bluuebunny/crossref_metadata_2025 huggingface dataset:
Iterative, constrained 3‐D image reconstruction of transmitted light bright‐field micrographs based on maximum likelihood estimation
Authors: WILLIS B. , ROYSAM B. , TURNER J. N. , HOLMES T. J.
Published: August 2011

We present several image reconstruction algorithms for generating three‐dimensional (3‐D) renderings of bright‐field micrographs that are founded on maximum likelihood estimation (MLE) theory. The basic principle of the algorithms is in estimating the values of the optical densities of the specimen. A computer simulation and initial experimental testing of a steepest ascent version of the algorithm is presented. The computer simulation demonstrates that the MLE algorithm has an advantage over previously used inverse filtering techniques in that it partially restores the zeroed Fourier components in the well‐known missing‐cone region. We present 3‐D reconstructions from real biological data to show the potential of the algorithm in practical applications.
Measurement of co‐localization of objects in dual‐colour confocal images
Authors: MANDERS E. M. M. , VERBEEK F. J. , ATEN J. A.
Published: August 2011

A method to measure the degree of co‐localization of objects in confocal dual‐colour images has been developed. This image analysis produced two coefficients that represent the fraction of co‐localizing objects in each component of a dual‐channel image. The generation of test objects with a Gaussian intensity distribution, at well‐defined positions in both components of dual‐channel images, allowed an accurate investigation of the reliability of the procedure. To do that, the co‐localization coefficients were determined before degrading the image with background, cross‐talk and Poisson noise. These synthesized sources of image deterioration represent sources of deterioration that must be dealt with in practical confocal imaging, namely dark current, non‐specific binding and cross‐reactivity of fluorescent probes, optical cross‐talk and photon noise. The degraded images were restored by filtering and cross‐talk correction. The co‐localization coefficients of the restored images were not significantly different from those of the original undegraded images. Finally, we tested the procedure on images of real biological specimens. The results of these tests correspond with data found in the literature. We conclude that the co‐localization coefficients can provide relevant quantitative information about the positional relation between biological objects or processes.
Unbiased volume estimation with coaxial sections: an application to the human bladder
Authors: CRUZ‐ORIVE L. M. , ROBERTS N.
Published: August 2011

A problem of current medical interest is how to estimate the volume of an object from coaxial sections. Such images can be produced in ultrasound scanning, when the planar scan sections all emanate from a common axis. Two unbiased volume estimators for this sampling regime, previously published without derivation, are derived and presented here in some detail. The estimators are based on an ancient theorem of Pappus of Alexandria (c. ad 320). One of the estimators was used to estimate the volume of urine in the bladder of six human volunteers. The case example was attractive because the corresponding volumes could be voided and directly measured after the scanning session, thus confirming the unbiasedness of the estimator used. Sample size guidelines are also given.
Computerized characterization of the geometry of real porous media: their discretization, analysis and interpretation
Authors: THOVERT J. F. , SALLES J. , ADLER P. M.
Published: August 2011

Real or reconstructed porous media can be discretized as a series of elementary cubes, which are filled either with a solid or a fluid phase. Various algorithms, based on pseudo‐diffusion processes, are proposed to determine the connected and percolating components of the pore space. The graph of the pore space is obtained by two different methods; the most efficient is based upon homotopic thinning. Topological characteristics, such as the number of loops, are derived. Systematic applications of these algorithms are illustrated on computer reconstructions of various sandstones.
Characterization of materials using surface and thin‐film analysis techniques
Authors: FITZGERALD A. G.
Published: August 2011

The application of X‐ray microanalysis, electron energy‐loss spectroscopy (EELS) and the surface analysis techniques of Auger electron spectroscopy (AES) and X‐ray photoelectron spectroscopy (XPS) to the characterization of materials is discussed. Examples of the application of these techniques to the characterization of amorphous films of silicon nitride, silicon carbide, hydrogenated carbon films and synthetic diamond films are described. Compositions obtained by each of these analytical techniques have been found to be in good agreement. The use of AES, XPS and EELS to investigate bonding and electronic structure in thin films and surfaces is discussed. Computer programs that have been developed to improve quantification in AES and XPS are described. These programs allow a comparison of matrix factor and final surface compositions obtained using different electron attenuation length corrections, back‐scatter corrections and photo‐ionization cross‐sections. Computer programs involving Monte Carlo simulations of X‐ray emission, which have been developed to enable quantification of the composition of thin films on thick substrates, are also described.
Infra‐red microspectroscopy as a complementary technique to electron‐probe microanalysis for the investigation of natural corrosion on potash glasses
Authors: COOPER G. I. , COX G. A. , PERUTZ R. N.
Published: August 2011

