bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of microstructural parameters of random spatial surface fractals by measuring chord length distributions
Authors: HERMANN H. , OHSER J.
Published: August 2011

An experimental procedure for determining the micro‐structural parameters of random spatial surface fractals is proposed. The procedure is based on chord length distribution measurements of planar sections of the investigated material at different linear resolutions, and comparison of the experimental results with the corresponding parameters of a well‐defined structure model. The variability of the model allows microstructures with arbitrary volume fraction and fractal dimension to be considered. Furthermore, both discrete and continuous scaling behaviour have been studied.
Calibration of an electron back‐scattering pattern set‐up
Authors: LASSEN N. C. KRIEGER , BILDE‐SØRENSEN J. B.
Published: August 2011

The determination of lattice orientations from electron back‐scattering patterns (EBSPs) in a scanning electron microscope (SEM) requires accurate knowledge of the position of the pattern centre and the source point to screen distance. This paper outlines a new procedure that enables the determination of these parameters for any given set‐up of the EBSP/SEM system. The calibration procedure simply requires the positions and indices of at least four poles in a pattern obtained from an arbitrary specimen, and eliminates the need for standard specimens or special attachments to the EBSP/SEM system. The pattern centre is shown to be located with a precision of approximately 0·5° and the source point to screen distance can be determined with a relative precision of approximately 0·5%.
Observation of biological materials by X‐ray photoelectron‐conversion contact microscopy
Authors: WATANABE N. , MATSUMURA T. , INAGAKI Y. , KINOSHITA K. , SHIMANUKI Y. , FURUYA K. , TAGUCHI T. , TANIGUCHI M. , KIHARA H.
Published: August 2011

Dried biological specimens, such as fossil diatoms, collagen, nerve tissue and spicule of Trepang, were observed by X‐ray photoelectron‐conversion contact microscopy. A spatial resolution of 0·2 μm was attained. The fossil diatom image shows a clear difference below and above the carbon K‐absorption edge (4·46nm).
Observation of the loss of the hydroxyapatite sixfold symmetry in a human fetal tooth enamel crystal
Authors: BRES E. F. , STEUER P. , VOEGEL J.‐C. , FRANK R. M. , CUISINIER F. J. G.
Published: August 2011

A deviation from the hydroxyapatite hexagonal symmetry of a human tooth enamel crystal observed by high‐resolution electron microscopy is reported. This symmetry deviation is characterized by: (1) ‘preferential’ planes that can be indexed as (100) with an intensity that differs from the (300) and the other {100} hexagonal equivalent planes; and (2) streaking of higher order reflections in the optical diffractogram of the image of the crystal. Computer simulations show that similar ‘preferential’ planes can also be observed at specific crystal tilt angles (and/or beam tilt and/or objective aperture misalignment) and at crystal thickness/microscope defocus values in images of hydroxyapatite crystals observed along the [0001] or [] zone axes. The streaking of higher order reflections in the optical diffractogram is related to a deformation of the crystal itself and does indeed show a symmetry deviation of the crystal under observation.
The adsorption staining technique applied to isolated premessenger ribonucleoprotein particles: A comparison with conventional techniques using electron microscope tomography
Authors: LÖNNROTH A. , SKOGLUND U. , TOKUYASU K. T. , DANEHOLT B.
Published: August 2011

A specific type of premessenger RNP particle, Balbiani ring granules from the dipteran Chironomus tentans, was biochemically isolated and visualized in three dimensions with electron microscope tomography. The particles were prepared for electron microscopy in three different ways: positively stained, negatively stained and adsorption‐stained (embedded in polyvinyl alcohol, PVA, and concomitantly stained). The results were compared with those obtained for RNP particles studied in situ in ultrathin sections of plastic‐embedded cells. The positively stained particles were compacted and heavily deformed with little or no internal structure. The negatively stained and the adsorption‐stained particles were well preserved; the outer contours and the central cavities of the particles were outlined. The internal structure, i.e. the folded 7‐nm elementary fibre, could not be recognized in the negatively stained particles. In the adsorption‐stained particles, however, the fibre was discernable, although not quite as distinctly demarcated as in the plastic‐embedded samples. We conclude that embedding in PVA with concomitant staining with uranyl acetate is a rapid method to obtain both good preservation and staining of isolated RNP particles. The PVA‐embedded particles were also found to be sufficiently resistant to irradiation to permit a comprehensive tilt‐series to be taken for electron microscope tomography.

ribonucleoprotein particles.
Methods for large data volumes from confocal scanning laser microscopy of lung
Authors: OLDMIXON E. H. , CARLSSON K.
Published: August 2011

