bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of lanthanide tracer methods in the study of mammalian pulmonary parenchyma and cardiac muscle by electron energy‐loss spectroscopy
Authors: FEHRENBACH H. , SCHMIEDL A. , BRASCH F. , RICHTER J.
Published: August 2011

Lanthanum (La) has widely been used as a tracer to study the integrity of plasma membranes. With conventional transmission electron microscopy (cTEM), the absence of electron scattering deposits from the cytoplasm has generally been assumed to reflect an intact cell membrane. However, the application of electron spectroscopic imaging (ESI) and electron energy‐loss spectroscopy (EELS) reveals that electron scattering deposits may be present which do not contain La. However, La could be detected in regions of pulmonary parenchyma and cardiac muscle that were devoid of electron scattering deposits. Therefore, to exclude misinterpretations based on cTEM the application of microanalytical techniques is strongly recommended for the study of the integrity of plasma membranes by means of La tracers. In addition, ESI and EELS are shown to distinguish between different tracers in simultaneous applications of La and terbium (Tb) which were used at the different faces of the pulmonary air‐blood barrier. The analysis of the distribution of both tracers which form electron scattering deposits, indistinguishable by cTEM, may help us to understand the different functional significances of cellular alterations of both cellular borders of the barrier.

As was shown for La, however, strictly controlled conditions are mandatory during the fixation procedure because an increase in the incubation time to more than 1 h in samples of pulmonary parenchyma may result in the occurrence of La deposits within the cytoplasm. In the absence of electron scattering deposits, the presence of La in glycogen granules and ribosome‐containing areas of various types of alveolar septal cells even after 15 min incubation indicates that the absence of deposits does not necessarily correspond to the absence of the tracer.
Quantitative analysis of the microstructure of the human cornea and sclera using 2‐D Fourier methods
Authors: VAEZY S. , CLARK J. I.
Published: August 2011

A two‐dimensional (2‐D) Fourier analysis was used to characterize the microstructure of the human cornea and sclera. The average centre‐to‐centre spacing of collagen fibrils was found to be 59 nm for the cornea and 285 nm for the sclera. These results agreed with those obtained by direct measurement using the electron micrographs, and those reported in the literature. The spatial order in the microstructure of the cornea was much greater when compared with that of the sclera. The results of the 2‐D Fourier analysis were consistent with the theory of transparency of the eye. The 2‐D Fourier analysis will be useful in quantitative characterization and analysis of the complex microstructure of biological cells and tissues in normal development and abnormal pathogenesis.
Reduced‐carrier single‐sideband microscopy: A powerful method for the observation of transparent microscopical objects
Authors: BRETSCHNEIDER F. , TEUNIS P. F. M.
Published: August 2011

The theoretical and practical properties of different forms of contrast formation in the microscope based on anaxial illumination are investigated: so‐called single‐sideband (SSB) techniques. The use of anaxial illumination in transmitted‐light microscopy is by itself a form of phase contrast (asymmetric illumination contrast, or AIC), but needs enhancement via a video circuit coupled to the microscope. The addition of a partially absorbing mask, known as a carrier‐attenuation filter (CAF), in a proper, conjugate plane in the microscope improves contrast substantially.

The imaging properties of this reduced‐carrier, single‐sideband imaging method (RC‐SSB)were tested using the transparent parts of a compact disc (CD); the tracks may be treated as small objects with a controllable phase shift. The results were compared both theoretically and experimentally with Zernike phase contrast and with Nomarski differential interference contrast.

The SSB technique has been shown to reveal transparent, submicrometre parts of living, unstained tissue, such as microvilli on sensory receptor cells of the transparent catfish, Kryptopterus. The high resolving power, together with the variable spatial‐frequency contrast enhancement, makes it a powerful technique for the imaging of in‐vivo subcellular details.
Fluorescence bleach rate imaging
Authors: BRAKENHOFF G. J. , VISSCHER K. , GIJSBERS E. J.
Published: August 2011

Bleach rate imaging on a (cooled) CCD can be easily achieved using a confocal microscope with bilateral scanning and detection coupled to a workstation; it is as easy as acquiring regular fluorescence images. Several analysis and display methods for bleach rate imaging are presented such as the bleach map (and its inverse) and a matrix‐based decomposition method for multi‐labelled specimens based on the bleach rate differences between the dyes used. With these tools, bleach‐rate‐based imaging can become a viable alternative to multiple labelling techniques for component identification in fluorescent specimens.
Calcium alginate encapsulation of small specimens for transmission electron microscopy
Authors: PAGE A. M. , LAGNADO J. R. , FORD T. W. , PLACE G.
Published: August 2011

A technique of encapsulating small objects in calcium alginate for further processing for transmission electron microscopy is described. Five methods are outlined which enable a variety of specimens including single cells (in suspension and on agar plates), small organisms and monolayers of tissue culture cells to be processed. A method for immunolabelling alginate‐entrapped material is also outlined.
Sequential treatment by phosphotungstic acid and uranyl acetate enhances the adherence of lipid membranes and membrane proteins to hydrophobic EM grids
Authors: BARNAKOV A. N.
Published: August 2011

