bluuebunny/crossref_metadata_2025_split huggingface dataset:
In situ elemental analysis and visualization in cryofixed nervous tissues: X‐ray microanalytical investigations of embryological and mature brain, inner ear, photoreceptors, muscle and muscle spindles. Comparison of preparation methods for analysis and visualization at cellular and subcellular levels
Authors: Wróblewski Romuald 
Published: August 2011

SummaryFor meaningful X‐ray microanalysis (XRMA) in biology and medicine, the development of preparative and quantitative methods has been necessary. The methods need to preserve close to in vivo distribution of diffusible ions with at the same time reasonable morphological preservation of the tissue.Analyses at low and middle microanalytical resolution are useful at the initial stages of an investigation or when data from large populations of samples have to be acquired. Cryomicrotomy, which makes it possible for the single cells within semi‐thin and thick cryosections examined by X‐ray microanalysis to be further characterized histochemi‐cally (enzyme and substrate content), has been adopted for several pathophysiological studies. The method is particularly suitable for the analysis of complex morphological tissues with many cell types as in the brain or sensory organs of the internal ear.For microanalysis at the subcellular level, we developed a preparative procedure based on the frozen fixed preparation which is freeze‐dried in vacuo at −80°C and then at the same temperature, without breaking the vacuum, impregnated with a low‐temperature Lowicryl‐type resin. The resin is polymerized by u.v. light. This method prevents redistribution of the ions in the tissue and retains the antigenicity of the tissue. A considerable number of cells can be analysed simultaneously and the elemental composition in different cell compartments can be compared due to the similar analytical conditions within the section.An alternative to thin plastic sections of freeze‐dried material is thin cryosections cut at −150°C and analysed at low temperatures. Although some methodological problems still exist in preparation of cryosections, this type of section is potentially the most useful in analysis of diffusible ions, especially calcium which in most biological systems is present in very low concentrations.New preparative techniques for XRMA brought severe problems in visualization of the specimens prepared by cryomethods. Charging, low contrast, mass loss and contamination, which are often negligible in conventional electron microscopy, have still to be solved in XRMA of cryoprepared specimens. However, the methods of semi‐thin and thick cryosectioning and low‐temperature embedding were successfully used for analysis of cells and organelles and for the study of fluids in restricted biological spaces such as the inner ear, muscle spindles and ventricles of the brain in rats. Accordingly, examinations which were impossible by micropuncture and ion selective techniques could be carried out by XRMA. New data on the developing embryological organs and maturation of biological fluids in small biological cavities were obtained.
Whole‐mount preparations of mouse lens epithelium for the fluorescent cytological study of actin
Authors: Liou W. 
Published: August 2011

SUMMARYA technique is given for the preparation of a sheet of epithelial cells from the capsule of the crystalline lens. A new method is described for fixation and staining with fluorescent phalloidin or actin antibody in order to localize the actin cytoskeleton in this tissue. Optical section of the preparation resolves such actin features as apical polygonal arrays, sequestered actin bundles, perinuclear actin aggregates, observed here for the first time, and filamentous networks in the basal region of the cell. This method is superior to previous ones in its ability to preserve actin‐abundant sectors distinctively.
Image analysis of Nissl‐stained neuronal perikarya in the primary visual cortex of the rat: Automatic detection and segmentation of neuronal profiles with nuclei and nucleoli*
Authors: Ahrens Peter , Schleicher Axel , Zilles Karl , Werner Lisa 
Published: August 2011

