bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fluorescent in‐situ hybridization to detect cellular RNA by flow cytometry and confocal microscopy†
Authors: Bauman Jan G. J. , Bayer Jan A. , van Dekken Herman 
Published: August 2011

SUMMARYA fluorescent in‐situ hybridization procedure was developed which is suitable for detection of specific cellular RNA in cells fixed in suspension. The procedure was originally developed for analysis of single‐cell suspensions by flow cytometry. The resulting fluorescent cells proved to have their 3‐D morphology prefectly preserved. The spatial distribution of specific ribosomal RNA and messenger RNA could then be analysed by confocal microscopy of individual cells. In the hybridization procedure, biotinylated single‐stranded RNA probes were used that were produced by transcription from cloned DNA fragments. Detection of poly‐(A)+ RNA was performed with a poly‐biotin‐d(U)‐tailed oligo‐d(T) probe. Bound probe was detected using streptavidin–fluorescein isothiocyanate. For flow cytometry, nuclear DNA was counterstained with di‐amidine‐2‐phenyl indol. Multi‐parameter flow cytometry was used to quantify the fluorescence intensity, i.e. the hybridization signal, of thousands of cells. Confocal scanning microscopy on individual cells revealed the intracellular distribution of the target RNA. The distribution of ribosomal RNA and poly‐(A)+ RNA mRNA in mouse bone marrow and the human leukaemia cell line HL60 cells was investigated. Ribosomal RNA was confined to the cytoplasm of the cells, although in many cells the nucleoli could also be distinguished. Poly‐(A)+ RNA in HL60 cells was found both in the cytoplasm and nucleus. A granular cytoplasmic distribution was seen in part of these cells.
Localization of ribosomal and telomeric DNA sequences in intact plant nuclei by in‐situ hybridization and three‐dimensional optical microscopy
Authors: Rawlins D. J. , Shaw P. J. 
Published: August 2011

SUMMARYWe have combined the use of three‐dimensional (3‐D) fluorescence microscopy and computer image processing of images with in‐situ hybridization to analyse the 3‐D organization of interphase nuclei in plants. In sections of root tips of Pisum sativum, using cDNA probes, we have shown that telomeres are arranged around the nuclear periphery and that the ribosomal genes in this species appear to exist in discrete, 3‐D domains.
Analysis by confocal scanning laser microscopy imaging of the spatial distribution of intermediate filaments in foetal and adult rat liver cells
Authors: Vassy Jany , Rigaut Jean Paul , Hill Anne‐Marie , Foucrier Jean 
Published: August 2011

SUMMARYConfocal scanning laser microscopy has been used to make three‐dimensional observations of the spatial distribution of cytoskeleton intermediate filaments in rat liver hepatocytes, at various stages during foetal development and in the adult. Single and double immuno‐labelling with fluorescein and Texas Red fluorescence have been used to study the intracellular spatial distribution of C18 cytokeratin and vimentin. Simultaneous confocal imaging with double‐fluorescence emission requires an image processing step for the correction of ‘contamination’ effects due to the overlap between fluorescein and Texas Red emission spectra.At the pre‐natal period (day 20 of gestation) each type of intermediate filament labelling is only present in a certain cellular category, C18 cytokeratin in hepatocytes and vimentin in mesenchymal cells. However, at the earliest developmental stages (day 12 of gestation), vimentin and cytokeratin seem to be found in the same type of cells, probably mesenchymal cells.Some striking developmental changes, associated with the differentiation of the liver parenchyma, are observed for both C18 cytokeratin and vimentin. In earlier foetal stages, C18 filaments are scarce, hazily labelled and randomly distributed inside the hepatocytic cytoplasm. Late during foetal development (days 18–20 of gestation), hepatocytic cytokeratin filaments are abundant, well individualized and sharply labelled. The hepatocytes are arranged in a muralium duplex architecture (two‐cell‐thick sheets) and the labelling intensity measured in the hepatocytic cytoplasm at the basal pole is double that measured at the sinusoidal pole, while, in the adult, hepatocytes are arranged in a muralium simplex architecture (one‐cell‐thick sheets) and cytokeratin filaments have a symmetrical distribution in relation to the nuclear region.
Three‐dimensional electron microscopy of entire cells
Authors: Bron Cph. , Gremillet Ph. , Launay D. , Jourlin M. , Gautschi H. P. , Bächi Th. , Schüpbach J. 
Published: August 2011

