bluuebunny/crossref_metadata_2025_split huggingface dataset:
Direct identification, and localization of Azospirillum in the rhizosphere of wheat using fluorescence‐labelled monoclonal antibodies and confocal scanning laser microscopy
Authors: SCHLOTER M. , BORLINGHAUS R. , BODE W. , HARTMANN A. 
Published: August 2011

SummaryAzospirillum is a soil bacterium with plant growth‐promoting potential. Strain‐specific monoclonal antibodies (mAbs) which bind to different antigenic determinants of the bacterial cell surface with high specificity for A. brasilense Sp7 were used to monitor this bacterium in the rhizosphere of wheat plants. The mAbs were marked directly with the fluorochromes fluorescein (FITC) or tetramethylrhodamine (TRITC). With the labelled mAbs and confocal scanning laser microscopy, an in situ identification and localization of this bacterium in root segments was possible.
A new method for investigating the undersurface of cell monolayers by scanning electron microscopy
Authors: RICHARDS R. G. , LLOYD P. C. , RAHN B. A. , GWYNN I. AP 
Published: August 2011

SummaryGlow discharge is commonly used for cleaning the inside of coating units and for cleaning hard surfaces before carbon or metal evaporation procedures. In this study it has been used to remove the embedding medium to reveal, for scanning electron microscope (SEM) study, the undersurfaces of Balb/c 3T3 fibroblastic cells that have been cultured on Thermanox discs and embedded in LR White resin. Ten to twenty‐minute ionization times were found to reveal the largest area of the undersurface without causing damage to the cells. Chemical etching of the resin was also shown to reveal the undersurface of the cells, but caused some damage, preventing successful re‐embedding for transmission electron microscopy, and at higher magnifications revealed less detail. A circular impression within the main outline of the cells was observed in many cells, which is considered to reflect the presence of a nucleus. The undersurfaces of most cells, after applying both methods of etching, displayed a number of very short processes. Subsequent transmission electron microscopy of ultrathin sectioned, re‐embedded, areas of the gold sputter‐coated blocks revealed the depth of ionization that had occurred and confirmed that the specimens observed in SEM were the undersurfaces of cells. This method can be modified to study the attaching surface of any organism to a substratum.
Origin and significance of the SEM cathodoluminescence from zircon
Authors: KOSCHEK G. 
Published: August 2011

SummaryIn geochronological investigations, zircons are used frequently as a geological clock because of the small amounts of radioactive U isotopes and their decay products, e.g. Pb, incorporated into the crystal structure. However, the U or Pb concentration of zircon is not only dependent on the radioactive decay. One factor is the presence of old, inherited zircon cores in younger igneous zircons. This polyphase structure often cannot be recognized by conventional imaging methods, such as light microscopy. Dating such zircons, can therefore lead to ambiguous age results.However, zircons can be selected for dating purposes using cathodoluminescence (CL) in the scanning electron microscope. It is shown that zircon phases of different ages can be identified by their specific CL properties. Moreover, conclusions on the origin and on the development of the inherited phases can be drawn by comparison with detrital zircons.The zircon CL is formed by the superpostion of several broad bands. The shape of the spectra is dependent on the zircon genesis. By fitting the spectra with Gaussian curves, the individual CL bands can be separated. Using this methodology, it is possible to trace back the essential parts of the zircon CL to a superpostion of the quartz and zirconia (ZrO2) CL. In addition, CL phenomena due to dysprosium impurities can be separated from intrinsic zircon CL properties.
Video camera calibration for optical densitometry
Authors: BALDOCK R. A. , POOLE I. 
Published: August 2011

SummaryAn efficient technique for calibrating video cameras to record optical density (OD) from microscopic images is described. The method corrects for variation over the field of the brightfield and darkfield intensities, does not assume a linear response of the camera to the incident intensity and requires a single calibration filter.
Empirically determined freezing time for quick‐freezing with a liquid‐nitrogen‐cooled copper block
Authors: BAATSEN P. H. W. W. 
Published: August 2011