SUMMARYAn image analysing procedure for the morphometric characterization of cortical neurons in Nissl‐stained brain sections is described. It consists of the automatic detection of cellular profiles and their compartments: cytoplasm, nucleus and nucleolus.The algorithm was designed to cope with the large morphological spectrum of cortical perikarya (e.g. geometrical properties of perikarya, staining intensities of cell compartments and nucleo‐plasmic area‐ratio) including pyramidal (Golgi‐category I) and non‐pyramidal (Golgi‐category II) neurons.Clusters of cells were separated and non‐neuronal structures (e.g. glia, endothelial cells) as well as tangential, non‐nucleolated sections through neuronal perikarya recognized and excluded from further analysis without requiring interactive procedures.The performance of the profile recognition procedure was evaluated using 426 nucleolated and non‐nucleolated profiles of different types of neurons in the primary visual cortex of the rat. Nucleolated profiles were recognized as such with a 91% accuracy, non‐nucleolated profiles were rejected correctly in 90% of cases.After automatic segmentation and selection of nucleolated neuronal profiles from the microscopic field, a large set of quantitative morphological features including geometrical, densitometrical and textural parameters can be measured using high power light microscopy. This permits quantitative morphometric characterization of different neuronal types. This procedure is the first part of a system for the automatic classification of Nissl‐stained cortical neurons.
3‐D image formation in high‐aperture fluorescence confocal microscopy: a numerical analysis
Authors: van der Voort H. T. M. , Brakenhoff G. J. 
Published: August 2011

SUMMARYThe imaging properties of a confocal fluorescence microscope are considered on the basis of a theoretical model. The model takes into account high‐aperture objectives, the polarization state of the excitation light and a finite detector pinhole. Electromagnetic diffraction theory of the field near focus as developed by Richards and Wolf is used to compute the optical properties of the model. These are shown to be dependent on the polarization of the light. With the resulting three‐dimensional point spread function we have studied the imaging of point, line and plane objects as a function of their orientation with respect to the confocal plane. In addition, the effect of the pinhole size on the image formation of these objects is discussed.The results indicate the necessity to take object orientation into account during image processing activities such as segmentation or analysis.
A fibre‐optic 3‐D microscope with high depth sensitivity
Authors: Fujii Y. , Yamazaki Y. 
Published: August 2011

SUMMARYA new fibre‐optic microscope with high depth resolution is proposed and experimentally realized. In this microscope, a pair of monomode optical fibre couplers are used to form a six‐port all‐fibre Michelson interferometer. A lock‐in amplifier is used to improve the depth sensitivity and to simplify the optical and electronic circuits. The phase sensing signal is applied to a mirror at one arm of the interferometer. The optimum resolution was about 0ċ4 nm. The transverse resolution of this microscope is determined by the core diameter of the monomode fibre (9 μm). This 3‐D microscope is quite stable to ambient mechanical disturbances, and can be applied to the 3‐D microscopy of various biological objects.
Image processing techniques for 3‐D chromosome analysis
Authors: Houtsmuller A. B. , Oud J. L. , van der Voort H. T. M. , Baarslag M. W. , Krol J. J. , Mosterd B. , Mans A. , Brakenhoff G. J. , Nanninga N. 
Published: August 2011

SUMMARY3‐D karyotype analysis is developing rapidly due to the availability of confocal microscopes and CCD video cameras, and the development of 3‐D processing techniques. Here, image enhancement and visualization techniques specifically designed for 3‐D karyotype analysis are described. To facilitate a good comparison between the different techniques, the same 3‐D image, obtained with a confocal scanning laser microscope (CSLM), of a mitotic prophase nucleus of a root‐tip cell of Crepis capillaris was used throughout. Besides well‐known stereoscopic presentation, another means of improving depth perception is shown, i.e. a solid modelling algorithm, which simulates the process of fluorescence.An interactive routine to dissect objects in the image is presented as an alternative for automated segmentation algorithms, which cannot be applied to closely apposed or merging objects.As an example of a convenient way to reduce the vast amount of data (2 Mbyte per image), a partly automated 3‐D cursor is presented in detail. This cursor is used to trace the central axes of chromosomes and record them as strings of Cartesian coordinates. The advantages of a computer graphics display, which facilitates real‐time rotation and hence is a powerful tool in studying 3‐D features of chromosomes, are also shown.
Analysing and sorting human chromosomes
Authors: Green Daryll K. 
Published: August 2011

