bluuebunny/crossref_metadata_2025 huggingface dataset:
Fractal characterization by frequency analysis. II. A new method
Authors: AGUILAR M. , ANGUIANO E. , PANCORBO M.
Published: August 2011

A new frequency analysis method, fractal analysis by circular average (FACA), and an image replication procedure are proposed that together produce accurate measurements of the fractal dimension of surfaces and profiles, eliminating Fourier transform artefacts which arise from the lack of periodic continuity in real surfaces and profiles.
Evaluation of the precision of systematic sampling: Nugget effect and covariogram modelling
Authors: THIOULOUSE J. , ROYET J. P. , PLOYE H. , HOULLIER F.
Published: August 2011

Systematic sampling designs are widely used in stereology. When an estimator of the total amount, Q, of the sampled variable is evaluated by such a procedure, the coefficient of error can be predicted by applying Matheron's theory of regionalized variables. To evaluate the accuracy of the estimate of Q, it is necessary to study the behaviour of the regionalized variable and to model its covariogram. Histological data with a low short‐range variability and agronomic data with a pronounced nugget effect provided the biological material for extreme case studies. Results show that the short‐range variability, if present, cannot be detected when only small samples are available. An underestimation of the coefficient of error is then to be expected. We propose several models of the covariogram, which can be used to test for the presence of a nugget effect. If a nugget effect is present, these models will provide better estimates of the coefficient of error. If there is no nugget effect a simplified method can be used and will provide reliable estimates of the coefficient of error.
Edge detection in petrographic images
Authors: STARKEY J. , SAMANTARAY A. K.
Published: August 2011

The automatic detection of mineral grain boundaries in images obtained from a polarized‐light microscope requires special techniques. Observations in both plane‐ and cross‐polarized light may be necessary and the section must be rotated relative to the plane of polarization of the microscope to see all the grain boundaries. In computer‐based microscopy this can be accomplished by the sequential accumulation of individual images captured from one microscope field of view with different polarizer orientations.

For real‐time implementation the sequential images are segmented individually by applying Canny's algorithm. A separable Gaussian mask is used for smoothing and a 3 times 3 convolution mask is used to generate 1‐pixel‐wide boundaries, which are located at the zero‐crossing of the second‐order derivative of the intensity gradient. The boundaries are extracted and accumulated in a composite image. The resulting composite image is a synoptic grain‐boundary image of the rock.
Development, quantitative performance and applications of a parallel electron energy‐loss spectrum imaging system
Authors: BOTTON G. , L'ESPÉRANCE G.
Published: August 2011

The development of a parallel electron energy‐loss spectrum imaging system is presented. The analytical performance of the imaging technique was investigated and the system applied to materials science problems. The system, which allows acquisition and storage of a parallel electron energy‐loss spectrum at each pixel of an image, was developed by interfacing a multichannel analyser and a microscope to a computer workstation. In the experimental conditions used for imaging, detection limits and quantification errors were large and varied as a function of spatial resolution and the range of chemical elements of interest in the image. Applications of this imaging technique in materials science showed that quantitative chemical information is provided by the system and that the use of relative thickness maps and detailed statistical analysis of the spectrum‐image allowed an unbiased interpretation of the images. As energy‐loss spectra are available after processing, spectroscopic information about the analysed material can be used to provide supplementary information.
Semiautomated methods for cancellous bone histomorphometry using a general‐purpose video image analysis system
Authors: HUFFER W. E. , RUEGG P. , ZHU J.‐M. , LEPOFF R. B.
Published: August 2011

Semiautomated methods are used to measure elongated, curved and complex branching profiles and isolated perimeter segments in monochrome video images with a general‐purpose analysis system. These methods are used to make the major primary measurements of bone histomorphometry. Accuracy and reproducibility of the image acquisition, processing and measurement system is documented by measuring a semicircular standard of known dimensions. Semiautomated applications of the Ar/Le method for measuring areas and perimeters, and calculating lengths and widths of osteoid seams, lengths of mineralization labels and mineral apposition rate, wall width, indirect measurements of eroded, osteoclastic and osteoblastic perimeters without tracing, and measurement of mineralized or total cancellous bone area and perimeter gave values comparable to measurements of the same parameters by tracing or grid counting techniques with equal or better reproducibility and much greater efficiency. Intraindividual variation in measuring multiple bone biopsies was comparable to that reported with current standard methods. Major sources of variability for semiautomated methods were image magnification and selection of profile edges by thresholding, and sources of variability for manual methods are image magnification, numbers of orthogonal intercepts, tracing speed and accuracy of the algorithm used to measure traced pixels. Semiautomated methods are accurate, reproducible and rapid methods suitable for bone histomorphometry.
Estimation of the directional distribution of spatial fibre processes using stereology and confocal scanning laser microscopy
Authors: MATTFELDT T. , CLARKE A. , ARCHENHOLD G.
Published: August 2011