Confocal scanning laser microscopy makes it possible to obtain series of optical sections in precise registration. Certain studies of lung parenchyma, however, require both the fine resolution obtainable with high‐numerical‐aperture (NA) objectives and the extensive fields of view that usually would be achieved only with low‐NA objectives. This article presents a technique that resolves this conflict by using a sequence of operations: (i) to correct intensity variations on individual sections due to non‐uniform illumination/detection characteristics of the microscope; (ii) to correct intensity variations between successive sections in a series due to, for example, depth‐related absorption or step changes in detector sensitivity; (iii) to adjust adjacent, overlapping stacks of sections to a common intensity level; and (iv) to fuse a group of such overlapping stacks into a single series of larger sections. This resulting stack may contain, for example, a complete cross‐section of an alveolar ductal unit about 500 μm or more in diameter at about 1‐μm pixel resolution.
Orthogonal‐plane fluorescence optical sectioning: Three‐dimensional imaging of macroscopic biological specimens
Authors: VOIE A. H. , BURNS D. H. , SPELMAN F. A.
Published: August 2011

An imaging technique called orthogonal‐plane fluorescence optical sectioning (OPFOS) was developed to image the internal architecture of the cochlea. Expressions for the three‐dimensional point spread function and the axial and lateral resolution are derived. Methodologies for tissue preparation and for construction, alignment, calibration and characterization of an OPFOS apparatus are presented. The instrument described produced focused, high‐resolution images of optical sections of an intact, excised guineapig cochlea. The lateral and axial resolutions of the images were 10 and 26 μm, respectively, within a 1·5‐mm field of view.
Applications of medium‐voltage STEM for the 3‐D study of organelles within very thick sections
Authors: BEORCHIA A. , HELIOT L. , MENAGER M. , KAPLAN H. , PLOTON D.
Published: August 2011

Scanning transmission electron microscopy at 300 kV enables the visualization of nucleolar silver‐stained structures within thick sections (3–8 μm) of Epon‐embedded cells at high tilt angles (–50°; + 50°). Thick sections coated with gold particles were used to determine the best conditions for obtaining images with high contrast and good resolution. For a 6‐μm‐thick section the values of thinning and shrinkage under the beam are 35 to 10%, respectively. At the electron density used in these experiments (100e—/Å2/s) it is estimated that these modifications of the section stabilized in less than 10 min. The broadening of the beam through the section was measured and calculations indicated that the subsequent resolution reached 100 nm for objects localized near the lower side of 4‐μm‐thick sections with a spot‐size of 5·6 nm. Comparing the same biological samples, viewed alternately in CTEM and STEM, demonstrated that images obtained in STEM have a better resolution and contrast for sections thicker than 3 μm. Therefore, the visualization of densely stained structures, observed through very thick sections in the STEM mode, will be very useful in the near future for microtomographic reconstruction of cellular organelles.
Spurious contributions from scattered electrons in thin‐film X‐ray microanalysis
Authors: LI Y. H. , CHEN Z. , LORETTO M. H.
Published: August 2011

Carbon films supported on half copper grids have been used in a JEOL 4000FX to investigate the source of Cu X‐rays generated when the electron probe is focused on the carbon film. Both horizontal and high take‐off angle detectors were used to collect the spectra with accelerating voltages at 200, 300 and 400 kV. Energy‐dispersive X‐ray results obtained with the electron beam focused either on the carbon film or to avoid the film have been compared. By tilting the copper grid along its cut edge, the contribution of X‐rays generated by electrons scattered by the sample has been distinguished from the contribution of specimen‐generated Bremsstrahlung. These observations, together with measurements made both with and without an objective aperture, show that the main spurious contribution is from the electrons, which interact with the lower objective pole‐piece, generating both backscattered electrons and high‐energy X‐rays which subsequently interact with the sample. This result is further confirmed by the observations on epitaxial CdTe and CdHgTe layers on a single‐crystal GaAs substrate.
Cerium methods for light and electron microscopical histochemistry
Authors: VAN NOORDEN C. J. F. , FREDERIKS W. M.
Published: August 2011

Methods based on the use of cerium to detect the activity of oxidases and phosphatases are rapidly expanding. Both classes of enzymes can be demonstrated with cerium at the electron and light microscopical level. Thein situdetection of H2O2production with cerium is another application that has great potential, particularly in experimental pathological research. The fine precipitate of the cerium‐containing final reaction product, cerium perhydroxide or cerium phosphate, enables a very precise localization. With such techniques, important advances have been made in cell biology, such as the discovery of new organelles, functional subcompartmentization of peroxisomes, tubular lysosomes and the elucidation of the function of extracellular ATPases.