SummaryA method is presented to determine freezing time empirically. The method is based on determining the amount of stretch of a skinned muscle fibre while it is being frozen. Freezing time, as determined with this method, lies in between 0·5 and 1·5 ms.
An application of scanned focused ion beam milling to studies on the internal morphology of small arthropods
Authors: YOUNG R. J. , DINGLE T. , ROBINSON K. , PUGH P. J. A. 
Published: August 2011

SummaryFor the first time a scanned focused ion beam of approximately 50 nm diameter has been used to prepare biological material. Small defined areas of the surface were removed by ion etching to allow examination of the underlying structures with a scanning electron microscope. Different milling procedures were carried out on two anatomical features in mites of the genus Halarachne (Halarachnidae: Mesostigmata). In the first, square holes were milled into the surface of the peritrematal plate to reveal the structure of the underlying respiratory peritrematal groove. In the second, transverse cuts were made across the shafts of the sensory sensilli which make up the sensory Haller's organ on tarsus I. This latter procedure revealed detail both within the core and walls of sensilli. Details of specimen preparation and milling procedures, as well as suitability and interpretation of results, are presented.
Variable‐depth electropolishing of TEM samples
Authors: LEONARD S. W. 
Published: August 2011

SummaryA variable‐depth electropolishing technique has been developed for transmission electron microscopy samples using copper as sample material. This was required for an experiment concerning the measurement of the variation of dislocation density with depth for ion‐implanted materials. The polishing technique was achieved by a two‐step process, involving the measurement and use of the polishing rate to polish to a specific depth and the application of a transparent cover to one side of the sample for back‐thinning. With this technique, any sample depth can be made accessible for observation with a transmission electron microscope and the method should be applicable to many different materials and electropolishers.
The effect of soft X‐radiation on myofibrils
Authors: BENNETT P. M. , FOSTER G. F. , BUCKLEY C. J. , BURGE R. E. 
Published: August 2011

SummaryMyofibrils, the contractile organelles from striated muscles, have been examined in the X‐ray microscope to determine the effect of radiation on their function and structure. Using X‐rays of energy 350–385 eV in the water window we find that after an exposure to 7·5 × 105 photons/μm2 (calculated to give an absorbed dose of 20000 Gy) the myofibrils will no longer contract. The use of the free radical scavenging agent, DMSO, gives some protection to the fibrils. It has also been found that after this much irradiation the fibrils lose up to 20% of their mass. Further substantial mass loss occurs on subsequent irradiation. After 25 times the loss‐of‐function exposure only 30% of the mass remains. Analysis of a series of images of the same myofibril covering this range of exposures shows that the mass is preferentially lost in some areas of the structure and consequently significant structural changes occur.
Improvements in the technique of vascular perfusion‐fixation employing a fluorocarbon‐containing perfusate and a peristaltic pump controlled by pressure feedback
Authors: ROSTGAARD J. , QVORTRUP K. , POULSEN S. S. 
Published: August 2011

SummaryA new, improved technique for whole‐body perfusion‐fixation of rats and other small animals is described. The driving force is a peristaltic pump which is feedback regulated by a pressure transducer that monitors the blood/perfusion pressure in the left ventricle of the heart. The primary perfusate‐fixative is composed of a blood substitute—13·3% oxygenated fluorocarbon FC‐75—in 0·05 M cacodylate buffer (pH 7·4) with 2% glutaraldehyde. The secondary perfusate‐fixative is composed of 2% glutaraldehyde in 0·05 M cacodylate buffer (pH 7·4) with 20 mM CaCl2. A double‐barrelled, self‐holding cannula is used to cannulate the heart; the outer and inner barrels of the cannula are connected to the peristaltic pump and to the pressure transducer, respectively. The tissue oxygen tension in the rat is monitored by a subcutaneous oxygen electrode. Measurements showed that tissue hypoxia/anoxia did not develop before or during the perfusion‐fixation. Thus, the technique permits study of specimens which do not exhibit fixation gradients and do not contain cells fixed in a state of asphyxia. This is substantiated by electron micrographs of cells from different organs, revealing new fine structural elements. By adding oxygenated fluorocarbon to glutaraldehyde perfusate‐fixatives, enough oxygen is made accessible for cellular respiration as well as for the oxygen‐consuming chemical reactions of glutaraldehyde with the tissue. Data on anaesthesia, operative manoeuvres, mechanical components of the system, preparation of fixatives and flow of the perfusate‐fixatives are furnished and discussed.
An unbiased and efficient procedure for 3‐D connectivity measurement as applied to porous media
Authors: ZHAO H. Q. , MACDONALD I. F. 
Published: August 2011