A simple procedure for screening by electron microscopic observations of conditions for the reconstitution of membrane proteins into lipid bilayers is described. This procedure consists of a 5–10‐s treatment of electron microscopic grids, to which the sample has already been applied, with 1% phosphotungstic acid before proceeding with final staining in uranyl acetate. The method substantially enhances the adherence of lipid membranes and membrane protein particles to hydrophobic collodion/carbon grids.
Stereological analysis of the spatial Poisson–Voronoi tessellation
Authors: HAHN U. , LORZ U.
Published: August 2011

Stereological model tests and parameter estimators for the spatial Poisson–Voronoi tessellation are discussed. The tests aim to discriminate the Poisson–Voronoi tessellation from more regular or more irregular tessellations. The power of the model tests under some special parametric alternative hypotheses is investigated by simulation. Among the tests considered, the most powerful test is based on the variance of the section cell areas. Various stereological estimators for the model parameter of the spatial Poisson–Voronoi tessellation are compared with respect to their bias and variance by means of a Monte–Carlo study. Formulae are given for variance prediction. An estimator based on vertex counting is found to be the best. Robustness is investigated by applying the estimators to Voronoi tessellations generated by other point process models.
The homogeneity of phase repartition in TiB2–Fe composites using variance and covariance analysis
Authors: MISSIAEN J. M. , CHAIX J. M.
Published: August 2011

The microstructure of a sintered TiB2–Fe material has been studied by image analysis. The dispersion of phases, with particular attention to porosity, is investigated using chord lengths, covariance functions and variance of volume fractions. An extension of the usual point covariance analysis is made by sampling series of 10 contiguous fields, and measuring the covariance in the direction of field alignment. A model of a multiscale structure is proposed to interpret the different transitions of the variogram as composition fluctuations at different scales. Boolean models are used to give a geometrical representation of these fluctuations: the dispersion of porosity can be seen either as 20–30‐μm pore clusters or as the result of TiB2 +Fe 30–50‐μm clusters, probably formed during the elaboration process. Complementary information is obtained from the evolution of measured variance versus field size: a model is proposed to analyse the shape of the experimental curves. At small scales, ‘short’ integral ranges are determined, also obtained from the covariance. At very large scale (> 300 μm), composition fluctuations of about 2% are detected.
Shape processing and analysis using the calypter
Authors: PIRARD E.
Published: August 2011

After presenting some elementary criteria that should be respected by any automatic shape analysis technique, this paper focuses on the importance of the binary image encoding method. Most image analysers simply use a raster image to represent a binary object. If, occasionally, a vectorial description is available, it is merely chosen for its performances in data compression. Data compression and shape analysis have different goals and usual methods cannot satisfy both. The calypter is a new descriptor vectorizing the shape as a set of maximal inscribed discs. It is the most efficient means of accomplishing Euclidean mathematical morphology transformations and allows for further developments in binary shape processing. A simple adaptive contour filtering technique is presented. The calypter offers local and global perception of shape characteristics. It permits complete automation of the morphometric roundness charts used in many laboratories and also generates new shape parameters. A case study of three sand populations is presented to show the pertinence of a new ‘equivalent roundness' parameter.
Optimizing the performance of confocal point scanning laser microscopes over the full field of view
Authors: ENTWISTLE A. , NOBLE M.
Published: August 2011

To examine many of the imaging capabilities of confocal scanning laser microscopes rapidly and reliably over the whole field of view three simple, easily prepared specimens are required: a mirror positioned on a carefully measured shallow gradient, a film of highly fluorescent material and a rectangular grid with a readily defined centre. Using these specimens the adjustment of any combination of confocal scanning laser visualization system and light microscope can be examined throughout the field of view. The effects of misalignment of the various subcomponents of a confocal scanning laser microscope on both the axial spread function of a plane and the shading pattern over the image field are described. Finally, where the design of the confocal optics permits, the three specimens can be used to facilitate the alignment of the various components to the optimal level achievable.
HREM image simulations for small particle catalysts on crystalline supports
Authors: YAO M.‐H. , SMITH D. J.
Published: August 2011