SUMMARYAnalysing and sorting human chromosomes by flow cytometry is a powerful tool in the hands of the molecular biologist. Because of the large number of chromosomes analysed by flow, typically 105 for each sample, estimates of the distribution of DNA throughout the chromosome complement of an individual can be made to within three megabase pairs. Rearrangements of DNA in this size range can often be clearly seen and measured by flow cytometry in situations where it was not obvious by traditional cytogenetics.The production of enriched samples of a particular chromosome by flow sorting and the subsequent construction of DNA libraries has played an important part in mapping genes to particular hereditary diseases. Techniques now exist which allow the hybridization of a few thousand sorted chromosomes with characterized or uncharacterized DNA probes to give relatively quick answers about specific chromosome genes. The process of obtaining a sorted chromosome sample from a growing population of peripheral blood lymphocytes or lymphoblastoid cells is compared with other methods of achieving similar results in molecular biological terms. Advances in flow cytometric techniques, which include slit‐scanning and hybridization of DNA probes in suspension, are likely to improve the enrichment quality of specific sorted human chromosomes.
Projection of topographic maps from curved brain surfaces into a plane
Authors: Rager Günter , Frei Bernhard , Kaup Burchard 
Published: August 2011

SUMMARYThe environment is represented in the brain by topographic maps. These maps and their interrelationship form a dominant feature in the brain, but unfortunately, they are often projected onto surfaces which are variously curved. It would be useful to be able to compare the topography on these surfaces quantitatively with the origin of the map. This comparison would be possible if the curved brain surfaces could be represented in planes. Therefore, it was necessary to develop a procedure for unfolding maps on convex structures like the retina and the optic tectum of the chick. For this purpose a unit sphere was constructed whose origin was close to the centre of gravity of the curved brain structure. The surface of this structure was then projected into the sphere. From its representation on the spherical surface it was unfolded into the plane using a polar azimuthal radially equidistant projection. Our method can safely be applied to convex structures only. However, it is not limited to the visual system. The mathematical procedure is formulated in such a way as to be easily implemented. It is exemplified by experiments on the optic tectum and the developing retina of the chick.
Assignment of two ultrastructures formed by a mixture of hexonamides using autoradiography and electron microscopy
Authors: Boettcher Christoph , Boekema Egbert J. , Fuhrhop Juergen ‐H. 
Published: August 2011

SUMMARYThe combined application of autoradiography and electron microscopy allowed the assignment of molecular components to individual micellar fibres in a mixed gel. Resolution was of the order of 0.1 μm. As a result, it was shown that bimolecular sheets of N‐dodecyl‐l‐mannonamide (= l‐Man‐12) completely separated from helical rods consisting of tritiated N‐octyl‐d‐gluconamide (= d‐Glu‐8). The method should be useful for the analysis of several other synthetic supramolecular systems.
Low‐temperature EDX microanalysis of halogenated copper phthalocyanine samples
Authors: McColgan P. , Chapman J. N. , Nicholson W. A. P. 
Published: August 2011

SUMMARYX‐ray microanalysis at temperatures near that of liquid nitrogen is used to determine the halogen content of small volumes of halogenated copper phthalocyanine pigments. Details of the low‐temperature stage used and the benefits conferred by cooling the sample are given. The experimental procedure adopted involved recording a series of spectra from the same area and extrapolating the measured compositions to zero dose. Factors affecting the accuracy of the analyses are discussed.
Unbiased estimation of curve length in 3‐D using vertical slices
Authors: Gokhale A. M. 
Published: August 2011

SUMMARYAn efficient sampling procedure is presented for estimation of total line length per unit volume Lv. It involves the following steps: (1) choose a vertical axis in the specimen, and cut the specimen to obtain VUR vertical slices of constant thickness Δ such that parallel planes of the slices contain the vertical direction; (2) observe the projected image of a vertical slice using transmission microscopy such that beam direction is perpendicular to the slice; (3) count the number of intersections of the projected images of the lineal features of interest with cycloid‐shaped test lines whose minor axis is perpendicular to the vertical axis. The expected value of the number of intersections per unit length prj is related to Lv as follows:Thus, Lv can be estimated from the measurements performed on the projected images of VUR vertical slices.
Time‐resolved cryo‐electron microscopy of vitrified muscular components
Authors: Lepault Jean , Erk Inge , Nicolas Gisèle , Ranck Jean‐Luc 
Published: August 2011