Natural corrosion crusts on potash—lime—silica glasses, that have been buried in soil for very long periods, were examined using infra‐red reflectance microspectroscopy and electron‐probe techniques. Individually these techniques are useful: infra‐red provides information on which functional groups are present, but is not good at distinguishing between cations; electron‐probe microanalysis reveals the composition of a sample, but cannot explain how the elements are combined. By using both techniques on the same sample areas the corrosion was shown to be composed of layers of amorphous silica gel with precipitates such as carbonate‐substituted calcium phosphate, calcium sulphate and manganese dioxide.
Theoretical calculation of probe size of low‐voltage scanning electron microscopes
Authors: XIMEN J. , SHAO Z. , LIN P. S. D.
Published: August 2011

Detailed investigating into the effects of spherical and chromatic aberrations, diffraction and the probe current allows the more general formulae for the optimized aperture and the minimum probe radius in low‐voltage scanning electron microscopes (LVSEMs) to be derived using both wave optics and geometric optics. The probe sizes for a diversity of electron sources in LVSEMs have been estimated, which may be useful for practical applications. The computed results indicate the possibility of achieving 1·5–2·0‐nm resolution at low voltages.
Electron microprobe study of the effect of long‐term vacuum storage of freeze‐dried cryosections on distribution of elements in cells
Authors: PELC R. , ŽIŽKA Z.
Published: August 2011

The distribution of elements in yeast cells was measured on freshly prepared, freeze‐dried cryosections and compared with the distribution obtained on the same sections after storage for 20 months in a vacuum below 2·6 kPa. The average concentration of phosphorus remained unchanged but was equalized throughout the cells, i.e. it migrated from vacuoles into the cytoplasm and mitochondria. Zinc remained preferentially localized in the vacuoles, but the ratio between vacuolar and cytoplasmic zinc concentrations decreased about three‐fold. Cytoplasmic and mitochondrial concentrations of potassium remained unchanged while partial release from the vacuoles and subsequently from the cells was observed. This resulted in a homogeneous distribution of potassium in the cells after 20 months and some of the vacuolar potassium appeared in spectra of formvar film measured several micrometres from the cells. A large increase in the sodium (from 160 to 360% more than in fresh sections), magnesium (from 110 to 200% more) and sulphur (from 70 to 350% more) contents was observed in all cellular compartments (except for vacuoles, where only a 20% increase in the magnesium content was observed), while chlorine was almost completely released from the cells. The limitations of the use of long‐term vacuum‐stored cryosections for electron micro‐probe analysis of cells are discussed.

yeast.
The study of fission track and other crystalline defects using confocal scanning laser microscopy
Authors: PETFORD N. , MILLER J. A.
Published: August 2011

Confocal scanning laser microscopy (SLM) is a technique that offers geologists a new way of studying structures in minerals at the submicrometre level. As an example we show how the non‐destructive nature of confocal SLM can be used to measure and count fission tracks (line defects formed by the spontaneous fission of238U) in the uranium‐bearing mineral apatite, and to provide information about the geometry and crystallographic orientation of fluid inclusions trapped inside apatite grains during crystallization. The technique also provides a means of studying the internal geometry of chemical zonation in minerals. The digitized nature of the SLM images makes them amenable to a variety of image analysis techniques, and we show how image analysis can be used to measure fission tracks in mica sheets and provide crude estimates of track dip. Finally, using a chemically etched mica sheet we show how confocal SLM can be used to provide a detailed near‐surface (1–5 μm) analysis of geological materials.
Evaluation of several common standards for the X‐ray microanalysis of thin biological specimens
Authors: PATAK A. , WRIGHT A. , MARSHALL A. T.
Published: August 2011

The question of the best type of standard to use for X‐ray microanalysis of thin biological specimens remains unanswered. Standards embedded in an organic matrix have the advantage that they resemble biological specimens, but their composition is generally not known exactly. We compared several standards and, surprisingly, inorganic binary salts sprayed onto a supporting film were the most suitable: they corresponded closely with several other methods using organic matrices; they were easily produced; and their composition is known. Glutaraldehydeurea aminoplastic resin thin sections and thin films containing dissolved salts were problematic. The composition of the polymer appears to be variable, and the thin films did not correspond with any other standard tested. Chelex100 bio‐standard beads and flakes loaded with accurately determined concentrations of ions, embedded in epoxy resin and thin sectioned, tended to correspond to the results obtained with the binary salts. However, the results from some bio‐standards were inexplicably aberrant. An epoxy resin standard was used for bromine, and was found to agree closely with the binary standards.
Computed three‐dimensional reconstruction of median‐eminence capillary modules: Image alignment and correlation
Authors: HIBBARD L. S. , GROTHE R. A. , ARNICAR‐SULZE T. L. , DOVEY‐HARTMAN B. J. , PAGE R. B.
Published: August 2011

Image alignment is an absolute requirement for three‐dimensional (3‐D) reconstruction from serial sections, and Fourier correlation is the most powerful way to compute alignments. The rotational and translational components of misalignment can be corrected by an iterative correlation procedure, but for images having significant differences, alignment can fail with a likelihood proportional to the extent of the differences. We found that translational correction was determined much more reliably when low‐pass filters were applied to the product transforms from which the correlations were calculated. Rotational corrections based on polar analyses of the auto‐correlations of the images instead of on the images directly contributed to more accurate alignments. These methods were used to generate 3‐D reconstructions of brain capillary modules from serial‐section mosaics of digitized transmission electron micrographs.
Applied stereology in materials science and engineering
Authors: LIU G.
Published: August 2011

