bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cryosectioning of plant material frozen at high pressure
Authors: Michel M. , Hillmann T. , Müller M. 
Published: August 2011

SUMMARYLow‐temperature electron microscopy combined with electron diffraction allows determination of the state of structural preservation as well as that of the solidified cellular water in frozen‐hydrated sections. Apple leaves frozen at high pressure and cryosectioned with glass knives covered with a very thin layer of frozen water exhibit a minimum of artefacts and are thin enough for structural analysis. The Zeiss 902 EM energy‐filtering microscope helps to improve the poor contrast of frozen‐hydrated sections by imaging in the zero‐loss mode. All sections analysed showed the electron diffraction pattern of vitreous water. This is ideal: the character of the liquid phase is preserved. A very high yield of good cryosections is achieved because the samples are vitrified and the friction on the glass knife surface is reduced by a thin ice layer.
A model for cryosectioning based on the morphology of vitrified ultrathin sections
Authors: Richter Karsten , Gnägi Helmut , Dubochet Jacques 
Published: August 2011

SUMMARYElectron microscopy of vitrified ultrathin sections allows cell ultrastructure to be studied in the hydrated state. Sectioning of the frozen material is, however, a limiting step, since the cutting forces cause severe mechanical deformation. In order to address this problem, we have investigated the surface of cryosections. It is shown that cryosections have two fundamentally different surfaces. One surface is rough, deformed by cutting‐induced deformation lines which are orientated perpendicular to the cutting direction. The other surface, in comparison, is not affected by those deformation lines. Except for knife marks it is smooth. In order to explain the observations, the following model is proposed. The rough relief corresponds to the former block face. Its roughness originates from material that is squeezed out of the section plane when the section is compressed in the cutting direction and bent away from the specimen block. The smooth section surface is the surface in contact with the knife during the sectioning. This contact keeps the surface smooth while imprinting the knife marks.
Application of high‐resolution scanning electron microscopy to biological macromolecules
Authors: Nakadera Takashi , Mitsushima Akira , Tanaka Keiichi 
Published: August 2011

SUMMARYThe development of ultrahigh‐resolution scanning electron microscopes (SEMs) has made the observation of biological macromolecules feasible, but adequate preparation methods have not yet been established. Although it has been possible to observe some molecules after they have been spread on a carbon substrate, this method has not proved suitable for other molecules which exhibit lower contrast, or are more susceptible to damage by the electron beam. In this study we have applied heavy‐metal impregnation methods using phosphotungstic acid, uranyl acetate, or osmium tetroxide mordanted by tannic acid. In addition, contamination due to the electron beam was reduced by improving the vacuum in the specimen chamber, and by the use of a heated specimen stage. Using these measures, haemocyanin, ferritin, apoferritin, thyro‐globulin and immunoglobulin M were successfully imaged. Ultrahigh‐resolution SEM seems likely to become an important means for studying the morphology of biological macromolecules.
A quantitative analysis of transparency in the human sclera and cornea using Fourier methods
Authors: Vaezy Shahram , Clark John I. 
Published: August 2011

SUMMARYCellular microstructure observed by scanning transmission electron microscopy (STEM) was analysed using Fourier methods. Fourier components of the density fluctuations in the sclera and the cornea of the human eye were quantified. The results show that the Fourier components responsible for the opacity of the sclera have sizes of the order of the wavelength of visible light. In the sclera, approximately 54% of the spectral energy of the density fluctuation falls in the range of 200–1100 nm (scattering range). In the cornea, approximately 24% of the total spectral energy falls in this range. The predominant Fourier components of the density fluctuations in the opaque sclera are approximately 300 nm in wavelength, whereas those of the transparent cornea are approximately 80 nm in wavelength. This method will be useful in quantitative analysis of microstructural changes accompanying biological phenomena such as normal development of transparency in the human lens, and abnormal loss of transparency during cataract formation.
The influence of specimen refractive index, detector signal integration, and non‐uniform scan speed on the imaging properties in confocal microscopy
Authors: Carlsson K. 
Published: August 2011