At the light microscopical level, the activity of enzymes can be quantifiedin situbecause the production of final reaction product in the cerium methods is proportional to enzyme activity in tissue sections or cells. Cerium precipitates have strong reflectance properties and this enables their use in confocal scanning laser microscopy. In the present review, the principles of cerium methods are outlined and applications in cell biology and pathology are discussed.
Imaging modes for bilateral confocal scanning microscopy
Authors: BRAKENHOFF G. J. , VISSCHER K.
Published: August 2011

The bilateral scanning approach to confocal microscopy is characterized by the direct generation of the image on a two‐dimensional (2‐D) detector. This detector can be a photographic plate, a CCD detector or the human eye, the human eye permitting direct visualization of the confocal image. Unlike Nipkow‐type systems, laser light sources can be used for excitation. A design called a carousel has been developed, in which the bilateral confocal scan capability can be added to an existing microscope so that rapid exchange and comparison between confocal and non‐confocal imaging conditions is possible. The design permits independent adjustment of confocal sectioning properties with lateral resolutions better than, or, in the worst case equivalent to, those available in conventional microscopy. The carousel can be considered as a stationary optical path in which certain imaging conditions, such as confocality, are defined and operate on part of the imaging field. The action of the bilateral scan mirror then extends this image condition over the whole field. A number of optical arrangements for the carousel are presented which realize various forms of confocal fluorescence and reflection imaging, with point, multiple point or slit confocal detection arrangements. Through the addition of active elements to the carousel direct stereoscopic, ratio, time‐resolved and other types of imaging can be achieved, with direct image formation on a CCD, eye or other 2‐D detectors without the need to modify the host microscope. Depending on the photon flux available, these imaging modes can run in real‐time or can use a cooled CCD at (very) low light level for image integration over an extended period.
An efficient method for calculating the least‐squares background fit in electron energy‐loss spectroscopy
Authors: BECKERS A. L. D. , GELSEMA E. S. , DE BRUIJN W. C.
Published: August 2011

In quantitative electron energy‐loss spectroscopy, an estimate has to be made of the unspecific spectral background to obtain the element‐specific contribution of an ionization edge. For energy losses above c. 100 eV, such a background estimate is commonly performed by calculating the least–squares fit of a power‐law model to a part of the pre‐edge region of the spectrum and by extrapolating this model to the region beyond the edge. Existing methods are cumbersome in terms of computation time and stability, or yield biased estimates. Computation efficiency becomes important when processing spectral‐image sequences. In this paper a new, computationally efficient numerical method is presented, which appears to be extremely robust in practice.
Endopolygalacturonase‐gold complex: Parameters influencing the labelling of pectic acid in the cell walls of flax hypocotyl
Authors: CABIN‐FLAMAN A. , JAUNEAU A. , MORVAN C. , DEMARTY M.
Published: August 2011

An enzyme/colloidal‐gold complex was prepared using a purified endopolygalacturonase (endoPGH, E.C. 3.2.1.15). Gold particles with a radius of 4·8 nm were prepared. To stabilize the gold particles, it was necessary to add a minimal amount of enzyme (approximately 0·5 μg) at a pH higher than its isoelectric pH. Under these conditions, the number of protein molecules adsorbed per gold particle was approximately one.

The parameters influencing the diffusion of the endoPGH‐gold complex to its target molecules were screened and tested through quantitative approaches: (i) the labelling efficiency q was calculated according to the equation for the diffusion of gold markers from the bulk solutions to target molecules and (ii) the labelling density N/S was measured by counting gold particles on electron micrographs.

The highest value of N/S was obtained by determining the optimal values for the parameters outlined in the equation for q. To obtain the highest value of N/S, it was not necessary to choose values for these parameters which gave high values for q.

The specificity of the probe towards homogalacturonic acid blocks was established through controls. The localization of these blocks was illustrated throughout the walls of different kinds of flax seeding cells.
Second‐order stereology of benign and malignant alterations of the human mammary gland
Authors: MATTFELDT T. , FREY H. , ROSE C.
Published: August 2011

The purpose of the present study was a quantitative characterization of the three‐dimensional arrangement of the epithelial component of benign and malignant alterations of the female breast by combining stereology with stochastic geometry. Twenty cases of fibrous mastopathy and 20 cases of invasive ductal mammary cancer were studied at the light microscopic level. Segmentation of the epithelial tissue component was performed with an image analyser. From the resulting binary images, unbiased estimates of the covariance C(r) and the intensity Vv of the epithelial volume component were obtained automatically by computer. From these data, estimates of the correlation function k(r), of the pair correlation function g(r), of the radial distribution function RDF(r) and of the reduced second moment function K(r) of epithelial volume were determined. The estimates of C(r) and RDF(r) differed between groups, but these functions depend on spatial pattern and Vv. As carcinomas showed a significantly higher epithelial volume density Vv than mastopathies, estimation of C(r) and RDF(r) alone did not permit a safe distinction between possibly different types of spatial arrangement of epithelium in the benign and malignant lesions. Analysis of the estimates of k(r), g(r) and K(r), which are not influenced by Vv, showed definite interaction between epithelial volume elements, with clustering at short distances and repulsion at long distances. In both groups, the null hypothesis of purely random arrangement of epithelium had to be rejected. The clearest distinction between groups was obtained by estimation of g(r), which showed that short‐range, tubular pattern as well as long‐range, lobular architecture are better preserved in benign than in malignant lesions. The low interindividual scatter of k(r), g(r) and K(r) indicates a high biological significance of spatial pattern, which is presumably under strict genetic control. Potential uses of the method are: (i) the identification of biomathematical models which could contribute to a better understanding of the growth processes involved, (ii) conditional simulation of the underlying three‐dimensional structures by computer, and (iii) supporting the diagnosis of mammary lesions with borderline histopathological appearance.
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