SummaryA new stereological technique to measure the mean genus (connectivity) per unit volume Gv of a porous medium is described and applied to a real sandstone sample. The technique is based on the ‘net volume tangent counts’ performed on disector samples, i.e. pairs of consecutive sections of an interconnected structure. It consists of a set of simple counting rules applied to the features on the sections of the structure and can be easily implemented manually. The applicability and efficiency of the procedure is evaluated by applying it to a Berea sandstone sample which has been studied previously using a network analysis approach by interactive three‐dimensional computer reconstruction. It is shown that the procedure yields results in good agreement with the network analysis result, but has the advantages that it is much easier to implement, is more flexible in how the data are collected, is more efficient, and is known to provide an unbiased estimate of the mean genus per unit volume of the whole structure.
Liquid substitution: A versatile procedure for SEM specimen preparation of biological materials without drying or coating
Authors: ENSIKAT H. J. , BARTHLOTT W. 
Published: August 2011

SummaryCertain liquids with a very low vapour pressure, such as glycerol or triethylene glycol, can be used to infiltrate biological specimens so that they may be observed in the scanning electron microscope (SEM) without drying. The conductive properties of the fluids allow specimens to be examined either uncoated or with very thin coatings. The advantages of liquid substitution include the retention of lipids, waxes, loose particles, and surface contaminants. Since the procedure does not require expensive equipment, it offers an alternative to critical point drying or cryo‐preparation. For certain types of specimens, liquid substitution may represent the best preparation procedure. In addition, the fluids themselves may be imaged directly in the SEM, or indirectly by cathodoluminescence following labelling with fluorochromes.
Conformational characterization of nucleosomes by principal component analysis of their electron micrographs
Authors: ZABAL M. M. Z. , CZARNOTA G. J. , BAZETT‐JONES D. P. , OTTENSMEYER F. P. 
Published: August 2011

SummaryOptimized fixation conditions were determined for protein‐protein and protein‐DNA crosslinking within calf‐thymus nucleosomes in low monovalent salt concentrations. Nucleosomes were examined without heavy‐atom staining by darkfield electron microscopy. The dimensions of these macromolecular complexes and those of HeLa core particles optimally fixed in divalent salt were analysed using principal component analysis. According to this analysis the structure of the calf‐thymus nucleosomes was best presented by a prolate ellipsoid. Particle images had average major and minor axis lengths of 14·1 and 10·5 nm, respectively. In contrast, the HeLa nucleosomes were best modelled by an oblate ellipsoid from the analysis of their images, which had average major and minor axes of 13·3 and 11·5 nm. The applicability of this multivariate statistical analysis to the interpretation of macromolecular images is illustrated and discussed.
Fractal characterization by frequency analysis. I. Surfaces
Authors: ANGUIANO E. , PANCORBO M. , AGUILAR M. 
Published: August 2011

SummaryA study of the quality and accuracy of the methods based on frequency analysis for the fractal characterization of surfaces as measured by scanning tunnelling microscopy (or profilometry) is made. The study is based on computer simulation of images of fractal surfaces. A discussion of the mathematical algorithms used for computer generation of fractal surfaces then follows. The main conclusion is that studies of fractal characterization by frequency analysis reported in previous papers in the STM field, as well as conclusions about the performance of the various methods, are doubtful. New methods for frequency analysis that in some cases produce more reliable results are proposed.
Reflectance in situ hybridization (RISH): detection, by confocal reflectance laser microscopy, of gold‐labelled riboprobes in breast cancer cell lines and histological specimens
Authors: LINARES‐CRUZ G. , RIGAUT J. P. , VASSY J. , De OLIVEIRA T. C. , De CREMOUX P. , OLOFSSON B. , CALVO F. 
Published: August 2011