SUMMARYRefraction of light in a specimen volume may cause aberrations that influence the imaging properties in confocal microscopy. In this paper the influence on three‐dimensional resolution and geometry is experimentally investigated for a uniform specimen volume. It is found that the depth resolution is more severely affected than the lateral resolution. This is unfortunate, because even under ideal conditions the depth resolution is lower than the lateral resolution. Lateral image geometry is little affected by the specimen refractive index, whereas the depth scale can be considerably elongated or compressed.The influence of a finite detector integration time is also considered. This can give a noticeable, but not particularly severe effect on the image resolution in the line‐scan direction. Because the integration time can be accurately controlled, a shorter integration time can be used when maximum resolution is essential, albeit at the price of a higher noise level.In scanning fluorescence microscopy a non‐uniform scan speed may give large variations in bleaching over the specimen surface. Experiments illustrate how serious such non‐uniform bleaching effects can be when a specimen area is repeatedly scanned, for example when recording optical serial sections.
Absorption and scattering correction in fluorescence confocal microscopy
Authors: Visser T. D. , Groen F. C. A. , Brakenhoff G. J. 
Published: August 2011

SUMMARYIn three‐dimensional (3‐D) fluorescence images produced by a confocal scanning laser microscope (CSLM), the contribution of the deeper layers is attenuated due to absorption and scattering of both the excitation and the fluorescence light. Because of these effects a quantitative analysis of the images is not always possible without restoration. Both scattering and absorption are governed by an exponential decay law. Using only one (space‐dependent) extinction coefficient, the total attenuation process can be described. Given the extinction coefficient we calculate within a non‐uniform object the relative intensity of the excitation light at its deeper layers. We also give a method to estimate the extinction coefficients which are required to restore 3‐D images. An implementation of such a restoration filter is discussed and an example of a successful restoration is given.
Working with the confocal scanning UV‐laser microscope: specific DNA localization at high sensitivity and multiple‐parameter fluorescence
Authors: Montag Markus , Kukulies Jörg , Jörgens Reinhard , Gundlach Heinz , Trendelenburg Michael F. , Spring Herbert 
Published: August 2011

SUMMARYThe use of fast‐staining DNA‐specific dyes such as DAPI or Hoechst 33342/33258 has been a major problem for confocal scanning laser microscopy (CSLM) studies of intranuclear chromatin organization. Moreover, the availability of a confocal ultraviolet scanning laser microscope configuration, which allows an excitation at wavelengths of 364 nm as well as 488, 514 and 543 nm, is a prerequisite for single as well as multiple fluorescence parameter studies, especially if these studies are concerned with the precise localization of intranuclear signals.Here we report the characteristics and application of a CSLM, which was adapted for UV‐excitation and therefore enables comparison of the spatial distribution of several types of signals within one preparation. In addition to multiple‐parameter studies, we have also investigated the sensitivity of the system with regard to the identification of the double‐stranded DNA of lampbrush chromosome loops in germinal vesicles of amphibian oocytes.
Accumulation of fluorescent non‐cationic probes in mitochondria of cultured cells: observations, a proposed mechanism, and some implications
Authors: Rashid Fiza , Horobin Richard W. 
Published: August 2011

SUMMARYCultured rat fibroblasts were exposed to millimolar concentrations of forty‐four non‐cationic fluorescent probes, of very varied physico‐chemical properties. Mitochondrial staining occurred with nineteen of these probes, nine of which were nominally anionic and ten nominally non‐ionic. All nineteen were in fact lipophilic weak acids. Using structural parameters these could be specified numerically as follows: electric charge ≤ 0; log P(less‐ionized form) < 0; and pKa ∼ 7. In addition to these structural variables, dye concentration and the time of exposure of cells to probes were significant factors for the staining of mitochondria. Accumulation of these compounds can be understood in terms of ion‐trapping of hydrophilic salts of lipophilic weak acids, due to the internal pH of respiring mitochondria being higher than the cytosolic pH. As a case example of the application of this approach, the mode of action of many inhibitors of mitochondrial anabolism is discussed in terms of the mechanisms introduced here.
Reflection electron imaging and spectroscopy studies of aluminium metal reduction at α‐aluminium oxide (0,1,2) surfaces
Authors: Wang Z. L. 
Published: August 2011