A general view of the role of stereology in materials science and engineering (MSE), is followed by a discussion of applied stereology for one‐, two‐ and multi‐phase structures, and by examples of stereology applications to MSE, such as the impact of stereology on the creation and evolution of modern high–strength, low‐alloy steels. The present state and prospects of applied stereology in MSE are informally discussed. It is demonstrated that, without stereology, materials science cannot evolve into a truly quantitative science, and that the needs of both materials scientists and materials engineers should be met by the continuing cooperation of those who develop stereological methods and those who apply them.

To solve such problems as quantifying the interaction between features of similar and different dimensions in two‐ or multi‐phase structures, a general parameter for volumetrical contiguity of second‐phase particles, a contiguity parameter for second‐phase particle profiles in grain‐boundary surfaces, and techniques termed ‘reversed stereology’ are proposed and discussed.
Contribution to microscopic analysis of birefringence in polarization‐maintaining optical fibres
Authors: SOVA M. , BERKA L. , ŜPUNDA J.
Published: August 2011

A direct microscopic method for determination of birefringence in polarization‐maintaining optoelectronic fibres has been developed. The fibre, approximately 0·15 mm in diameter, is embedded in a suitable resin (epoxy). A thin section (approximately 3 mm in thickness) is then cut perpendicular to the fibre axis, ground and polished. Birefringent patterns (isochromatic fringes) can be seen and photographed using a light‐polarizing microscope with appropriate magnification. A method for performing a quantitative analysis of birefringence in real optoelectronic fibres using a macroscopic model is indicated.
Viruses accumulate spontaneously near droplet surfaces: A method to concentrate viruses for electron microscopy
Authors: JOHNSON R. P. C. , GREGORY D. W.
Published: August 2011

Virus particles suspended in a drop of water tend to concentrate at or near its surface with the air. The concentrated, and probably more purified, particles may then be collected on a film‐coated grid for negative staining and electron microscopy. This is a useful method, simpler than others (e.g. high‐speed centrifugation, Lyphogel, or precipitation by (NH4)2SO4) which are used to process clinical specimens for diagnosis where virus particles may be too dilute in the original sample. It is shown, by freeze‐fracturing for electron microscopy, that Orf virus particles do accumulate at and just below the surfaces of drops. Various physical effects which may cause the particles to accumulate are considered. Results from a computer model suggest that Brownian motion alone could be adequate to transport a useful quantity of the particles in the body of a 2‐mm‐diameter hemispherical drop to its surface if the particles do not clump and if they remain trapped at the surface when they reach it. In practice, transport by Brownian motion is likely to be augmented by swirling, convection and other effects within drops.
New infiltration method permits use of freeze substitution for preparation of wood tissues for transmission electron microscopy
Authors: RISTIC Z. , ASHWORTH E. N.
Published: August 2011

A new infiltration method was developed for preparation of wood tissues for transmission electron microscopy (TEM) using freeze substitution. The method included treatment of freeze‐substituted samples with sodium phosphate buffer before infiltration with epoxy resin. Samples from red osier (Cornus sericea L.) and flowering dogwood (C. florida L.) were studied to assess the quality of the infiltration method. The method produced well‐embedded samples with good trimming and cutting qualities. To our knowledge, this is the first report on preparation of wood tissues for TEM using freeze substitution.
Thermal oxidation stages of copper studied by STM in flowing argon
Authors: GARCÍA‐CANTÚ R. , ALVARADO J. J. , SOLORZA O.
Published: August 2011

Scanning tunnelling microscopy (STM) images under flowing argon are reported for successive stages of thermal oxidation of copper at room temperature. These STM images show zones with high tunnelling current peaks, more directly related to oxide growth than to the roughness of the topography.
Unbiased estimation of human body composition by the Cavalieri method using magnetic resonance imaging
Authors: ROBERTS N. , CRUZ‐ORIVE L. M. , REID N. M. K. , BRODIE D. A. , BOURNE M. , EDWARDS R. H. T.
Published: August 2011

The classical methods for estimating the volume of human body compartments in vivo (e.g. skin‐fold thickness for fat, radioisotope counting for different compartments, etc.) are generally indirect and rely on essentially empirical relationships — hence they are biased to unknown degrees. The advent of modern non‐invasive scanning techniques, such as X‐ray computed tomography (CT) and magnetic resonance imaging (MRI) is now widening the scope of volume quantification, especially in combination with stereological methods. Apart from its superior soft tissue contrast, MRI enjoys the distinct advantage of not using ionizing radiations.