SUMMARYA method for reflectance in situ hybridization (RISH) is presented. The importance of the method is demonstrated by results obtained on cytological and histological breast cancer specimens.Scattering reflectance signals from 1‐nm colloidal‐gold particles after RNA/RNA in situ hybridization, using digoxigenin‐labelled riboprobes, were detected by confocal scanning laser microscopy.The mRNA expression of two ras‐related genes, rho B and rho C, was analysed in human histological breast cancer specimens and in human breast cancer cell lines. Horizontal (x, y) and vertical (z) optical sections after three‐dimensional imaging were used for visualization.A marked heterogeneity (between individual cells and between specimens) was noted for the expression of the rho B gene, both in cytological and in histological samples. On the other hand, rho C was always expressed and showed no heterogeneity.This method allows the identification of several cellular constituents in an heterogeneous tissue structure, as demonstrated by the simultaneous detection of rho B (or rho C) by reflectance and of DNA, cytokeratin and/or vimentin by fluorescence.
Some remarks on the accuracy of surface area estimation using the spatial grid
Authors: SANDAU K. , HAHN U. 
Published: August 2011

SUMMARYA set of three line grids in three orthogonal directions is called a spatial grid. This spatial grid can be used for surface area estimation by counting the number of intersection points of a surface with the grid lines. If direction and localization of the spatial grid are suitably randomized, the expectation of this number is proportional to the surface area of interest. The method was especially developed for cases where the surface to be measured is embedded in a medium, which is the usual case in microscopical applications, and where a stack of serial optical sections of the surface is available.The paper presents an improvement of an earlier version of the counting rule for intersection points. Furthermore, if the direction of sectioning is not uniform random, a bias results. This bias is calculated for a disc as a perfectly anisotropic object. A generalization of the estimator is considered by introducing a weighted mean instead of the usual arithmetic mean. The variance due to the randomized direction is investigated depending on the weights, and the minimum of this variance is derived. The relationship between the covariogram and the variance of the surface area estimated with the spatial grid is considered.
Volume‐weighted mean particle volume estimation using different measurement methods
Authors: ARTACHO‐PÉRULA E. , ROLDÁN‐VILLALOBOS R. 
Published: August 2011

SUMMARYThe use of the ‘point‐sampled intercepts’ method on histopathological material is evaluated for the main purpose of comparing different methods of intercept length measurements. The volume‐weighted mean nuclear volume—nuclear —of the carcinoma of the ampulla of Vater is calculated using three methods for measuring intercept lengths: a semiautomatic image analyser, an equidistant Io‐ruler and a logarithmic ‐ruler. Rulers of several classes and lengths are used, and the results of the nuclear estimations are compared. The equidistant Io‐ruler is more accurate than the logarithmic ‐ruler. With a greater number of ruler classes and with adjustment of ruler length to the greatest nuclear intercept lengths, the systematic deviation from the true value of the nuclear is smaller. The nuclear parameter has great power to differentiate intestinal carcinoma from normal tissue.
Automated tracing and volume measurements of neurons from 3‐D confocal fluorescence microscopy data
Authors: COHEN A. R. , ROYSAM B. , TURNER J. N. 
Published: August 2011

SUMMARYThree‐dimensional (3‐D) image analysis algorithms and experimental results that demonstrate the feasibility of fully automated tracing of neurons from fluorescence confocal microscopy data are presented. The input to the automated analysis is a set of successive optical slices that have been acquired using a confocal scanning laser microscope. The output of the system is a labelled graph representation of the neuronal topology that is spatially aligned with the 3‐D image data. A variety of topological and metric analyses can be carried out using this representation. For instance, precise measurements of volumes, lengths, diameters and tortuosities can be made over specific portions of the neuron that are specified in terms of the graph representation. The effectiveness of the method is demonstrated for a set of sample fields featuring selectively stained neurons. Additional work will be needed to refine the method for unsupervised use with complex data involving multiple intertwined neurons and extremely fine dendritic structures.
Algorithms for automated characterization of cell populations in thick specimens from 3‐D confocal fluorescence microscopy data
Authors: ROYSAM B. , ANCIN H. , BHATTACHARJYA A. K. , CHISTI M. A. , SEEGAL R. , TURNER J. N. 
Published: August 2011