SUMMARYCleaved α‐aluminium oxide (0,1,2) surfaces are studied using combined techniques of scanning reflection electron microscopy and reflection electron energy‐loss spectroscopy. An α‐aluminium oxide (0,1,2) surface can terminate with a layer of either oxygen or Al atoms depending on where the bonds break. Aluminium metal can be reduced only from the domains initially terminated with oxygen by an intensive electron beam. The interpretation of this damage effect is based on the desorption process of oxygen atoms after Auger decay. The resistance to beam damage of the domains initially terminated with Al is found to be due to the protection of the surface adsorbed amorphous oxygen layer.
Images of 16S ribosomal RNA by scanning tunnelling microscopy
Authors: Flamion Pierre‐Jacques , Cachia Claire , Schreiber Jean‐Pierre , David Thierry , Lesniewska Eric , Goudonnet Jean‐Pierre 
Published: August 2011

SUMMARYWe report the use of scanning tunnelling microscopy (STM) to study surface topographies of complex nucleic acid structures. From low‐resolution STM images of uncoated 16S ribosomal RNA, we demonstrate the possibility of determining several objective parameters (molecular mass and radius of gyration) in order to characterize and identify the molecules observed. These parameters were compared with values obtained by other physical methods and the radius of gyration was found to be the most reliable. At high resolution, it was possible to measure the main dimensions of selected V‐form particles more precisely than with electron microscopy. Images of the more compact form have been also obtained that show different domains in the macromolecular structure.
Application of scanning electron microscopy (SEM) and microbead techniques to study the localization of p24 and p18 antigens of HIV‐1 on the surface of HIV‐1‐infected H9‐lymphocytes
Authors: Dennin Reinhard H. , Beyer Axel 
Published: August 2011

SUMMARYImmunofluorescence staining techniques at present, when applied to follow the expression of HIV‐1‐specific antigens on infected cells, only give the information that the antigens detected are localized in the outer region of the membrane of the infected cell. We therefore set up a procedure using magnetic polystyrol particles coated with antibodies specific for the HIV‐1 antigens under study, in combination with scanning electron microscopy. We were able to demonstrate that p24 and p18 structural antigens are clearly expressed on the surface of HIV‐1‐infected H9 lymphocytes. This means that there was no steric hindrance for structures of cell‐like size specific for HIV‐1 antigens to interact with their target antigens. Other antigens may be hidden in membrane structures and are therefore inaccessible, for example, to the beads used here, which were of a similar size to antigen‐specific cells in vivo. The results of this model system must be seen with respect to the interaction of antigen‐specific cell‐mediated immunity with full antibody‐dependent cellular cytotoxicity, or without cytotoxic T lymphocytes, the mediator function of antibodies.
Visualization of ultraviolet microbeam irradiation sites with uranyl acetate
Authors: Czaban B. Barbara , Forer Arthur 
Published: August 2011

SUMMARYA method which enables visualization of the position of the focused ultraviolet (UV) light (260–300 nm) in a UV microbeam apparatus is described. The UV light is focused onto uranyl acetate which fluoresces. The fluorescence is of low intensity, but can be visualized using a TV‐camera sensitive to low light levels. This is a simple, quick, and objective way to locate the position of the focused UV light in UV microbeam.
High‐pressure scanning electron microscopy of insulating materials: A new approach
Authors: FARLEY A. N. , SHAH J. S. 
Published: August 2011