The imaging conditions for electron microscope studies of supported ultrafine particle catalysts have been investigated by multislice simulations. Images of Pt and ReO4 particles ranging from 0·4 to 2·3 nm in size were simulated in both plan view and profile view with a rutile (TiO2) support. It was shown that particle visibility varied greatly with the objective lens defocus. Optimum defocus was not favourable for supported particles in plan view since the ultrafine supported particles were least visible at this defocus. Underfocusing, especially at defoci corresponding to half‐spacing fringes in the TiO2 support, led to improved visibility and resolution of the supported particles. Although the structure and shape of supported ultrafine particles should be resolved better with a 400‐kV high‐resolution electron microscope, their detectability is poorer than with a 200‐kV instrument. An ReO4 cluster should be detectable at 200 kV on TiO2 supports up to 5 nm in thickness, whereas it is only likely to be detectable at 400 kV on supports up to 3 nm in thickness. The simulations confirmed that optimum defocus is most favourable for imaging supported particles in profile view. Atomic information for particles as small as a 13‐atom Pt cuboctahedral cluster should be resolvable with a 400‐kV instrument. The crystalline Ti monolayer observed on surfaces of Pt particles, which could explain the mechanism known as SMSI, was simulated as an example of profile imaging.
Scanning microphotolysis: A new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging
Authors: WEDEKIND P. , KUBITSCHECK U. , PETERS R.
Published: August 2011

The fluorescence photobleaching method has been widely used to study molecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high‐resolution, three‐dimensional imaging. A new technique, scanning microphotolysis (Scamp), combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a sufficiently powerful laser and a novel device, the ‘Scamper’. This consisted essentially of a filter changer, an acousto‐optical modulator (AOM) and a computer. The computer was programmed to activate the AOM during scanning according to a freely defined image mask. As a result almost any desired pattern could be bleached (‘written’) into fluorescent samples at high definition and then imaged (‘read’) at non‐bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patterns. Experiments with living cells concerning dynamic processes in cytoskeletal filaments and the lateral mobility of membrane lipids suggest a wide range of potential biological applications. Thus, Scamp offers new possibilities for the optical manipulation and analysis of both technical and biological microsystems.
Light microscopical evaluation of pyrite oxidation of heap leached samples from a Pittsburgh seam coal
Authors: IRDI G. A. , BOOHER H. B.
Published: August 2011

The U.S. Department of Energy and the U.S. Bureau of Mines conducted experiments to determine the feasibility of reducing the pyritic sulphur content of a conventionally cleaned Pittsburgh seam coal by heap leaching. Two identical heaps, one indoor and one outdoor, were constructed, and sprayed with recycled rainwater/leachate for approximately 200 days. Lump samples were selected prior to initiation of heap leaching activities and from both heaps after 5‐ and 11‐month intervals for microscopic (petrographic) examination. These examinations revealed no significant differences in the occurrence of pyrite oxidation between the two heaps. These observations were consistent with the results from the chemical analyses that showed no significant differences in pyritic sulphur removal between the indoor and outdoor heaps. Samples from the starting coal were also selected and examined to see how different pyrite morphologies behaved during artificial weathering. Dendritic and framboidal pyrite forms appeared to be the more reactive forms.
An atomic force microscope for cytological and histological investigations
Authors: MARIANI T. , MUSIO A. , FREDIANI C. , SBRANA I. , ASCOLI C.
Published: August 2011

An atomic force microscope (AFM) specifically designed for cytological and histological studies and able to operate on the same scale of the highest optical magnification is described. The AFM is a non‐invasive instrument; it operates on samples which do not require any kind of treatment and it can produce information that supplements and completes the information given by traditional microscopical methods. The apparatus has been used to image fixed human chromosomes and to investigate the action of trypsin during the staining for banding. First results showed that banding patterns very similar to G‐banding pre‐exist to staining and to trypsin treatment in human metaphase chromosomes, and that the trypsin treatment induces a structural collapse in the chromatin. The instrument was also used on thin sections of plant tissue and gave promising results. Experience confirmed that AFM is a suitable tool for this kind of investigation, and proved the importance of developing AFM microscopes specifically designed for routine use in cytology and histology, conceived for non‐specialized users.
Unstained and in vivo fluorescently stained bacterial nucleoids and plasmolysis observed by a new specimen preparation method for high‐power light microscopy of metabolically active cells
Authors: KELLENBERGER E. , VAN DER KAMP C. KELLENBERGER
Published: August 2011

Microscope slides were coated with a layer of gelatin, the thickness of the gelatin increasing linearly along the long axis. The bacterial suspension is applied to the dried gelatin and covered by a coverslip. The medium is absorbed by the gelatin and thus the cells applied against the coverslip. By this method, cultures of concentrations below 108 cells/ml provide statistically relevant numbers for observation without prior concentration steps. It is easier to apply than the existing methods for the observation of bacterial nucleoids by phase contrast imaging. Because the cells are maintained in growing conditions the method is useful for the vital fluorescence DAPI‐staining of various bacterial species and for observations of plasmolysis and its reversal at different physiological conditions and extracellular osmolalities. The previously generally assumed view that the plasmolytic changes of the cell morphology are immediate upon the hyperosmotic shock and are rapidly repaired when the cell is able to metabolize actively was confirmed; this is in contrast to some recent claims.
Fibre optic scrambling in light microscopy: A computer simulation and analysis
Authors: REITZ F. B. , PAGLIARO L.
Published: August 2011