Azospirillum 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

Glow 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

In 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

An 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

A 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

For 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

A 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

Myofibrils, 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

A 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

A 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

Certain 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

Optimized 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

A 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

A 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

A 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

The 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

Three‐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

Methods 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

Digital 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

Evidence 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.
A particle segmentation method based on nucleation and growth of the background
Authors: ZHENG XIN , KOLB MAX
Published: August 2011

A new algorithm for image segmentation is proposed, which is capable of extracting particles in the presence of noise and background fluctuations. It begins with the detection of small regions belonging to the background, called background nuclei, and then lets these nuclei grow to become the entire background. Edge information and region information of the image are used simultaneously.
Atomic force microscopy of freeze‐fracture replicas of rat atrial tissue
Authors: KORDYLEWSKI L. , SANER D. , LAL R.
Published: August 2011

Atomic force microscopy (AFM) has provided three‐dimensional (3‐D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well‐established technique of transmission electron microscopy (TEM) was performed. Freeze‐fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large‐scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3‐D surface contour) and TEM (2‐D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.
Calibration of the scanning (atomic) force microscope with gold particles
Authors: XU S. , ARNSDORF M. F.
Published: August 2011

Scanning force microscopy (SFM) holds great promise for biological research. Two major problems that have confronted imaging with the scanning force microscope have been the distortion of the image and overestimation in measurements of lateral size due to the varying geometry and characteristics of the scanning tip. In this study, spherical colloidal gold particles (10, 20 and 40 nm in diameter) were used to determine (1) tip parameters (size, shape and semivertical angle); (2) the distortion of the image caused by the tip; and (3) the overestimation or broadening of lateral dimensions. These gold particles deviate little in size, are rigid and have a size similar to biological macromolecules. Images of the colloidal gold particles by SFM were compared with those obtained by electron microscopy (EM). The height of the gold particles as measured by SFM and EM was comparable and was little affected by the tip geometry. The measurements of the lateral dimensions of colloidal gold, however, showed substantial differences between SFM and EM in that SFM resulted in an overestimate of the lateral dimensions. Moreover, the distortion of images and broadening of lateral dimensions were specific to the SFM tip used. The calibration of the SFM tip with mica provided little clue as to the type of distortion and the amount of lateral broadening observed when the larger gold particles were scanned. The SFM image also depended on the orientation of the tip with respect to the specimen. Our results suggest that quantitative SFM imaging requires calibration to identify and account for both the distortions and the magnitude of lateral broadening caused by the cantilever tip. Calibration with gold particles is fast and nondestructive to the tip. The raw imaging data of the specimen can be corrected for the tip effect and true structural information can be derived. In summary, we present a simple and practical method for the calibration of the SFM tip using gold particles with a size in the range of biomacromolecules that allows: (1) selection of a cantilever tip that produces an image with minimal distortion; (2) quantitative determination of tip parameters; (3) reconstruction of the shape of the tip at different heights from the tip apex; (4) appreciation of the type of distortion that may be introduced by a specific tip and quantification of the overestimation of the lateral dimensions; and (5) calculation of the true structure of the specimen from the image data. The significance is that such calibration will permit quantitative and accurate imaging with SFM.
Contrast in the transmission mode of a low‐voltage scanning electron microscope
Authors: GOLLA U. , SCHINDLER B. , REIMER L.
Published: August 2011

The contrast thicknesses (xk) of thin carbon and platinum films have been measured in the transmission mode of a low‐voltage scanning electron microscope for apertures of 40 and 100 mrad and electron energies (E) between 1 and 30 keV. The measured values overlap with those previously measured for E (≥ 17keV) in a transmission electron microscope. Differences in the decrease of xk with decreasing E between carbon and platinum agree with Wentzel‐Kramer‐Brillouin calculations of the elastic cross‐sections. Knowing the value of xk allows the exponential decrease ∝ exp(—x/xk) in transmission with increasing mass‐thickness (x = ρt) of the specimen and the increasing gain of contrast for stained biological sections with decreasing electron energy to be calculated for brightfield and darkfield modes.