SUMMARYBiological objects may be arrested in defined stages of their activity by fast freezing and may then be structurally examined. If the time between the start of activity and freezing is controlled, structural rearrangements due to biological function can be determined. Cryo‐electron microscopy shows great potential for the study of such time‐dependent phenomena. This study examines the actin polymerization process using cryo‐electron microscopy of vitrified specimens. Actin filaments are shown to undergo a structural change during polymerization. In the early stages of the polymerization process (t < 2 min), filaments exhibit a pronounced structural variation and frequently show a central low‐density area. In the later stages of the polymerization, F‐actin‐ADP filaments have a more uniform appearance and rarely display a central low‐density area. These findings, analysed on the basis of a previously proposed polymerization model, suggest that polymerization intermediates (F‐actin‐ATP and more probably F‐actin‐ADP‐Pi) and filaments at steady state (F‐actin‐ADP) have different structures. To investigate the physiological relevance of these results at the cellular level, the potential of cryo‐substitution in preserving the structure of muscular fibre was assessed. Optical diffraction patterns of relaxed and contracted frog cutaneous muscle are similar to the corresponding X‐ray diffraction patterns. The resolution of the images extends to about 7 nm. These results show that dynamic study of muscle contraction is possible using cryo‐substitution.
The characterization of microstructures in the atom probe
Authors: Hetherington M. G. , Miller M. K. 
Published: August 2011

SUMMARYMathematical descriptions of typical microsctructures which are observed in the atom probe are constructed from the theory of Markov chains. Short‐range ordering and clustering, overlapping and non‐overlapping particles and spinodal decomposition can all be modelled using this formalism. The models are used to calculate the important autocorrelation function.
Atomic force microscopy of a hydrated bacterial surface protein
Authors: Wiegräbe Winfried , Nonnenmacher Martin , Guckenberger Reinhard , Wolter Olaf 
Published: August 2011

SUMMARYThe protein surface layer of the bacterium Deinococcus radiodurans (HPI layer) was examined with an atomic force microscope (AFM). The measurements on the air‐dried, but still hydrated layer were performed in the attractive imaging mode in which the forces between tip and sample are much smaller than in AFM in the repulsive mode or in scanning tunnelling microscopy (STM). The results are compared with STM and transmission electron microscopy (TEM) data.
Validation of convection‐limited cooling of samples for freeze‐fracture electron microscopy
Authors: Bailey Stuart M. , Zasadzinski Joseph A. N. 
Published: August 2011

SUMMARYRapid freezing is the most important step in sample preparation for freeze‐fracture and other cryotechniques for electron microscopy. A simple heat transfer model is experimentally validated to show that convection from the cryogen to the specimen is the limiting step in rapid freezing of small samples [Biot modulus, (hd/k) < 1] by measuring cooling rates in a variety of samples, materials, and cryogens. In comparison to the commonly accepted conduction‐limited model, the convection‐limited model predicts, and our experiments show, that cooling rates are proportional to the surface area to volume ratio, independent of the sample thermal conductivity, and inversely proportional to the product of sample density and heat capacity. We show that almost any material can be frozen at similar rates if the sample thickness, the cryogen, and the method and velocity of contact with cryogen are similar. Liquid ethane or propane cooled to liquid nitrogen temperature are shown to give the best results.
A preparation method of specimens of the fungus Penicillium chrysogenum for ultrastructural and immuno‐electron microscopical studies
Authors: Müller Wally H. , van der Krift Theo P. , Knoll Gerd , Smaal Erik B. , Verkleij Arie J. 
Published: August 2011