Optical fibres bent in two mutually perpendicular planes have proven useful for randomizing illumination in light microscopes. These optical scramblers can increase the resolution and/or contrast obtained with several modes of light microscopy. Here, computer simulations are used to investigate several parameters affecting light randomization in curved optical fibres in order to further the theoretical basis for scrambler design. Light passing through 90° bends of optical fibre of varying radii of curvature was modelled by ray tracing in two dimensions, and scrambling mechanisms were observed. The effects of varying the position and angle of entry of light on the phase and direction of propagation of the emergent light were determined. It was found that (a) thorough scrambling does not necessarily require high numerical aperture (NA) entry of light into the fibre, (b) considerable order persists after a single 90° bend of an idealized fibre and (c) a higher degree of scrambling (at the cost of transmission efficiency) is achieved in more tightly curved fibres. The pathlength variations introduced by scrambling proved smaller than typical laser coherence lengths, requiring temporal scrambling (vibrating the fibre).
Edge detection, three‐dimensional cell boundary reconstruction and volume and surface area estimation from differential interference contrast images
Authors: KENYON C. M. , YANAI M. , MACKLEM P. T.
Published: August 2011

Three‐dimensional (3‐D) cell morphology is important for the understanding of cell function and can by quantified in terms of volume and surface area. Differential interference contrast (DIC, or Nomarski) imaging can enable cell edges to be clearly visualized in unstained tissue due to the slight difference in refractive index between aqueous media and cytoplasm. DIC is affected in only one direction ‐ the direction of the optical shear. A 1‐D edge detector was used in that direction with a scale length equal to that of an in‐focus edge to highlight cell boundaries. By comparison with the signal from the edge detector on an out‐of‐focus slice, the in‐focus slices could be segmented and, after noise suppression, cell outlines obtained. A voxel paradigm was used to calculate cell volume and differential geometry was used for surface area estimation. We applied this approach to obtain 3‐D dimensional information by optical sectioning of motile Amoeba proteus.
Estimation of the transport properties of polymer composites by geodesic propagation
Authors: BRÉMOND R. , JEULIN D. , GATEAU P. , JARRIN J. , SERPE G.
Published: August 2011

The purpose of this study is to estimate the transport coefficients (diffusion, permeability) of polymer composites from two‐dimensional images of these media (polyethylene/polyamide alloys) obtained by the scanning electron microscope. The main characteristics of the media investigated are a wide morphological variability (nodular and lamellar media) and transport properties that are very different according to the phase. The computation tools used must accordingly have a wide range of validity. The idea of the proposed algorithm is to calculate the distance from an edge of the image, with a modified distance according to the phase crossed. The results obtained are compared with those obtained by finite difference integration of Fick's laws, and with permeability measurements of hydrocarbons in the materials.
Intracellular localization of the antitumour drug adriamycin in living cultured cells: A confocal microscopy study
Authors: MESCHINI S. , MOLINARI A. , CALCABRINI A. , CITRO G. , ARANCIA G.
Published: August 2011

The intracellular distribution of the anthracyclinic antibiotic adriamycin in living cultured cells has been investigated by confocal microscopy.

In human melanoma cells (M14), adriamycin was localized inside the nuclei. When adriamycin‐treated M14 cells were allowed to recover in drug‐free medium, a complete efflux of the drug from the nucleus was revealed. In recovered cells, a weakly fluorescent signal was observed in the perinuclear region. When M14 cells were recovered in a medium containing colcemid, a microtubule depolymerizing agent, the drug transport from the nucleus to the cell periphery appeared to be inhibited, suggesting that the microtubule network is strongly involved in drug transport mechanisms. In multidrug‐resistant (MDR) cells the intracellular location of adriamycin was shown to be noticeably different from that of the parental wild‐type cells. In particular, in resistant human breast carcinoma cells (MCF‐7), adriamycin appeared to be exclusively located within the cytoplasm whereas the nuclei were shown to be completely negative. When adriamycin treatment was performed in association with MDR revertants, such as Lonidamine (inhibitor of the energy metabolism) or verapamil (inhibitor of the P‐glycoprotein efflux pump), a marked enhancement of the cytoplasmic signal was observed in resistant cells. Under these conditions, adriamycin appeared concentrated in the perinuclear region, but the nuclei were still negative. Confocal microscopy proved to be a very useful method for the study of the intracellular transport of fluorescent substances, such as anthracyclinic antibiotics, and for the investigation of the multidrug resistance phenomenon in tumour cells.
Refractive‐index‐induced aberrations in two‐photon confocal fluorescence microscopy
Authors: JACOBSEN H. , HÄNNINEN P. , SOINI E. , HELL S. W.
Published: August 2011