By a proper landmarking and control of the scanner couch, an adult male volunteer was scanned exhaustively into parallel systematic MR ‘sections’. Four compartments were defined, namely bone, muscle, organs and fat (which included the skin), and their corresponding volumes were easily and efficiently estimated by the Cavalieri method: the total section area of a compartment times the section interval estimates the volume of the compartment without bias. Formulae and nomograms are given to predict the errors and to optimize the design. To estimate an individual's muscle volume with a 5% coefficient of error, 10 sections and less than 10min point counting (to estimate the relevant section areas) are required. Bone and fat require about twice as much work. To estimate the mean muscle volume of a population with the same error contribution, from a random sample of six subjects, the workload per subject can be divided by √6, namely 4 min per subject. For a given number of sections planimetry would be as accurate but far more time consuming than point counting.
SPARK, a kernel of software programs for spatial reconstruction in electron microscopy
Authors: LANZAVECCHIA S. , BELLON P. L. , SCATTURIN V.
Published: August 2011

SPARK, an acronym for ‘SPAtial Reconstruction Kernel’, is the nucleus of a software library being developed for the three‐dimensional (3‐D) reconstruction of objects observed by the electron microscope. A unifying concept is used: the Fourier transform, known in several central sections, is resampled to obtain a 3‐D Cartesian grid, which is inverted by a fast Fourier transform. This technique is used for both single‐axis tilting (of 2‐D periodic layers or of isolated objects) and for the random conical‐tilt technique. The principles that make it possible to recover Cartesian grids in the two different geometries are illustrated and some preliminary results are reported. SPARK resamples the Cartesian grids with the use of a fast and efficient algorithm of Shannon interpolation developed by the authors.

Compared to back‐projection techniques the method shows a considerable improvement in execution time with no sacrifice in accuracy; it therefore allows the effects of a variety of parameters in a given reconstruction to be scrutinized in a reasonable time. Some new possibilities and future extensions of the library are briefly outlined.
Quantitative acoustic microscopy of individual living human cells
Authors: BRIGGS G. A. D. , WANG J. , GUNDLE R.
Published: August 2011

The elastic properties of cells can be measured with microscopic resolution by acoustic microscopy. By measuring the waveform of very short pulses, the thickness, and the acoustic velocity, impedance and attenuation can be determined from the two separate signals reflected from the top and the bottom of the cell.
Spatial distribution of curve length: Concept and estimation
Authors: ROBERTS N. , CRUZ‐ORIVE L. M.
Published: August 2011

The length of a curvilinear feature, such as a dendrite tree of a neuron, can, in principle, be estimated by the recent, non‐invasive method of total vertical projections (TVPs). Curve length is a measure of size, but it reports nothing about curve shape. The shape of a tree‐like structure can be described to some extent by the distribution of branch length in properly defined regions of three‐dimensional (3‐D) space. A definition of curve length distribution in three dimensions is proposed and implemented here on a human neuron. The relevant 3‐D regions overlap after projection, and therefore the TVPs method cannot be used directly to estimate the corresponding feature lengths. However, using the ANALYZETM software system running on a SUN®SPARC workstation, dendrite subsets sitting in predefined regions of space were rendered in different colours and measured separately by the TVPs method using a cycloid test system. In combination with non‐invasive image acquisition and processing techniques, the length distribution concept is likely to be useful in the metrical analysis of either microscopic or macroscopic arborizations in a wide variety of contexts, including living cells and organisms.
Image sharpness and contrast transfer in coherent confocal microscopy
Authors: OLDENBOURG R. , TERADA H. , TIBERIO R. , INOUÉ S.
Published: August 2011

Confocal microscopes provide clear, thin optical sections with little disturbance from regions of the specimen that are not in focus. In addition, they appear to provide somewhat greater lateral and axial image resolution than with non‐confocal microscope optics. To address the question of resolution and contrast transfer of light microscopes, a new test slide that enables the direct measurement of the contrast transfer characteristics (CTC) of microscope optics at the highest numerical aperature has been developed. With this new test slide, the performance of a confocal scanning laser microscope operating in the confocal reflection mode and the non‐confocal transmission mode was examined. The CTC curves show that the confocal instrument maintains exceptionally high contrast (up to twice that with non‐confocal optics) as the dimension of the object approaches the diffraction limit of resolution; at these dimensions, image detail is lost with non‐confocal microscopes owing to a progressive loss of image contrast. Furthermore, we have calculated theoretical CTC curves by modelling the confocal and non‐confocal imaging modes using discrete Fourier analysis. The close agreement between the theoretical and experimental CTC curves supports the earlier prediction that the coherent confocal and the incoherent non‐confocal imaging mode have the same limit of resolution (defined here as the inverse of the spatial frequency at which the contrast transfer converges to zero). The apparently greater image resolution of the coherent confocal optics is a consequence of the improved contrast transfer at spacings which are close to the resolution limit.
A light‐emitting diode light standard for photo‐ and videomicroscopy
Authors: BEACH J. M. , DULING B. R.
Published: August 2011