SUMMARYThe digital processing of serial electron‐microscope sections containing laser‐induced topographical references allows a three‐dimensional (3‐D) reconstruction of entire cells at a depth resolution of 40–60 nm by the use of novel image analysis methods. The images are directly processed by a video‐camera placed under the electron microscope in TEM mode or by the electron counting device in STEM mode. The deformations associated with the cutting of embedded cells are back‐calculated by new computer algorithms developed for image analysis and treatment. They correct the artefacts caused by serial sectioning and automatically reconstruct the third dimension of the cells. Used in such a way, our data provide definitive information on the 3‐D architecture of cells. This computer‐assisted 3‐D analysis represents a new tool for the documentation and analysis of cell ultrastructure and for morphometric studies. Furthermore, it is now possible for the observer to view the contents of the reconstructed tissue volume in a variety of different ways using computer‐aided display techniques.
A combination of topographical contrast and stereoscopy for the reconstruction of surface topographies in SEM
Authors: Beil W. , Carlsen I. C. 
Published: August 2011

SUMMARYThe reconstruction of three‐dimensional surface topographies can be done in two principally different ways: conventional stereoscopy and ‘shape from shading’. In conventional stereoscopy the depth information is obtained from two perspective views of the specimen. For that, perspectively corresponding features have to be identified in both views to determine depth from perspective shift. Conventional stereoscopy normally results in a relatively sparse set of irregularly distributed points whose elevations are known precisely. The shape‐from‐shading approach determines the local surface orientation from the local surface luminosity. Over a limited range of surface inclinations the emission of secondary (SE) and back‐scattered (BSE) electrons depends uniquely on the angle between electron beam and local surface normal of the specimen. Shape from shading uses this relationship to determine the surface normal with multiple detectors mounted in different take‐off directions. Contrary to conventional stereoscopy shape from shading yields depth information from each surface point, but this method is less accurate than stereoscopy.In this paper we propose a combination of both approaches, in which the dense, but less accurate results from shape from shading are used to fill the gaps in the sparsely distributed, but very accurately known, depth information obtained from stereoscopy.
Methods for obtaining high quality stereoscopic images of microscopic objects
Authors: Sroczyński S. F. 
Published: August 2011

SUMMARYOver 100 years ago Ernst Abbe developed the so‐called method of half‐moon diaphragms, placed in the exit pupil plane of a binocular microscope, for stereoscopic viewing of microscopic objects. Simplicity of the method being a great advantage, the obvious disadvantages were reduced resolution, azimuthal errors of imaging and failure to control plasticity of the images viewed. Using modern microscopic optics it is possible to avoid these complications, and a complete control of the plasticity of the stereoscopic image as perceived by the observer is achievable while working with medium, strong and the strongest microscope objective lenses. A number of formulae, a nomogram, and a set of tables for calculation of optimal settings are presented in order to promote a range of practical applications, including photomicrography.
Ultrastructural preservation of nuclei and chromatin: improvement with low‐temperature methods
Authors: Horowitz R. A. , Giannasca P. J. , Woodcock C. L. 
Published: August 2011