The effect of refractive index mismatch on the image quality in two‐photon confocal fluorescence microscopy is investigated by experiment and numerical calculations. The results show a strong decrease in the image brightness using high‐aperture objectives when the image plane is moved deeper into the sample. When exciting at 740 nm and recording the fluorescence around 460 nm in a glycerol‐mounted sample using a lens of a numerical aperture of 1·4 (oil immersion), a 25% decrease in the intensity is observed at a depth of 9 μm. In an aqueous sample, the same decrease is observed at a depth of 3 μm. By reducing the numerical aperture to 1·0, the intensity decrease can be avoided at the expense of the overall resolution and signal intensity. The experiments are compared with the predictions of a theory that takes into account the vectorial character of light and the refraction of the wavefronts according to Fermat's principle. Advice is given concerning how the effects can be taken into account in practice.
Optoelectronic detector probes for scanning near‐field optical microscopy
Authors: DANZEBRINK H. U.
Published: August 2011

A brief explanation of the optoelectronic probe concept and a comparison between the implementation of passive waveguide probes and optoelectronic probes in scanning near‐field optical microscopy (SNOM) is presented. The first probe realizations using cleaved semiconductor crystals and the work at present in progress using microfabricated Si pyramids are described. These crystals with evaporated metal electrodes forming a slit aperture with subwave‐length dimensions work as metal–semiconductor–metal photodetectors. Their optical detection behaviour is investigated by measuring the intensity distribution of a laser focal point. Measurements where the external bias voltage is changed show that it is possible to modify the detection behaviour of the device because of the varying depletion widths. The last part of the article describes a concept where pyramidal probes should function simultaneously as sensors for scanning force microscopy (SFM) to measure topography and as optoelectronic probes for scanning near‐field optoelectronic microscopy (SNOEM).
LIP‐model‐based three‐dimensional reconstruction and visualization of HIV‐infected entire cells
Authors: GREMILLET P. , JOURLIN M. , PINOLI J.‐C.
Published: August 2011

This paper presents a global solution from acquisition to visualization for the three‐dimensional reconstruction of cell sections. Original techniques are proposed for the correct handling of the geometrical section distortions, and a new interpretation based on the logarithmic image processing (LIP) model is applied in order to create normalized grey‐level sections where these are missing. Finally, a new method for generating a mesh of triangles to describe the envelope of the reconstructed cell is proposed, as well as a visualization mixing image synthesis and grey‐level information. The product allows the user to explore the reconstructed cellular block in any desired direction, by showing user‐defined grey‐level sections inside the block mixed to a synthetic view of the cell envelope.
A simple method for processing individual oocytes and embryos for electron microscopy
Authors: NOGUÉS C. , MARTÍ M. , BOADA M. , PONSÁ M.
Published: August 2011

A simple method for handling individual specimens that must be processed either for scanning or transmission electron microscopy studies is described. For scanning microscope processing, dehydration is carried out with samples enclosed in small cages made from TAAB capsules in which top and bottom are substituted by plankton nets, and for transmission electron microscopy, samples are pre‐embedded in agarose. This procedure significantly reduces mouth pipetting, dissecting microscope observations, is less labour intensive and, most importantly, reduces sample loss.
A novel method of Z‐contrast imaging in STEM applied to double‐labelling
Authors: TICHELAAR W. , FERGUSON C. , OLIVO J.‐C. , LEONARD K. R. , HAIDER M.
Published: August 2011

A novel method of Z‐contrast imaging in the scanning transmission electron microscope (STEM) is presented. The technique relies on the element dependence of the angular distribution of the scattered electrons, and is realized with a detector consisting of a set of concentric rings. It is possible to discriminate 9‐nm colloidal gold and silver specifically distributed on thin sections. In addition to this practical work, numerical evaluations are used to assess the method. With two smaller markers, this approach will be useful in discriminating closely spaced antigenic sites when steric hindrance occurs with double‐labelling using probes of different sizes.
High‐pressure freezing of cell suspensions in cellulose capillary tubes
Authors: HOHENBERG H. , MANNWEILER K. , MÜLLER M.
Published: August 2011

A procedure for efficient cryoimmobilization of large volumes of cell suspensions or micro‐organisms by high‐pressure freezing is described. This procedure uses transparent, porous cellulose capillary tubes with an inner diameter of 200 μm, into which the suspensions are drawn by capillary action. The tubes are processed by high‐pressure freezing and freeze‐substitution as if they were tissue samples. Centrifugation of suspensions at low temperatures is no longer necessary and cryopreparation is greatly facilitated.

A very high yield of adequately frozen specimens is obtained due to the constant, defined sample geometry. This approach can also be used to process suspensions by conventional chemical fixation, eliminating the need to embed pellets in low‐melting‐point agarose, for example, prior to chemical fixation.