Fibrous structures like polymers, glass fibres, muscle fibres and capillaries are important components of materials and tissues. A spatial fibre process is the union of smoothly curved or linear one‐dimensional features of finite length, arranged in an unbounded three‐dimensional reference space according to some random mechanism. Design‐based stereology was combined with confocal scanning laser microscopy to study samples of fibre‐reinforced composites, which were considered as realizations of not necessarily isotropic fibre processes. The methods enable the unbiased estimation of the intensity and of the directional distribution of spatial fibre processes from arbitrarily directed pairs of registered parallel optical sections a known distance apart. The directions of fibres sampled by a frame of observation on the reference plane are estimated from the coordinates of the intersection points of the fibres with both optical planes using confocal scanning laser microscopy. The true directional distribution of the fibre process is estimated by weighting each measured direction by the reciprocal of its chance of being sampled, which can be inferred from the data. The concept of complete directional randomness for uniformly and independently distributed spatial directions is introduced. The cumulative distribution function of the angular distances between different directions and other exact relations are derived for complete randomness of vectorial and axial directions. A Monte Carlo method is constructed to test spatial fibre processes, whose fibres have negligibly small curvature, for complete directional randomness. Confocal scanning laser microscopy was used to study the angular distribution of glass fibres in a polymer composite which was subjected to increasing hydrostatic extrusion. The hypothesis of complete directional randomness had to be rejected for all samples with 1% probability of error. The directional distribution was of the bipolar type, with the principal axis directed parallel to the axis of extrusion. Progressive stretching of the material increased the degree of anisotropy of the glass fibres. Although presented for an application in polymer physics, the methods are general and may also be applied in biological investigations.
Tip artefacts in scanning force microscopy
Authors: SCHWARZ U. D. , HAEFKE H. , REIMANN P. , GÜNTHERODT H.‐J.
Published: August 2011

Since its invention in 1986, scanning force microscopy (SFM) has experienced great success as a characterization method for topography on small scales. In spite of the enormous potential of the method, it is limited by the quality of the tip used for probing the surface topography. Convolutions of non‐ideal tip shapes with the real topography and tip bending, flexing and jumping effects produce artefacts in the resulting images.

A brief description of the preparation and characteristics of the most commonly used SFM tips is given. A variety of different artefacts originating from tip properties is presented and illustrated with selected scanning force micrographs. Methods to minimize tip artefacts in SFM images are described.
Contribution of energy‐filtering TEM to the detection of calcium: Application to mast cells
Authors: HOROYAN M. , SOLER M. , MARTIN J. M. , BENOLIEL A.‐M. , FRATERNO M. , PASSEREL M. , KATCHBURIAN E. , BONGRAND P. , FOA C.
Published: August 2011

The ultrastructural distribution and quantification of calcium in mast cells prepared by anhydrous processing was investigated by energy‐filtering transmission electron microscopy (EFTEM) using a Zeiss 902 electron microscope. Optimal conditions for calcium detection were determined using inorganic (calcium phosphate) and organic (calcium‐loaded chelex beads) standards with known amounts of calcium. Electron energy‐loss spectroscopy (EELS) revealed calcium at the L2,3 edge and also at the M2,3 edge for all specimens examined. Comparison with X‐ray microanalysis confirmed the results obtained with EELS. Electron spectroscopic imaging (ESI) was applied for mapping calcium both in standards and in cells and we showed that mast cell granules were the main site of calcium localization. Although, results have shown that a combination of analytical techniques is required to obtain reliable results.
Morphometric assessment of surface conformation as an estimate of roughness
Authors: SILAGE D. A. , BARAN G. R.
Published: August 2011

One of the standard methods for assessing the roughness of a material subjected to wear uses the surface arithmetic means and root‐mean‐square deviation. However, these parameters often do not provide a qualitative assessment of the difference in materials worn under the same conditions of load and elapsed time. The profile and surface roughness parameters are frequently inconsistent. Such measurements are also required to determine the wear characteristics of various materials under different conditions. A morphometric assessment of wear characteristics, based on the surface area fraction of localized deviations in the surface texture and stress fractures, is provided, and clearly indicates the observed difference.
Method for the study of the three‐dimensional orientation of the nuclei of myocardial cells in fetal human heart by means of confocal scanning laser microscopy
Authors: USSON Y. , PARAZZA F. , JOUK P.‐S. , MICHALOWICZ G.
Published: August 2011