A light calibration system consisting of a compact light‐emitting diode (LED) source with feedback control of intensity is described. The source is positioned in the focal plane of the microscope objective and produces flat‐field illumination of up to 31 μW. The source can be easily used to determine the performance of microscope optics and camera response. It can also be used as a standard light source for calibration of experimental systems. Selectable light intensities are produced by controlling the LED input power via a feedback circuit consisting of a photodiode that detects output light intensity. Spectral coverage extends between 550 and 670nm using green, yellow and red LEDs mounted side by side, which are selected individually. The LED chips are encapsulated in plastic diffusers which homogenize the light, and a flat field of illumination is obtained through a thin 1‐mm‐diameter aperture positioned directly over each chip. Provision is made for insertion of Ronchi rulings over the aperture to enable measurements of contrast modulation in a uniform field. The light may be pulse‐modulated to assess camera response times and the device can be synchronized with video frames. Narrow bandpass interference filters can be placed between the objective lens and the LED source to produce monochromatic light without affecting the spacing of controlled light intensities since emission spectra do not shift appreciably over the range of LED powers chosen in this design. Results of tests using controlled light intensity and uniform illumination are presented.
Scanning luminescence X‐ray microscopy: Imaging fluorescence dyes at suboptical resolution
Authors: JACOBSEN C. , LINDAAS S. , WILLIAMS S. , ZHANG X.
Published: August 2011

Scanning luminescence X‐ray microscopy is based on the use of the very small focused probe of a scanning X‐ray microscope to stimulate visible light emission from phosphors and dyes. Using an undulator X‐ray source and a Fresnel zone plate to produce a focused X‐ray probe, images of P31 phosphor grains with a resolution of 50–75 nm have been obtained, and luminescence from polystyrene spheres loaded with 50–100 μmol/g of fluorescent dye has been imaged. The resolution was not limited by the focused X‐ray probe (the microscope has imaged features at 36‐nm spacing in transmission mode) but by dark noise and the low net efficiency of the luminescence detection system used for this investigation. This technique may make it possible to image dye‐tagged sites of biochemical activity at the resolution of the X‐ray microscope in wet, unsectioned, and unfixed cells, especially with soft X‐ray optimized dyes. Because the image is formed from the detection of signal against a dark background, calculations suggest that the radiation dose for luminescence imaging of dye‐tagged features should be 2–22 times lower than it is in transmission X‐ray microscopy. A possible extension of the technique for three‐dimensional imaging at the transverse resolution of the X‐ray microscope is described, where visible light collection optics might be used to obtain submicrometre axial resolution.
A device for picking up ultrathin serial sections
Authors: MEYER E. P. , DOMANICO V. J.
Published: August 2011

Reconstruction from serial sections is often required to understand the architecture of biological tissue. The sampling of serial sections calls for an enormous amount of patience and skill. Sections are cut with a diamond knife, sampled in a trough and must then be collected on grids for examination with the electron microscope (EM). This last step, in which the section ribbons have to be aligned with the EM grid, is especially difficult as both hands have to work in perfect co‐ordination. A simple manually operated or motorized mechanical device has been designed which facilitates the collection of EM section ribbons.
A compact Schwarzschild soft X‐ray microscope with a laser‐produced plasma source
Authors: HORIKAWA Y. , NAGAI K. , MOCHIMARU S. , IKETAKI Y.
Published: August 2011

A compact Schwarzschild soft X‐ray microscope using a laser‐produced plasma soft X‐ray source has been developed. The laser‐produced plasma source, which is small but of high brilliance, has made it possible to use the soft X‐ray microscope in a small laboratory. The microscope is composed of a Schwarzschild objective and a grazing incidence mirror condenser. Image contrast for biological specimens in soft X‐ray regions is investigated briefly. It is possible to observe the fine structures of a thin specimen at a wavelength of 15 nm; at this wavelength high‐contrast images of biological specimens have been obtained with a single laser shot of pulse width of 8 ns at a resolution of 0·3 μm. The resolution of the system is limited by the detector.
An investigation of substrate and sample preparation effects on scanning tunnelling microscopy studies on xanthan gum
Authors: WILKINS M. J. , DAVIES M. C. , JACKSON D. E. , ROBERTS C. J. , TENDLER S. J. B.
Published: August 2011

Scanning tunnelling microscopy is developing as an important biophysical tool for the molecular imaging of biological material. In this study, the effect of sample deposition technique and substrate employed on the resultant images of a microbial polysaccharide is investigated. Scanning tunnelling microscopy topographs of xanthan gum, pipette deposited and spray deposited onto highly orientated pyrolytic graphite and mica substrates are presented. The use of pipette deposition of the aqueous sample solution is shown to result in a xanthan network. In contrast, images of isolated xanthan molecules are obtained when spray deposition with glycerol is employed. The effect of these deposition techniques on the macroscopic distribution of sample material across substrates is shown using confocal fluorescence microscopy.
Effects of noise and anisotropy on the determination of fractal dimensions
Authors: RUSS J. C.
Published: August 2011