SUMMARYA new SEM technique for imaging uncoated non‐conducting specimens at high beam voltages is described which employs a high‐pressure environment and an electric field to achieve charge neutralization. During imaging, the specimen surface is kept at a stable low voltage, near earth potential, by directing a flow of positive gas ions at the specimen surface under the action of an electric bias field at a pressure of about 200 Pa. In this way charge neutrality is continuously maintained to obtain micrographs free of charging artefacts. Images are formed by specimen current detection containing both secondary electron and backscattered electron signal information. Micrographs of geological, ceramic, and semiconductor materials obtained with this method are presented. The technique is also useful for the SEM examination of histological sections of biological specimens without any further preparation.A simple theory for the charge neutralization process is described. It is based on the interaction of the primary and emissive signal components with the surrounding gas medium and the resulting neutralizing currents. Further micrographs are presented to illustrate the pressure dependence of the charge neutralization process in two glass specimens which show clearly identifiable charging artefacts in conventional microscopy.
A simple ethanol‐based freeze‐substitution technique for marine invertebrate embryos which allows retention of antigenicity
Authors: Campbell Stephen S. , Crawford Bruce J. , Reimer Corinne L. 
Published: August 2011

SUMMARYA simple technique has been developed for flash freezing and freeze substituting small (0.5 mm) marine embryos, which effectively preserves both cellular and extracellular components, using inexpensive equipment that is readily available in most laboratories. To achieve this, embryos of the starfish, Pisaster ochraceus, were isolated on copper freeze‐fracture EM grids. The embryos were then rapidly frozen by plunging the grids into a supercooled liquid cryogen, and stored in liquid nitrogen. Freeze substitution was carried out by placing the specimens in sealed vials containing anhydrous ethanol at −90°C for 4–5 days. Following substitution, the specimens were passively warmed to −20°C over 2 h and then to room temperature over a further 2 h. They were then embedded in either JB4 for light microscopy or Epon or LR White resins for transmission electron microscopy. Four different liquid cryogens, Freon 12, ethane, propane, and nitrogen slush, were tested. Freezing in propane, the best cryogen of the four, gave good preservation of the embryonic cells but poor preservation of the extracellular matrix (ECM). To overcome this, the embryos were exposed to four cryoprotective agents, dimethylsulphoxide, glycerol, ethylene glycol and propylene glycol prior to freezing, and the results were assessed. The experiments demonstrated that good preservation of both cells and ECM could be achieved by adding 15% propylene glycol in sea water to the embryos prior to freezing in propane. Material preserved in this manner not only gave excellent morphological results, but the antigenicity of both native antigens of ECM components and antibodies to which the animals had been exposed in vivo were retained. The application of this technique to other tissues and embryos should prove useful in many future studies.
Surface areas of basolateral membranes in renal distal tubules estimated by vertical sections
Authors: Mathiasen Flemming Ø. , Gundersen Hans Jøorgen G. , Maunsbach Arvid B. , Skriver Elisabeth 
Published: August 2011

SUMMARYThe surface area of the Na, K‐ATPase‐rich basolateral membranes in the medullary thick ascending limb of the distal tubule in the rabbit kidney was determined stereologically using the method of ‘vertical sections’, whereby unbiased surface estimates are obtained by imposing a cycloid test‐lattice on micrographs of ultrathin sections cut roughly longitudinal to the tubular axis.The unbiased estimate of the surface area of basolateral membranes per tubule length in the thick ascending limb was 1.45 × 106 μm2/mm. The results are compared with previous surface area measurements in this segment of the tubule and discussed with respect to the contributions from all sampling levels to the real biological variation. An optimized sampling scheme with a roughly fourfold reduction in workload is suggested.
An approach based on two‐dimensional graph theory for structural cluster detection and its histopathological application
Authors: Kayser K. , Sandau K. , Paul J. , Weisse G. 
Published: August 2011

SUMMARYAn approach based on graph theory is described for detecting clusters of cells in tissue specimens (two‐dimensional space). With a set of discrete basic elements (cell nuclei) having several measurable features (area, surface, main and minor axis of best‐fitting ellipses) a graph is defined as having attributes associated with edges. Different minimum spanning trees (MSTs) can be constructed using different weight functions on the attributes (attributed MST). Analysis of the MST and of an attributed MST by use of a decomposition function allows detection of image areas with similar local properties. These clusters, which are then clusters of the tree, describe, for example, partial growth in different directions in a case of a human fibrosarcoma assuming that tumour cell nuclei are homogeneous with respect to their configuration and size. The model allows the separation of clusters of tumour cells growing in different directions and the approximation of the different growth angles. This decomposition also allows us to create new (higher) orders of structure (cluster tree).
The significance of 3‐D transfer functions in confocal scanning microscopy
Authors: Sheppard C. J. R. , Gu Min 
Published: August 2011