SUMMARYThe ultrastructure of chromatin has been examined in nuclei prepared by a variety of low‐temperature methods. Embedding glutaraldehyde (GA)‐fixed nuclei in Lowicryl K4M or K11M following dehydration by the progressive lowering of temperature (PLT) method, or in K11M following spray freezing and freeze substitution (FS), produces chromatin fibres that have, in situ, a diameter close to the in vivo state, and show internal structural details consistent with patterns of nucleosome packing previously observed only in preparations of isolated fibres. This is a temperature‐dependent effect; fibres conventionally dehydrated and embedded in Lowicryl at 0°C or in conventional epoxy resin at 60°C have lower and less uniform diameters, and lack internal structural details. Of the techniques used, spray freezing followed by FS resulted in the most notable improvement over conventional methods. Inclusion of GA during FS of rapidly frozen, unfixed nuclei in methanol does not result in cross‐linking of nuclear proteins. In acetone, however, cross‐linking by GA occurs at — 45°C, or at lower temperatures if the water content of the acetone‐based FS media is kept deliberately high. Substitution regimes employing GA alone or in combination with uranyl acetate and/or osmium tetroxide do not result in fibre morphologies comparable to either prefixed or unfixed nuclei substituted in additive‐free substitution media. Whole fibroblasts show excellent preservation of nuclei and the nuclear/cytoplasmic interface after spray freezing followed by FS and low‐temperature embedding.
Morphogenesis of the brain as studied by 3‐D computer graphics simulation
Authors: Fujita Setsuya 
Published: August 2011

SUMMARYMorphogenesis of the brain is analysed by 3‐D computer graphics simulation. The initial form of the brain, i.e. a neural tube, is created in the computer as a small tubular figure in 3‐D Cartesian coordinates which are subsequently transformed into curvilinear coordinates by a set of growth functions. By this method the morphological development of the human brain is simulated. The same computer program can be applied to simulate the development of the brains of mouse, rat and monkey simply by changing the parameters slightly. The result of the analysis reveals that the smaller brains are produced by faster growth of the matrix cell population at the onset of development, coupled with faster rate of retardation of the growth during ontogeny. Thus, smaller brains in smaller animals are produced in a shorter period during their ontogeny.
Fluorescence histochemical techniques for catecholamines as tools in neurobiology
Authors: Felten David L. , Felten Suzanne Y. , Sladek John R. , Notter Mary D. , Carlson Sonia L. , Bellinger Denise L. , Wiegand Stanley J. 
Published: August 2011

SUMMARYFormaldehyde‐induced and glyoxylic‐acid‐induced fluorescence histochemistry permits the tissue localization of catecholamines in the central nervous system (CNS) and peripheral nervous system (PNS), and in culture. Counterstains such as ethidium bromide provide excellent background identification of specific innervated regions in both the CNS and the periphery. Use of fluorescence histochemistry with immunocytochemistry can elucidate catecholamine‐peptide relationships. Gelatin‐ink perfusion used with fluorescence histochemistry permits the investigation of neuro‐vascular relationships and documentation of vascular and parenchymal compartmentation of innervation. Combined use of fluorescence histochemistry and retrograde tracing methods demonstrates the specific cellular sources of innervation of target regions. Micropunch neurochemical analysis provides quantitative data for correlation with fluorescence histochemistry within a target region of innervation, and micro‐spectrofluorometric analysis provides a semi‐quantitative evaluation of the amount of fluorophore within a target region or within specific subcellular compartments such as the cell body or terminals.
Receptor autoradiography and 2‐deoxyglucose techniques in neuroscience
Authors: Wiederhold K.‐H. , Palacios J. M. 
Published: August 2011

SUMMARYThe use of the receptor autoradiography and 2‐deoxyglucose (2‐DG) techniques in neuroscience are reviewed. Receptors and other binding sites can be visualized autoradiographically in microtome tissue sections after labelling with radioligand in vivo or in vitro. Autoradiograms are generated by apposition of the labelled tissue to photographic emulsions. Combined with computerized image analysis, this technique can be used to analyse and quantify the microscopic distribution of receptors and receptor alterations associated with lesions or disease in human and animal tissues. The 2‐DG technique permits microscopic analysis of modifications in brain glucose utilization induced by physiological and pharmacological manipulations. Limitations of these techniques and attempts to optimize their resolution are also discussed.
Estimating the surface density of fissures in clay soil from deterministic parallel sections
Authors: Scott G. J. T. , Webster R. , Nortcliff S. 
Published: August 2011