Measurement of the fractal dimension of surfaces imaged by the scanning tunnelling microscope, atomic force microscope or similar instruments can be performed using several different algorithms. Dimensional analysis—plots of log (perimeter) vs. log (area)—are compared to Korcak and slit‐island methods, which also employ a horizontal plane section, and to Fourier analysis of the two‐dimensional array of elevation values. The effects of surface anisotropy and instrument noise on each of these measurement techniques is investigated.
Directional analysis of planar fibre networks: Application to cardboard microstructure
Authors: MOLCHANOV I. , STOYAN D. , FYODOROV K.
Published: August 2011

This paper discusses the problem of determining suitable roses of intersections for systems of planar thick fibres which cross and overlap. A planar Boolean model with long rectangular (or similar) grains is suggested as an appropriate mathematical model. It leads to a statistical estimator for the rose of intersections of the system of fibre spines. The method is used to analyse the microstructure of the outer layer of two samples of cardboard.
The observation of large magnetite (Fe3O4) crystals from magnetotactic bacteria by electron and atomic force microscopy
Authors: FARINA MARCOS , KACHAR BECHARA , LINS ULYSSES , BRODERICK RAYMOND , DE BARROS HENRIQUE LINS
Published: August 2011

Magnetite crystals inside coccoid magnetotactic bacteria found in lagoons near Rio de Janeiro city were examined by electron microscopy (EM) and atomic force microscopy (AFM). For AFM, ultrathin sections of bacteria embedded in Epon resin were etched with an ethanolic NaOH solution and observed both in the height and in the force modes. Comparative electron microscope images were useful for identifying crystalline reliefs in the etched sections. Different situations representing particular arrangements of crystal chains were observed by AFM. The majority of the bacteria examined presented unusually large magnetite crystals which remained strongly attached in linear chains even after the laboratory procedures for their isolation. This behaviour is different from all other biogenic magnetite crystals isolated so far. It is suggested that this attachment is due to the strong field between individual crystals as well as to the contact areas, which are the largest observed until now. The correct identification of a particular topography by AFM as a crystal relief may be critical when crystals are not aligned in chains; in these cases the linear dimensions and the presence of well‐defined edges and faces are important features to be taken into account. Characterization of the crystal faces is important for the study of magnetotactic micro‐organisms since the crystalline habits seem to be species‐specific. Observation of etched sections proved to be a helpful approach for crystal relief observation, especially when small amounts of bacteria were available.
Quantitative analysis of variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) of cell/substrate contacts
Authors: BURMEISTER J. S. , TRUSKEY G. A. , REICHERT W. M.
Published: August 2011

Variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) allows controlled variation of the illumination depth with the potential of measuring both membrane/substrate separation distances and sizes of focal contacts. VA‐TIRFM images are collected from well‐spread bovine aortic endothelial cells (BAEC) stained with a membrane‐bound carbocyanine dye. Quantitative determination of absolute membrane/substrate separation distances and individual focal contact area are attempted using a simplified model of TIRFM optics. For angles slightly greater than the critical angle of 64°, both the dorsal and ventral membranes were illuminated, while images excited above 66° illuminated only focal contacts. Above 74° the fluorescence of focal contacts was dominated by background noise. Direct application of the simplified optical model without accounting for background intensity was unsatisfactory. However, correction for background fluorescence and nonlinear regression of the untransformed data over the working range yielded focal contact separation distances of 24 ± 13 nm. Focal contact areas estimated by TIRFM (1·3 ± 0·7 μm2) agreed closely with areas observed by immunofluorescence staining of vinculin (1·5 ± 0·3 μm2).
A simple calibration for routine section thickness measurements using current density ratios
Authors: HENG Y. M. , OTTENSMEYER F. P. , ARSENAULT A. L. , SIMON G. T.
Published: August 2011

A method to calibrate current density ratios for the determination of specimen thickness is presented. This method uses a tilt series from a single noncrystalline specimen to create different thicknesses; these are used to generate data points to establish the relationship between specimen thickness and current density ratio. The actual specimen thickness at 0° tilt was determined to an accuracy of 5 nm by a parallax method. From the calibration curves obtained, we observed that the current density ratio was sensitive to relative thickness changes on the same section of less than 1 nm when a 50‐μm objective aperture was used.
A cryoglue to mount vitreous biological specimens for cryoultramicrotomy at 110K
Authors: RICHTER K.
Published: August 2011

Mixtures of ethanol, 2‐propanol and 2‐butanol can be used as a cryoglue to mount vitrified biological specimens for ultrathin cryosectioning. Brought directly from room temperature to a cutting support at 140K in the cold chamber of a cryoultramicrotome, these alcohols stiffen to a viscous and gluey consistency allowing the attachment or embedding of a vitreous biological sample. The mass hardens at lower temperatures fixing the sample well for microtomy. With ethanol: 2‐propanol (2:3), samples are applied at 140 K and ultrathin cutting can be done at 115 K.
The use of the stable isotope 44Ca in studies of calcium incorporation into dentin
Authors: LUNDGREN T. , ENGSTRÖM E. U. , LEVI‐SETTI R. , LINDE A. , NORÉN J. G.
Published: August 2011