The preparation procedure is demonstrated with suspensions of nematodes, paramecia and bacteria.
Nucleotide and protein distribution in BrdU‐labelled polytene chromosomes revealed by ion probe mass spectrometry
Authors: LEVI‐SETTI R. , CHABALA J. M. , SMOLIK S.
Published: August 2011

Detailed chemical maps of BrdU‐labelled polytene chromosomes of Drosophila melanogaster, obtained by imaging secondary ion mass spectrometry, reveal separately the distribution of DNA and proteins in the chromosomes. The thymidine‐analogue BrdU within the chromosomal DNA is localized by detecting the Br− secondary ion signal, while both nucleic acid and protein content are mapped through the abundantly emitted CN− signal. This novel approach supercedes, and helps explain the origin of, the banding patterns that are observed by conventional staining techniques. The high spatial resolution and chemical and isotopic sensitivity of this technique should enhance the localization of specific genes by in situ hybridization in mitotic chromosomes.
Application of back‐scattered electron imaging to the study of the lichen‐rock interface
Authors: WIERZCHOS J. , ASCASO C.
Published: August 2011

In the study of the lichen‐rock interface, light microscopy, scanning electron microscopy (SEM) in secondary emission mode and transmission electron microscopy are the most commonly used techniques. As these methods have some limitations, there is a need to explore other techniques for observation of the lichen‐substrate interface. One of the most promising methods is the application of SEM in the back‐scattered electron (BSE) emission mode.

The thallus ofAspicilia intermutans(Nyl.) Arn. growing on granitic rock was examined by SEM in BSE mode. The detailed preparation of transverse sections of the lichen‐rock contact zone is presented. The BSE scanning images of the lichen‐rock interface obtained present new insights into the ultrastructural features of the biological components, providing more information about the biogeophysical and biogeochemical weathering of rock.
Semi‐automated measurement of true chord length distributions and moments by video microscopy and image analysis
Authors: OLDMIXON E. H. , BUTLER J. P. , HOPPIN F. G.
Published: August 2011

The distribution of the lengths of airspace chords in pulmonary parenchyma characterizes many architectural features of the alveoli and alveolar ducts. Laborious to obtain manually, the distributions and density functions may be acquired semi‐automatically by video microscopy, digitization and image processing. The accuracy of the estimation is influenced by the microscopical methods and also by the techniques used (i) to convert the digitized grey‐scale picture to a two‐valued image, (ii) to collect the chord lengths and (iii) to compensate for finite field widths. The last problem arises because some chords are completely visible within a field while others are only partially seen, since one of the two air‐tissue boundaries lies outside the field of view. This error systematically biases the observed distribution. This paper contains solutions to hardware, software and analytic problems encountered while developing the capability to measure airspace chord length density functions semi‐automatically. Formulas for estimating the true chord length density function from samples of observed chord lengths are presented. Also given are formulas for the estimation of the first and second moments of the true chord length distribution from the means of observed chord lengths. These techniques of image preparation and analysis should be suitable for characterizing particle, grain or cell size distributions, especially where many profiles fall partially outside the field of view.
A simple algorithm to measure the volume‐weighted and number‐weighted mean volume of particles
Authors: LAROYE G. J. , GRANT K.
Published: August 2011

An algorithm is presented which offers an alternative approach for measuring volume‐ and number‐weighted mean volume and standard deviation of particles. Using a computer‐assisted manual method the following intermediate steps are performed automatically: generation of linear probes emanating from the sampling point of the object and intersecting the profile periphery, measurement of their lengths, and measurement of the area of the transect required for estimating the standard deviation of the volume‐weighted mean volume. By first tracing manually the outline of the periphery of the object with a cursor, on a magnetic tablet or on an image acquired into the computer with a video camera, the location of all pixels of the periphery is registered and the area of the transect is measured concurrently. The computer is informed of the coordinates of the selection point in the uniform random (UR) sampling grid by clicking the cursor. All ensuing operations are automatic. In the case of isotropic UR (IUR) sections the algorithm traces a series of uniform systematic random linear probes between the sampling point and the object profile periphery emanating from this selection point, radiating at angular intervals of 29–30° to the periphery. In the case of vertical sections, similar lines are generated at intervals where the sine of the angle changes by a value of 0·33. The volume‐weighted mean volume of the object is estimated from the average of all the products , where l represents the length of each individual random linear probe. As the periphery is traced, the algorithm can automatically determine the area of the cross‐section of the object, from which the standard deviation of the volume‐weighted mean volume can be calculated. Some elements of the above algorithm are also used for the measurement of the number‐weighted mean volume. The latter procedure is facilitated using an acoustic vertical depth monitor attached to the microscope. The impact of truncation (‘lost caps’) on the precision of the measurements is discussed. The algorithm is of particular use in light microscopy for measuring cell nuclei by direct visual inspection of the microscopic field using a side‐arm mirror assembly interfaced with a magnetic tablet.
Microspectroscopic measurement of the optical properties of rat liver in the visible region
Authors: SEIYAMA A. , CHEN S.‐S. , KOSAKA H. , SHIGA T.
Published: August 2011