SUMMARYIn much clay soil the pore space consists of 3‐D networks of narrow, approximately planar fissures. The density of the interface between fissures and solid soil is important; a method for its estimation from the horizontal and vertical cuboid samples that are taken conventionally is described here.Undisturbed samples are impregnated with resin containing a fluorescent dye. Parallel plane faces are cut, and the exposed surfaces are photographed under u.v. light. Pairs of superimposed photographic negatives are projected onto a screen, and the lateral displacement of each interface from one section to the next is measured at points of intersection of the interface with linear probes through the soil. These distances are used to infer the local orientation of the interfaces, which are then used with the point counts to estimate the interface areas.The method was applied to subsoil of the Windsor series. The interface density was estimated to be 9.69cm2/cm3 from one horizontal and one vertical sample. Point‐counting with isotropic uniform random linear probes applied to five randomly orientated samples from the same site gave an estimate of 8.78cm2/cm3. It seems probable that the error associated with the new method is no greater than that associated with the standard method.
Study of the early growth and branching of Streptomyces tendae by means of an image processing system
Authors: Reichl U. , Buschulte T. K. , Gilles E. D. 
Published: August 2011

SUMMARYA method is described for directly observing submerged growth of streptomycetes, which can be easily adapted for investigations of filamentously growing fungi. The use of an image processing system allowed a fast and accurate calculation of important morphological parameters. Most of the images can be analysed on‐line. Some results are presented for early growth and branching of Streptomyces tendae. It seems that mechanisms for growth of streptomycetes were the same for both solid and liquid media.
Measurements of cells in culture by scanning acoustic microscopy
Authors: Litniewski Jerzy , Bereiter‐Hahn Jürgen 
Published: August 2011

SUMMARYA novel method allowing the determination of thickness, attenuation and impedance of both living and fixed cells in culture using a single image recorded with a scanning acoustic microscope is described. The method is based on the recording of the image data locating the surface of the cell far below the geometric focus. In this way only the normal incident acoustic wave is used for image formation.Higher values of impedance and attenuation coefficients were found in the cell periphery than in the central part of the cell. This phenomenon is suggested to be due to the different organization of cytoskeletal elements.
Three‐dimensional dynamic morphometry on a diffraction pattern in the focal plane
Authors: Ishizaka S. 
Published: August 2011

SUMMARYA new type of morphometry, which shortens the scanning time of precise observation by confocal microscopy, has been investigated.To analyse the 3‐D distribution of active sites on a living cell, microspheres of the same size were immunologically marked on specific sites on a cell. Incident coherent light was scattered on the microspheres and the scattered light from each microsphere superimposed upon each other giving a diffraction pattern of the examined cell. Several series of interference fringes were generated on the diffraction pattern, according to the phase difference of the microspheres. These interference fringes on the 2‐D diffraction pattern enable the (relative) 3‐D positions of the microspheres to be determined.3‐D dynamic morphometry on a 2‐D diffraction pattern with interference fringes speeds up imaging in confocal microscopy.
A method for evaluating microscope objectives to optimize performance of confocal systems
Authors: Cogswell C. J. , Sheppard C. J. R. , Moss M. C. , Howard C. V. 
Published: August 2011

SUMMARYA method for evaluating the performance of microscope objectives on two types of confocal scanning optical microscope is presented. Of these two confocal microscope types, off‐axis beam‐scanning systems are found to require microscope objectives which have been corrected for flatness of field as well as for spherical aberration and astigmatism in order to obtain maximum axial and laterial resolution. In the case of on‐axis specimen‐scanning microscopes, less highly corrected objective lenses (not corrected for flatness of field) may in practice prove to have superior resolving properties.
Automated image segmentation and serial section reconstruction in microscopy
Authors: Moss V. A. , Jenkinson D. McEwan , Elder H. Y. 
Published: August 2011