The incorporation into rat incisor dentin of two calcium isotopes, the stable 44Ca and the radioactive 45Ca, was studied using secondary ion mass spectrometry (SIMS) stepscanning and imaging, and autoradiography, respectively. The results demonstrated a time‐dependent incorporation of the calcium isotopes into the mineral phase of dentin. With the SIMS step‐scanning, detecting 44Ca, the ion yield was high in the odontoblasts 2 min after intravenous injection. After 10 min a marked increase in signal intensity was found at the dentin mineralization front. This result was consistent with those obtained by 45Ca autoradiography; a peak of incorporation occurred 10 min after injection of the isotope. Likewise, localization of 44Ca to the mineralization front could be demonstrated 10 min after injection by SIMS imaging. In images obtained at earlier intervals, no such increase in ion yield could be detected. The results show that the nonradioactive, stable isotope 44Ca can be used as a marker for biomineralization in a similar way to radioactive 45Ca.
Minimizing sample evaporation in the environmental scanning electron microscope
Authors: CAMERON R. E. , DONALD A. M.
Published: August 2011

The ElectroScan environmental scanning electron microscope (ESEM) enables wet samples to be observed by eliminating air but allowing water vapour into the sample chamber. However, evaporation from, and condensation on, the sample may occur during the pumpdown sequence used to reach this state, which means that the sample may not be in its natural state when viewed if due care is not taken. In this paper, the pumping system of the ESEM is described mathematically and expressions are derived for the evaporation and condensation. This treatment is then used to calculate the optimum pumpdown sequence. The importance of using the optimized procedure is illustrated by micrographs of fat emulsions.
Resolution in nonlinear laser scanning microscopy
Authors: DEITCHE J. , KEMPE M. , RUDOLPH W.
Published: August 2011

The lateral and depth resolution of nonlinear microscopy was studied systematically. Nonlinear microscopy can be classified into several categories depending on the coherence properties of the process that generates the imaging signal from the illuminating light, on whether a single‐ or a two‐beam geometry is used, and whether the optical setup is Type I or Type II. An evaluation of the imaging equations shows that (i) lateral and depth resolution improve with increasing nonlinearity, (ii) the differences between coherent and incoherent imaging diminish, and (iii) nonlinear imaging allows depth discrimination in Type I microscopy.
The influence of tissue processing on quantitative histopathology in breast cancer
Authors: LADEKARL M.
Published: August 2011

Objective grading of breast cancer by morphometry has been suggested for improving the precision of the prognostic prediction. However, the tissue components evaluated might be influenced by variations in the processing, reducing the clinical value. In the present study, the impact of the period of fixation, of the acidity of the fixative and of the embedding medium was investigated by allocating tissue samples from 27 surgical breast cancer specimens systematically randomly to different modes of processing. The volume‐weighted mean volume of cancer cell nuclei, v̄V(nuc), was estimated using the method of point‐sampled intercepts on vertical sections. In addition, estimates of the mean nuclear profile area, āH(nuc), the nuclear volume fraction, VV(nuc), the nuclear profile density, ND, and the mitotic profile frequency, MF, were obtained.

The quantitative histopathological estimates were stable with respect to the investigated variables of the tissue processing. No significant differences were found when comparing the estimates obtained in samples from five tumours fixed in formalin at pH 5·0, 6·0, 7·0, 7·4 and 8·0, respectively. Similarly, no significant correlations between the estimates and the period of formalin fixation (24 h, 3 days and 3 months) were found in samples from five other tumours. However, the v̄V(nuc) was 13% larger (2p = 0·004) and the mean ND 17% smaller (2p = 0·04) in hydroxyethyl‐methacrylate‐embedded samples from 17 tumours as compared to paraffin‐embedded samples. Thus, the shrinkage observed in paraffin seems to affect nuclei less than tissue.
A novel application of microsphere perfusion and scanning electron microscopy to the identification of pulmonary arterioles in guinea‐pig and rabbit lungs
Authors: WALKER D. C. , HOSFORD S. , MACKENZIE A.
Published: August 2011

In arterioles of the lung the intravascular blood pressures are lower than in comparable vessels in the systemic circulation and the arteriole walls are thinner. Therefore, it is very difficult to distinguish between arterioles and venules of the same size using scanning electron microscopy. This study describes a novel application of latex microsphere perfusion and scanning electron microscopy which distinguishes between pulmonary arterioles and venules on the basis of endothelial cell morphology. Microspheres, 90 and 45 μm in diameter, were perfused into the arterial side of the pulmonary circulation of guinea‐pig and rabbit lungs. Scanning electron microscopy of the arterioles on both sides of the lodged microspheres indicated that the endothelial cells are spindle shaped. In contrast, the endothelial cells of equal diameter venules are polygonal. Furthermore, the nuclei of the arteriolar endothelial cells were significantly (P = 0·019) narrower than those of endothelial cells in venules of equal diameter. Finally, it was observed that the differences between arteriole and venule endothelial cells persisted distally to the capillaries.
Köhler illumination in the TEM: Fundamentals and advantages
Authors: BENNER G. , PROBST W.
Published: August 2011

Köhler illumination is the most favourable design for the illumination path of an electron microscope with a condenser objective lens. The new illumination system of the EM 910 and EM 912 OMEGA allows both wide area (Köhler) illumination for TEM operation and spot illumination for analytical investigations. Compared to conventional systems and objective lenses with a condenser mini lens, this system offers many advantages. In addition to the homogeneous, highly coherent and parallel illumination of every point in the specimen, it offers advantages for selected area diffraction and spot scan mode.