Microspectroscopy is used to investigate optical properties of haemoglobin‐free perfused rat liver. Visible spectra of 20 μm diameter spot size were measured in transmission and/or reflection modes as a function of the thickness (< 1200 μm) of the liver‐edge. Optical density (OD) in transmission mode increased with the increasing liver thickness, whereas in reflection mode OD decreased but became almost constant above a certain thickness (c. 600 μm) of the liver. The Kubelka‐Munk (KM) two‐flux model, with a minor modification, was applied successfully to the analysis of the changes in OD as a function of the thickness. This approach estimates the KM absorption coefficient (EKM), KM scattering coefficient (SKM) and effective penetration depth (δeff) of the liver. The optical properties were similar to reported values, obtained with different methods.
Quantitative water mapping of cryosectioned cells by electron energy‐loss spectroscopy
Authors: SUN S. Q. , SHI S‐L. , HUNT J. A. , LEAPMAN R. D.
Published: August 2011

A direct technique based on electron energy‐loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been developed to map subcellular distributions of water in frozen‐hydrated biological cryosections. Previously, methods for water determination have been indirect in that they have required the cryosections to be dehydrated first. The new approach makes use of spectrum‐imaging, where EELS data are collected in parallel at each pixel. Several operations are required to process the spectra including: subtraction of the detector dark current, deconvolution by the detector point‐spread function, removal of plural inelastic scattering and correction for the support film. The resulting single scattering distributions are fitted to standard reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the darkfield or brightfield image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variations in water content. Reference spectra have been recorded from the major biomolecules (protein, lipid, carbohydrate, nucleic acid) as well as from vitrified water and crystalline ice. It has been found that quantitative results can be obtained for the majority of subcellular compartments by fitting only water and protein reference spectra, and the accuracy of the method for these compartments has been estimated as ± 3·5%. With the present instrumentation the maximum allowed dose of 2 × 103 e/nm2 limits the useful spatial resolution to around 80 nm for ± 5% precision at a single pixel. By averaging pixel intensities a value of 56·8% with a precision of ± 2·0% has been determined for the water content of liver mitochondria. The water mapping technique may prove useful for applications to cell physiology and pathophysiology.
Hybrid scanning transmission electron/scanning tunnelling microscope system for the preparation and investigation of biomolecules
Authors: KNAPP H. F. , WYSS R. , HÄRING R. , HENN C. , GUCKENBERGER R. , ENGEL A.
Published: August 2011

A hybrid scanning transmission electron/scanning tunnelling microscope vacuum system is introduced, which allows freeze drying and metal coating of biological samples and their simultaneous observation by scanning transmission electron microscopy and scanning tunnelling microscopy (STM). Different metal coatings and STM tips were analysed to obtain the highest possible resolution for such a system. Bovine liver catalase was used as a test sample and the STM results are compared to a molecular scale model.
Backscattered electron imaging of the undersurface of resin‐embedded cells by field‐emission scanning electron microscopy
Authors: RICHARDS R. G. , GWYNN I. AP
Published: August 2011

In this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c 3T3 fibroblasts were cultured on either 13‐mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field‐emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 μm, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values to give ‘sections' of information from increasing depths within the sample. At 3–4 keV only structures a very short distance into the material were observed, essentially the areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metals were intensely bound became more evident. The technique allowed stepwise ‘sectional’ information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light‐microscope‐based system.
Tomographic reconstruction of the cross‐sectional refractive index distribution in semi‐transparent, birefringent fibres
Authors: WEDBERG T. C. , WEDBERG W. C.
Published: August 2011

Optical diffraction tomography (ODT) is used to reconstruct the complex refractive index distribution in cross‐sections of semi‐transparent, birefringent fibres. The selected fibres were polymer and animal fibres of either circular or non‐circular cross‐section with average thicknesses in the range 8–110 μm. This choice of samples was made to illustrate the imaging capabilities of ODT, and also to demonstrate some potential applications of the technique. The images representing the reconstructed refractive index distributions have a spatial resolution of about 2 μm, and show noticeable image contrast for refractive index variations of about 0·001. The ODT reconstructions compare well with refractive index information provided with the samples, and with scanning electron micrographs of cross‐sections of the same fibre samples. From these results it appears that ODT can be used to reconstruct the complex refractive index distribution in cross‐sections of semi‐transparent, birefringent fibres.
Imaging periodic surface relief structures
Authors: SHERIDAN J. T. , KÖURNER T. O.
Published: August 2011

Because of shadowing, multiple scatter and polarization effects, the interpretation of images of gratings with fine periods, isolated deep structures, and multiple scattering volume objects is seriously complicated. In this paper a review of the methods used to model such effects is presented. Periodic surface relief gratings are of particular current importance because of the possibility of producing calibration samples using them. Several examples which illustrate electromagnetic volume effects are examined. General trends which help in validating the use of Fourier‐transform‐based scalar transmittance theory are then indicated. The angular spectrum approach, which can be used, together with a scatter function generated using the rigorous electromagnetic theory, to calculate coherent, partially coherent and confocal images of volume objects, is also discussed.
Forbidden light scanning near‐field optical microscopy
Authors: HEINZELMANN H. , HECHT B. , NOVOTNY L. , POHL D. W.
Published: August 2011

Near‐field optics (NFO) opens the door to light microscopy techniques with resolutions well beyond the diffraction limit. The richness of optical investigations is now applicable on a near‐molecular level. Among the novel scanning near‐field optical microscopy (SNOM) schemes, the most prominent representatives are aperture SNOM and scanning tunnelling optical microscopy (STOM or PSTM).