SUMMARYA technique for automatic 3‐D reconstruction of specifically stained features in televised serial histological sections has been developed using an image analyser. Images which included these features were individually converted to binary images, compiled, displayed to show the 3‐D morphology and used to quantify the structure. The criteria necessary for producing valid reconstructions and the problems associated with the manipulation of images of fine detail, particularly those containing several thousand features, are illustrated by examples from skin.
Detection of doubly stained fluorescent specimens using confocal microscopy
Authors: Mossberg Karin , Ericsson Maria 
Published: August 2011

SUMMARYStudies of doubly stained specimens were performed with a confocal scanning microscope. The instrument used provides the possibility of making separate detections of the fluorescent dyes. The optimal choice of excitation wavelengths and optical filters are discussed. The fluorphores that were used are Lucifer Yellow, Texas Red, fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate.
Methodical aspects of 3‐D reconstruction of chromatin architecture in mouse trophoblast giant nuclei
Authors: Montag Markus , Spring Herbert , Trendelenburg Michael F. , Kriete Andres 
Published: August 2011

SUMMARYPrecise 3‐D reconstruction of the spatial organization of murine trophoblast giant‐cell chromatin is a prerequisite for detailed investigations on the fine structural changes in chromatin‐fibre organization during the trophoblast endomitotic cell cycle. It appears very likely that sequential fine structural changes in the chromatin arrangement are concomitant with the changes in the gene expression pattern of these cells during the early phase of murine gestation.The complex intra‐nuclear chromatin organization of mouse trophoblast giant nuclei was investigated in permanent tissue preparations which had been stained with a DNA‐specific dye. The spatial chromatin arrangement was examined in fluorescence with a confocal scanning laser microscope. Serial optical sections were recorded and subjected to a computer‐based 3‐D reconstruction method which is suitable even for very complex biological structures.
3‐D model of vascular network in rat skin obtained by stereo vision techniques
Authors: Kim N. H. , Aggarwal S. J. , Bovik A. C. , Diller K. R. 
Published: August 2011

SUMMARYThe quantitative analysis of the depth of injury, penetration of therapeutic agents in tissues, and the regeneration of vascular patency after a graded degree of thermal injury requires a knowledge of the shape and spatial configuration of the vascular networks in the tissue. We have applied computational stereo vision techniques to describe the 3‐D configuration of microvessels in full thickness rat skin vascular casts produced by perfusion of Yellow Microfil latex solution through the aorta. The principal concern is to describe the 3‐D structure of vascular networks using a set of 3‐D space curves. This representation is computed by integrating monocular and binocular processing; the 2‐D curve representation of blood vessels computed through monocular analysis is integrated with disparity data to yield a space curve representation for each vessel. A connection diagram is also computed to indicate the connections existing among the computed space curve representations.
Towards light microscopic imaging of hydrated ‘native’ ribosomal RNA genes
Authors: Spring H. , Trendelenburg M. F. 
Published: August 2011

SUMMARYInvestigations of the higher‐order structure of ‘native’ genes as well as antibody localization to defined gene chromatin areas require elaborate light microscopic imaging of the chromatin structure of interest, such as visualization of unfixed and unstained gene transcription units.Since all our present structural information on the, relatively, small nucleolar rRNA genes has been obtained using transmission electron microscopy of spread genes following fixation, staining and complete dehydration, this type of EM specimen was our first specimen for LM imaging. Using Nomarski differential interference contrast video microscopy, it was found that spread EM chromatin can be sufficiently visualized by light microscopy to allow precise correlation with the conventional TEM image.In addition, it is demonstrated that video‐enhanced LM can provide enough contrast enhancement for rapid visualization of spread ‘native’ rRNA genes in slightly fixed, fully hydrated, unstained gene chromatin.