A series of three‐dimensional image analysis tools are used to measure the three‐dimensional orientation of nuclei of myocardial cells. Confocal scanning laser microscopy makes it possible to acquire series of sections up to 100 μm inside thick tissue sections. A mean orientation vector of unit length is calculated for each segmented nucleus. The global orientation statistics are obtained by calculating the vectorial sum of the nuclear unit vectors. The final orientation is expressed by a mean azimuth angle, an elevation angle and a measure of the angular homogeneity. The method is illustrated for two different regions of the myocardium (interventricular septum and papillary muscle) of a normal human fetal heart. This quantitative method will be used to assess and calibrate the information provided by polarized light microscopy.
Severe shrinkage of Microfil during tissue clearing with the Spalteholz technique
Authors: MØLLER J. , ROBERTSEN K. , HANSEN E. S.
Published: August 2011

The effect of two commonly used tissue clearing methods on the morphology of vascular casts formed by Microfil, a silicone rubber injection compound, was investigated. Microfil undergoes extreme shrinkage when the casted tissue is cleared by the alchohol‐methyl salicylate clearing technique. No shrinkage is observed when the alternative glycerin clearing method is used. The alcohol‐methyl salicylate clearing technique should be avoided in studies employing Microfil.
Influence of zero‐loss filtering on electron optical phase contrast
Authors: HIRSCH P. , REIMER L.
Published: August 2011

Complex scattering amplitudes are used to calculate the phase contrast of colloidal gold particles. Comparison of measurements of the phase contrast intensity at the centre of the gold particle as a function of defocus for unfiltered and zero‐loss filtered images demonstrates the increase in phase contrast achieved by zero‐loss filtering even for a thick carbon substrate film. The granulation of amorphous germanium films is measured by the spatial rms (root mean square) values of image intensity in a defocus series.
Image‐EELS: Simultaneous recording of multiple electron energy‐loss spectra from series of electron spectroscopic images
Authors: KÖRTJE K.‐H.
Published: August 2011

A new approach for element microanalysis with energy‐filtering transmission electron microscopy (EFTEM) is presented which was accomplished with the CEM 902 electron microscope (Zeiss, Germany). This method is called Image‐EELS, because it is a synthesis of electron energy‐loss spectroscopy (EELS) and electron spectroscopic imaging (ESI). Series of energy‐filtered images at increasing energy losses are recorded from one area with a TV camera. In a second step the intensity of selected regions in the image stack is measured with an image analysis system and plotted as a function of the energy loss. Thus many spectra from different objects can be calculated from one image series and compared with each other. The spatial resolution of EELS is considerably enhanced, the noise is decreased because many pixels from irregular objects are integrated, and the information from ESI can be analysed as a function of the energy loss.
Kinetics of intracellular Ca2+ concentration changes and cell contraction of electrically stimulated cardiomyocytes as analysed by automated digital‐imaging microscopy
Authors: SCHNEIDER H. , FALLERT M. , WACHSMUTH E. D.
Published: August 2011

Enzymatically disaggregated, electrically stimulated cardiomyocytes from adult rats were examined by television‐mediated vital microscopy for intracellular Ca2+ concentration and contractile activity. Using an inverted microscope in the epifluorescence mode, the Ca2+ signal was imaged with a low‐light‐level CCD camera and traced by means of the intracellular concentration of the fluorescent complex of Ca2+ with its indicator Fluo‐3. Using the transmitted‐light mode, cardiomyocytes that were not loaded were imaged with a conventional CCD camera with automatic gain control and traced by length measurements. Optical images of at least 40 cardiomyocytes per batch of cells from one heart were recorded in up to 20 microscopic fields of observation on videotape within 20 min. They were consecutively analysed by a personal computer installed with an image analysis card at a time‐resolution of 20 ms, employing a discrete convolution operation, filtering and threshold setting for fluorescence measurements, and contour description and vectorial analysis for length measurements. Frames of fluorescent images were corrected for the halo effect caused by the increase in the Ca2+‐dependent fluorescence signal after electrical stimulation. The cell contraction had to be measured in the transmission mode without Fluo‐3 due to the inhibition caused by the intracellular Fluo‐3. The following coefficients of variation (V) were determined: Vfluorescence < 0·033 and Vtransmission < 0·003 for the precision of measurement, and Vfluorescence < 0·05 and Vtransmission < 0·04 for the reproducibility. The system was validated with isoprenaline and ouabain as agents to modify the Ca2+‐signal and the contraction. The response of cardiomyocytes of various rats to electrical stimulation, with respect to amplitude and its time point, had a V < 0·08 for both the Ca2+‐signal and the contraction.
Particle‐surface interaction in thin vitrified films for cryo‐electron microscopy
Authors: CYRKLAFF M. , ROOS N. , GROSS H. , DUBOCHET J.
Published: August 2011