SUMMARYA combination of cryofixation without pre‐treatment, freeze‐substitution and low‐temperature embedding was used to prepare specimens of Penicillium chrysogenum for electron microscopy. To produce specimens which are thin enough for appropriate cryofixation, the P. chrysogenum colonies were grown between dissected‐dialysis tubing on an agar plate, which in addition allowed longitudinal sectioning. In contrast to classical chemical fixation, this preparation procedure resulted in excellent preservation of ultrastructure. Furthermore, the penicillin biosynthetic enzyme acyltransferase could be unequivocally located by immunogold labelling, indicating a preservation of antigenic properties of the specimen. Labelling density was not conspicuously affected when using different freeze‐substitution media, but it was reduced after embedding in Epon 812.
Solute‐enriched surface layers and X‐ray microanalysis of thin foils of a commercial aluminium alloy
Authors: Park J. K. , Ardell A. J. 
Published: August 2011

SUMMARYThin foils of the commercial Al alloy 7075 were subjected to X‐ray microanalysis in a Jeol 100 CX TEMSCAN using a modified double‐tilting stage. The ratio of the Cu Kx and Al Kx peaks, Icu/IAl, was found to decrease rapidly with increasing foil thickness, t, in regions thinner than about 200 nm. This was attributed to the formation of surface layers enriched in Cu during electropolishing, consistent with the findings of other investigators. An equation predicting the variation of IB/IA with t in an A‐rich alloy in which B (or additional solute elements) is dilute is derived for a sample with surface layers enriched in B. The derivation predicts that IB/IA should vary linearly with t−1, and that the intercept of such a plot should be positive. It is suggested that consistency with this prediction serves as a criterion for true surface enrichment. Our data on Cu obey these predictions, and further analysis indicates that the layers on both surfaces are enriched by a factor of about 5.6. The ratio of the intensities of the Zn Kx and Al Kx X‐ray peaks was found to be nearly independent of foil thickness to t≅400 nm. This result, combined with additional analysis, indicates that the apparent enrichment observed by other investigators is most likely due to the influence of instrumental factors.
Staining sections of water‐miscible resins 2. Effects of staining‐reagent lipophilicity on the staining of glycol‐methacrylate‐embedded tissues
Authors: Horobin Richard W. , Gerrits Peter O. , Wright David J. 
Published: August 2011

SUMMARYGlycol methacrylate (GMA) sections of animal tissues were stained with a group of twenty‐seven reagents of very varied chemical characteristics. The artefactual background staining of the resin was found to be dependent on the hydrophilic/lipophilic character of the staining reagent, as estimated from the logarithm of its octanol‐water partition coefficient (log P). Intense background staining occurred with lipophilic stains, whose log P>2. In keeping with this, use of GMA semi‐permeable membranes for enzyme histochemistry failed to give staining when using a lipophilic substrate, probably because the substrate was trapped in the membrane. An analysis of other routine histochemical stains—in terms of the probable occurrence of high resin background staining and low tissue sensitivity—is made. A numerical guide is provided to help avoid artefacts resulting from hydrophobic and size effects. Note: small, hydrophilic reagents (log P <0; molecular weight < 550 Da) are least likely to show either type of artefact. Conversely, reagents which are lipophilic, or/and of intermediate size (log P > 2; 550 < ionic weight < 1000 Da), give strong background staining.
Extended electron energy‐loss fine structure and selected‐area electron diffraction studies of small palladium clusters
Authors: Diociaiuti M. , Picozzi P. , Santucci S. , Lozzi L. , De Crescenzi M. 
Published: August 2011

SUMMARYExtended electron energy‐loss fine structure (EXELFS) and selected‐area electron diffraction (SAED) techniques have both been applied to the study of the crystalline structure of Pd clusters of average diameters ranging from bulk to 24 Å. The combined use of these techniques gives complementary information about the crystalline structure of Pd clusters. Both techniques show the same lattice parameter expansion, about 4% for the smallest Pd cluster, with respect to the bulk. The EXELFS analysis performed on the Pd‐M4,5 edge shows a sizeable increase of structural disorder in the smallest cluster. SAED gives additional information about the Pd bulk sample, showing the occurrence of crystalline regions about 50 Å in diameter.