SUMMARYMethods are presented for the automated, quantitative and three‐dimensional (3‐D) analysis of cell populations in thick, essentially intact tissue sections while maintaining intercell spatial relationships. This analysis replaces current manual methods which are tedious and subjective.The thick sample is imaged in three dimensions using a confocal scanning laser microscope. The stack of optical slices is processed by a 3‐D segmentation algorithm that separates touching and overlapping structures using localization constraints. Adaptive data reduction is used to achieve computational efficiency. A hierarchical cluster analysis algorithm is used automatically to characterize the cell population by a variety of cell features. It allows automatic detection and characterization of patterns such as the 3‐D spatial clustering of cells, and the relative distributions of cells of various sizes. It also permits the detection of structures that are much smaller, larger, brighter, darker, or differently shaped than the rest of the population. The overall method is demonstrated for a set of rat brain tissue sections that were labelled for tyrosine hydroxylase using fluorescein‐conjugated antibodies. The automated system was verified by comparison with computer‐assisted manual counts from the same image fields.
Measurement of absolute tracer concentrations in tissue sections by using digital imaging fluorescence microscopy. Application to the study of plasma protein uptake by the arterial wall
Authors: WEINBERG P. D. , WINLOVE C. P. , PARKER K. H. 
Published: August 2011

SUMMARYDigital imaging fluorescence microscopy (DIFM) of tissue sections was used to quantify uptake of labelled plasma proteins by the arterial wall. Several aspects of the measuring system were investigated so that absolute tracer concentrations and their local variation could be derived from digitized images. These investigations may be relevant to other studies employing DIFM.Nonlinearities were found to arise from offsets in the video digitizers, from background fluorescence and stray light within the microscope and from the transfer characteristics of the intensified CCD camera. Camera gain controls showed complex behaviour. Camera output fell substantially for several hours after switching on and was affected by room temperature. Large spatial variations in response were caused by the geometry of the microscope optics and by the image intensifier. However, the ratios between areas were not affected by light intensity or camera gain settings. Measured intensities were independent of the depthwise location of fluorophores within tissue sections but they were affected by the emission from objects outside the measuring area. Photobleaching of tracer varied significantly over the range of excitation intensities and durations used but was not concentration dependent. Methods used to correct these effects and obtain a uniform, linear and constant relationship between concentration and grey level are described.Using the system and appropriate corrections, in vivo uptake of sulphorhodamine‐B‐labelled serum albumin by the rabbit aortic wall was investigated. Results obtained for the mean uptake of tracer and its local variation were quantitatively similar to those previously obtained with nonmicroscopic methods.
Structural features of cell walls from tomato cells adapted to grow on the herbicide 2,6‐dichlorobenzonitrile
Authors: WELLS B. , McCANN M. C. , SHEDLETZKY E. , DELMER D. , ROBERTS K. 
Published: August 2011

SUMMARYEvidence from high‐resolution images of primary cell walls suggests that the cell wall is constructed from at least two independent yet coextensive fibrous networks, one based on cellulose/hemicellulose and the other on pectin. The ability to analyse the structure of each of these networks in isolation has been hampered by a lack of suitable biological material such as mutants. However, the recent use of the cellulose‐synthesis inhibitor 2,6‐dichlorobenzonitrile (DCB) that prevents the formation of the cellulose‐xyloglucan network while allowing the pectin network to form a functional wall offers the unique opportunity of studying at least the pectin network independently. A range of electron microscopy techniques and a novel spectroscopy method are used to study the walls from tomato suspension cells adapted to growth on DCB. Measurements of the minimum cell wall thickness derived from thin sections of dehydrated walls show that the marked reduction in level of the cellulose/hemicellulose network affects neither the thickness of the wall formed, nor the apparent spacing of pectin molecules. However, images obtained by the fast‐freeze, deep‐etch, rotary‐shadowed (FDR) replica technique show that the three‐dimensional architecture of these pectin‐rich walls is very different from that of nonadapted walls. Fourier transform infrared (FTIR) microspectroscopy data and immunogold‐labelling studies provide additional evidence that supports the previous biochemical data.