New experimental and theoretical work has to be performed to study the phenomena specific to NFO. One such example is the angular dependence of light emission in aperture SNOM. The detection of radiation at angles greater than the critical angle of total internal reflection αc = arcsin(n−1), where n is the sample refractive index, can represent a microscopy scheme that combines the respective advantages of both aperture SNOM and STOM. Recent experiments have demonstrated the expected exponential dependence of light intensity on gap width (for fixed emission angle α > αc). The decay length as a function of α is in agreement with the Fresnel description of the evanescent field when total reflection occurs at an interface. These investigations were additionally motivated by calculations based on the multiple multipole method.
Automated correction of linear deformation due to sectioning in serial micrographs
Authors: JANSSON T. , GUSTAVSSON T. , RYDMARK M. , BERTHOLD C. H. , PASCHER R. , SKOGLUND T.
Published: August 2011

This paper describes an objective and automatic method for detection and correction of sectioning deformations in digitized micrographs, as well as an evaluation of the method applied to light and electron microscopic images of semi‐thin and ultra‐thin serial sections from brain cortex. The detection is based on matching of image subregions and the deformation model is bi‐linear, i.e. two first‐order polynomials are used for modelling compression/expansion in perpendicular directions. The procedure is applicable to prealigned serial two‐dimensional sections and is primarily aimed at three‐dimensional reconstruction of tissue samples consisting of a large number of cells with random distribution and morphology.
Microfractography of granitic rocks under confocal scanning laser microscopy
Authors: MONTOTO M. , MARTÍNEZ‐NISTAL A. , RODRÍGUEZ‐REY A. , FERNÁNDEZ‐MERAYO N. , SORIANO P.
Published: August 2011

Scanning laser microscopy, in the confocal mode (CSLM) has been applied to a granitic rock to characterize its fissure space. The technique provides a unique three‐dimensional picture of the rock microfractography. CSLM is unique in observing fine details of the fractographic network (connectivity, tortuosity, etc.), its geometry and its relation to other rock‐forming components.

The fractographic images with standard fluorescence microscopy are compared with those obtained with CSLM. The examples presented emphasize the advantages of CSLM: three‐dimensional visualization of the microfractographic network, crack connectivity, automatic evaluation of direction and slope of fissures.

These studies are related to the migration of radionuclides in the geosphere. The relations between potentially water‐conducting open fissures, and the rock‐forming minerals provide a means of modelling the ‘radionuclide retardation mechanism’, a security factor in their definitive storage in rock masses.
In‐situ monitoring of fibre and matrix deformation in fibre/polymer composites
Authors: FAVRE J.‐P. , AUVRAY M.‐H. , SIGETY P. , LÉVĚQUE D. , BRIANÇON C.
Published: August 2011

Three methods, namely microphotoelasticimetry, Raman spectroscopy and surface microgrids, are currently used at ONERA on fibre‐reinforced polymers to measure the in‐situ fibre or matrix deformation. They provide the materials scientist with valuable indications on the early damage growth. Recent results are given and information about the limitations of the methods are indicated.
The effect of microstructure on flow promotion in resin transfer moulding reinforcement fabrics
Authors: GRIFFIN P. R. , GROVE S. M. , GUILD F. J. , RUSSELL P. , SUMMERSCALES J.
Published: August 2011

The resin transfer moulding (RTM) process is becoming increasingly important for the manufacture of continuous fibre‐reinforced thermosetting resin matrix composites. The RTM process is a closed mould technique which reduces volatile emissions relative to traditional hand lay‐up methods. The fibres, generally as several layers of fabric, are prepared as a preform and laid in the closed mould. The resin is injected, at one or more points, and flows through the mould to form the finished product. In the manufacture of high‐performance composite structures, the flow of resin is constrained by the high volume fraction of reinforcement fibres required to achieve the performance. Commercial fabrics are becoming available which are woven with specially designed mesoscale architecture to promote flow of the resin. The flow rates in a series of such fabrics have been studied. The microstructures of the resulting composites have been examined using brightfield optical microscopy. A Quantimet image analyser was used to quantify the structures on both the mesoscale and the microscale. The flow rate has been shown to be related to the presence of both large and more modest sized pore space in the reinforcement architecture.