Combined with the electron optical selection of a condenser aperture, this illumination system provides the flexibility necessary to achieve optimum illumination for the specimen.
Optimal conditions for the use of correspondence analysis for element determination in EELS images
Authors: GELSEMA E. S. , BECKERS A. L. D. , DE BRUIJN W. C.
Published: August 2011

Correspondence analysis is used for the determination of element localization and element concentration in electron energy–loss imaging. The introduction of a new method for the quantification of element concentration embedded in the protocols of correspondence analysis may be regarded as a useful replacement of the power–law estimation technique, especially in regions of the spectrum where the latter method cannot be used. Conditions for the optimum use of the method are established.
Quantitative electron spectroscopic imaging in bio‐medicine: Methods for image acquisition, correction and analysis
Authors: BECKERS A. L. D. , DE BRUIJN W. C. , GELSEMA E. S. , CLETOM‐SOETEMAN M. I. , VAN EIJK H. G.
Published: August 2011

Many questions about the metabolism of specific elements in the human body might be answered if elemental concentrations could be measured in situ in cells. With electron energy‐loss spectroscopic imaging (ESI), concentrations can potentially be determined with high spatial resolution. The theory of the quantification procedure has already been derived. Many practical instrument‐related problems, however, have to be solved. In the current research an energy‐filtering TEM is used and the image‐acquisition chain is examined in detail. Quantification requires images to be recorded over a large dynamic range. To solve this problem, the use of optical attenuation filters has been introduced. The use of the combination of a scintillator screen and a TV‐camera as a detection system has consequences for the processing of the data. Corrections for the camera photometric sensitivity and, to some extent, for shading are necessary. Further consequences of such a detection system for the correction of the element a‐specific spectral background and element detection are discussed. The derived methodology is tested in several ways and finally applied for the quantitative analysis of iron in liver parenchymal cells of a porphyria cutanea tarda patient.
Effect of specimen thickness and carbon content on the carbon peak region in EEL spectra
Authors: DOOR R. , HÄBERLE K. D. , MARTIN R.
Published: August 2011

Amorphous carbon films of different thicknesses and combinations of carbon films with CaF2 films of different thicknesses were used to create a standard reference library of EEL spectra. The relationship between the size and shape of the carbon peak region and the low‐loss part of the spectrum was determined. The steepness of the background of the CK edge, the size of the carbon peak and the plasmon shoulder on top of the carbon peak were investigated as a function of the thickness of the films and of the relative amount of carbon after normalization of the zero‐loss peak. The minimum at the CK edge and the maximum of the carbon peak were considered as examples for a modelling of the carbon peak region in different specimens. Methodological aspects which affect the accuracy of the measurement (characteristic lines of the detector, calculation of specimen thickness, method of t/λ measurement, mass loss, gain change correction, normalization of the spectra, energy resolution) were examined. The results indicate that modelling of the whole carbon peak region, considering its dependence on mass thickness and carbon concentration, should be possible from our standard reference spectra without use of the low‐loss region. The use of modelled standard reference spectra for background extrapolation in biological specimens with unknown calcium content is proposed.
Application of recording and processing of energy‐filtered image sequences for the elemental mapping of biological specimens: Imaging‐Spectrum
Authors: LAVERGNE J.‐L. , FOA C. , BONGRAND P. , SEUX D. , MARTIN J.‐M.
Published: August 2011

Computerized energy‐filtered transmission electron microscope (EFTEM) permits the recording and the processing of energy‐filtered images, allowing a part of an electron energy‐loss spectrum for each picture element to be obtained. This method, called ‘Imaging‐Spectrum’, uses a Zeiss CEM902 coupled to several image analysis systems. The actual configuration records sequences of 48 images, 256 × 256 pixels, in steps of the energy loss, ΔE. Processing these sequences results in part of a core‐loss EELS‐spectrum for each pixel. This approach produces elemental maps with a short processing time. We have implemented three kinds of background calculation for the image subtraction. The influence of the irradiation dose and of the energy selecting slit width on the quality of the spectra is investigated. The method is applied to the analysis of some biological specimens (pericellular coat behaviour during adhesion between macrophages and red blood cells and location of calcite microcrystals in dental pulp cells). The Imaging‐Spectrum method appears to be suitable for the analysis of large areas.