and (ii) a conventional microscope when set up properly can have much better light sensitivity than a confocal microscope.
Effect of refraction on optical microscopic measurement of internal blood‐vessel diameter and its correction
Authors: Chen Eric Q. , Lam Chan F. , Periasamy Ammafi 
Published: August 2011

SUMMARYIn optical microscopic measurement of internal blood‐vessel diameters, the effect of refraction must be taken into account to ensure accuracy of the result. This effect is discussed and an analytical correction formula derived. Phantom blood vessels with known internal and external diameters were used to test the validity of the correction formula. The errors obtained prior to correction were reduced significantly after correction.
Characterization of biological macromolecules by combined mass mapping and electron energy‐loss spectroscopy
Authors: Leapman R. D. , Andrews S. B. 
Published: August 2011

SUMMARYThe combination of scanning transmission electron microscopy (STEM) and parallel‐detection energy‐loss spectroscopy (EELS) was used to detect specific bound elements within macromolecules and macromolecular assemblies prepared by direct freezing. After cryotransferring and freeze‐drying in situ, samples were re‐cooled to liquid nitrogen temperature and low‐dose (about 103 e/nm2) digital dark‐field images were obtained with single‐electron sensitivity using a beam energy of approximately 100 keV and a probe current of approximately 5 pA. These maps provided a means of characterizing the molecular weights of the structures at low dose. The probe current was subsequently increased to about 5 nA in order to perform elemental analysis. The 320 copper atoms in a keyhole limpet haemocyanin molecule (mol.wt = 8 MDa) were detected with a sensitivity of ± 30 atoms in an acquisition time of 200 s. Phosphorus was detected in an approximately 10‐nm length of single‐stranded RNA contained in a tobacco mosaic virus particle (mol.wt = 130 kDa/nm) with a sensitivity of ± 25 atoms. Near single‐atom sensitivity was achieved for the detection of iron in one haemoglobin molecule (mol.wt = 65 kDa, containing four Fe atoms). Such detection limits are only feasible if special processing methods are employed, as is demonstrated by the use of the second‐difference acquisition technique and multiple least‐squares fitting of reference spectra. Moreover, an extremely high electron dose (about 1010 e/nm2) is required resulting in mass loss that may be attributable to ‘knock‐on’ radiation damage.
The use of polarization analysis in the quantification of fluorescent emission: general principles
Authors: Entwistle A. , Noble M. 
Published: August 2011

SUMMARYFluorescent emission and fluorophore concentration are only linearly related below particular concentrations of fluorophore. Theoretically, analysis of fluorescent polarization might allow identification of situations in which local concentrations of fluorophore are above the range of linear response. Using a confocal scanning laser microscope, we demonstrate that progressive depolarization of fluorescent emission from fluorescein and fluorescein analogues occurs over the concentration range where linearity is lost. Critically, depolarization of emission is first seen at concentrations of fluorophore slightly below those at which linearity is lost. Thus, polarization analysis can be used to determine whether the local concentration of fluorophore is such that quantitative analysis can be carried out.
The isector: a simple and direct method for generating isotropic, uniform random sections from small specimens
Authors: Nyengaard Jens R. , Gundersen Hans Jørgen G. 
Published: August 2011

SUMMARYThe very simple and strong principle of vertical sections devised by Baddeley et al. has been a major advance in stereology when any kind of anisotropy is present in the specimen under study. On the other hand, some important stereological estimators still require isotropic, uniform random sections. This paper deals with a simple technique for embedding specimens in rubber moulds with spherical cavities. After the embedding, any handling of the resulting sphere independent of the specimen will induce isotropy of the final histological sections.
Molecular histochemistry and plant biology: a review
Authors: Harris N. 
Published: August 2011

SUMMARYThis review considers some of the recent advances in the applications of molecular histochemical techniques to the analysis of the temporal and spatial patterns of gene expression associated with differentiation and development in plant tissues. A range of methods is outlined, and those for tissue preparation and the isotopic and non‐isotopic labelling of probes for in situ hybridization studies are discussed, taking account of the various approaches which can be used for the localization of specifically bound probes.An overview of some successful applications of molecular histochemistry includes the elucidation of the roles of homeotic genes in floral biology, the ontogeny and differentiation of the photosynthetic apparatus, and aspects of seed development, including storage protein gene regulation during embryogenesis.
Vacuolar sequestration of fluorescent probes in plant cells: a review
Authors: Oparka Karl J. , Hawes Chris 
Published: August 2011

SUMMARYThe use of fluorescent probes as indicator and tracer molecules is becoming an important aspect of plant cell biology. In many cases the dye, whether introduced directly into the cytosol or sequestered by the cell from its external environment, is preferentially transferred to the vacuole. In the light of increasing evidence for endocytosis in plant cells, the sequestration of high‐molecular‐weight fluorescent dextrans and the membrane‐impermeant dye Lucifer Yellow‐CH into the vacuole has been cited as evidence supporting the presence of a fluid‐phase endocytic pathway. In this review we consider these recent reports of vacuolar sequestration in the light of new evidence arising on the mechanisms underlying dye uptake.
Wavelength considerations in confocal microscopy of botanical specimens
Authors: Fricker M. D. , White N. S. 
Published: August 2011

SUMMARYWe have used a multiple‐laser confocal microscope with lines at 325, 442, 488, 514 and 633 nm to investigate optical sectioning of botanical specimens over a wide range of wavelengths. The 442‐nm line allowed efficient excitation of Chromomycin A3, with minimal background autofluorescence, to visualize GC‐rich heterochromatin as an aid to chromosome identification. Sequential excitation with 442‐ and 488‐nm light enabled ratio imaging of cytosolic pH using BCECF. The red HeNe laser penetrated deep into intact plant tissues, being less prone to scattering than shorter blue lines, and was also used to image fluorescent samples in reflection, prior to fluorescence measurements, to reduce photobleaching. Chromatic corrections are more important in confocal microscope optics than in conventional microscopy. Measured focus differences between blue, green and red wavelengths, for commonly used objectives, were up to half the optical section thickness for both our multi‐laser system and a multi‐line single‐laser instrument. This limited high‐resolution sectioning at visible wavelengths caused a loss in signal. For ultraviolet excitation the focus shift was much larger and had to be corrected by pre‐focusing the illumination. With this system we have imaged DAPI‐stained nuclei, callose in pollen tubes using Aniline Blue and the calcium probe Indo‐1.