SUMMARYThe three‐dimensional (3‐D) transfer function is a useful concept for describing image formation in confocal scanning microscopy. From it we can derive the corresponding 2‐D transfer function for in‐focus imaging. In confocal transmission this can be derived analytically. The 1‐D transfer function for on‐axis imaging, which can be expressed in an analytical form even for confocal fluorescence with differing wavelengths of excitation and fluorescence, can be derived from the 3‐D transfer function. The 2‐D transfer function for in‐focus imaging in confocal fluorescence microscopy with a finite‐sized detector is also presented, which is shown to exhibit sign changes and can therefore result in reversals of image contrast.
The double disector: unbiased stereological estimation of the number of particles inside other particles
Authors: Marcussen Niels 
Published: August 2011

SUMMARYA double‐disector method for obtaining estimates of the number of particles inside other particles, illustrated by an estimation of the average cell number in a glomerulus, has been developed. The method is suitable for paraffin‐embedded tissue because it does not require knowledge about the section thickness. Most importantly, the estimate is absolutely unaffected by tissue shrinkage. The average number of cells in a human glomerulus is 2850 whereas in rats it is 580.
Geostatistical and morphological methods applied to three‐dimensional microscopy
Authors: Conan V. , Gesbert S. , Howard C.V. , Jeulin D. , Meyer F. , Renard D. 
Published: August 2011

SUMMARYThree‐dimensional (3‐D) images of osteocyte lacunae were examined on a confocal microscope. Both geostatistical and morphological processing techniques were used to improve and to analyse them. By a geostatistical approach, this study aims at improving 3‐D confocal images before any further image processing. Optimized linear filters, which take account of the second‐order statistics and the 3‐D structure of the data, allow for the removal of imperfections such as noise and/or blur due to the axial convolution, and interpolate voxels on a face‐centred cubic grid from an initial cubic grid. An application of this technique to 3‐D biological images is demonstrated. In a second step, a 3‐D binary image is digitized and cleaned with 3‐D morphological filters. The standard 3‐D measurements cannot be applied in this case, since all osteocytes cut the border of the field. For this reason a 3‐D Boolean model has been adjusted, from which it is possible to derive all useful information on the repartition and the morphology of the osteocytes.
Interpretation of electron microscope autoradiographs: correction for cross‐fire when membrane components are highly labelled
Authors: Hejblum Gilles , Mary Jean‐Yves , Morice Vincent , Aurengo Andre 
Published: August 2011

SUMMARYThe interpretation of electron microscope autoradiographs concerns the estimate of the relative concentration of a radiolabelled molecule in subcellular compartments. Because of the radiation spread, an autoradiographic grain underlying a subcellular compartment cannot be directly assigned to the latter. Observed data must therefore be corrected for cross‐fire (CF). We studied the efficiency of several methods designed for the CF correction when membrane components possess a high concentration of labelling, resulting in a high proportion of CF. Grains generated from simulated sources were subjected to the CF method, to an original method based on the simulated annealing optimization algorithm, and to the expectation‐maximization algorithm. The expectation‐maximization algorithm appears to be clearly superior to the two other methods. Nevertheless, the variances of the estimates were higher than expected. Since no analytical expression of the estimates is available, an objective comparison of the labelling in different compartments is not possible with a standard test. Consequently, the interpretation of autoradiographs in electron microscopy is still an open problem.
Observation of rhombic facets on platinum wire after heating in air using scanning tunnelling microscopy
Authors: Hoffmann‐Millack Britta , Roberts Clive J. , Steer William S. 
Published: August 2011

SUMMARYEvidence is provided for the formation of surface faceting due to heating in air at 1160 K. For the first time diamond‐shaped facets with angles of 60 and 120° have been observed on the surface of a sample of ultrapure platinum wire in air using scanning tunnelling microscopy. The maximum extension of the facets is about 1000 Å.