The concentration of particles in thin vitrified films of suspensions is described as a function of various parameters such as the type of particles observed, the time the sample is left on the grid and the effect of different washing procedures. The thin films are prepared for cryo‐electron microscopy by the classical, single‐side blotting method or by blotting both sides of the grid simultaneously. The two‐side blotting method gives the most faithful representation of the bulk solution. The single‐side blotting method results in particles preferentially adsorbing to the non‐blotted surface. This has the advantage that the concentration of particles in the thin vitrified film is higher than in the original suspension. The energy involved in adhesion of particles to the surface seems to be generally small. In most cases, it does not cause significant deformation of the particles or of the surface of the film. However, there are cases, as for example with lipid vesicles, where the particles are broken as a result of adsorption.

Since particles remain adsorbed to the air‐liquid interface, it is possible to wash or dialyse the solution directly on the grid with negligible loss of particles. This represents a very rapid and handy method for micro‐dialysis. A thin film is then formed by blotting the specimen and vitrified by rapid cooling.
Preparation of cultured airway smooth muscle for study of intracellular element concentrations by X‐ray microanalysis: Comparison of whole cells with cryosections
Authors: WARLEY A. , CRACKNELL K. P. B. , CAMMISH H. B. , TWORT C. H. C. , WARD J. P. T. , HIRST S. J.
Published: August 2011

Methods for growing and preparing smooth muscle cells, isolated from rabbit trachealis, for X‐ray microanalysis studies are presented. The cells are grown on Pioloformcovered gold grids supported on Thermanox coverslips. This provides a growth‐compatible substrate which is easy to handle and is easily incorporated into routine cell culture studies. The cells are analysed as whole mounts after removal of growth medium by washing, followed by cryofixation and freeze drying. The effects of different washing media (0·3msucrose, 0·15mammonium acetate and distilled water) on cytoplasmic elemental content are discussed. A method for growing the cells as monolayers and mounting the cryofixed monolayers for cryosectioning is also given. Comparison of elemental concentrations in the cytoplasm of distilled‐water washed cells with those of the cytoplasm of cryosectioned cells obtained from the same animal showed good agreement between values obtained from the two preparative procedures. These methods are therefore easily applied to the study of changes in intracellular element concentrations which may be important in understanding the mechanisms of proliferation which lead to increased airway smooth muscle mass in persistent severe asthma.
The measurement of intracellular antigens and DNA by multiparametric flow cytometry
Authors: CAMPLEJOHN R. S.
Published: August 2011

The aim of this paper is to provide a strategy for measuring intracellular antigens combined with DNA content in cells or nuclei. A series of protocols are included which enable the majority of such antigens to be labelled and further information is provided for cases in which the standard methods prove to be inadequate. The basic principles of cell permeabilization/fixation are described, thus explaining how methods can be divided into three basic categories: (a) alcohol fixation with or without detergent pretreatment; (b) paraformaldehyde fixation followed by permeabilization with alcohol or detergents; (c) permeabilization of unfixed cells. The preparation of nuclear suspensions from paraffinembedded material is described and the possibilities and problems of staining such suspensions for nuclear antigens are discussed. Examples of results obtained with the detailed protocols are given for staining with antibodies directed against proliferating cell nuclear antigen (PCNA), Ki‐67 antigen and Ki‐Sl antigen. Details of published studies of a variety of intracellular antigens are given in two tables. The power of multiparametric flow cytometry in the study of cell proliferation, differentiation and response of cells to damage is underlined.
Direct imaging in a water layer of human chromosome fibres composed of nucleosomes and their higher‐order structures by laser‐plasma X‐ray contact microscopy
Authors: KINJO Y. , SHINOHARA K. , ITO A. , NAKANO H. , WATANABE M. , HORIIKE Y. , KIKUCHI Y. , RICHARDSON M. C. , TANAKA K. A.
Published: August 2011

X‐ray contact microscopy with a 300‐ps‐duration laser‐plasma X‐ray source has been used to image hydrated human chromosomes. Clearly imaged are individual nucleosomes and their higher‐order particles (superbeads), elementary chromatin fibrils c. 30 nm in diameter and their higher‐order fibres of various sizes up to c. 120 nm in diameter. The results demonstrate that X‐ray microscopy is now capable of opening a new path of investigation into the detailed structures of hydrated chromosome fibres in their natural state.
High‐performance X‐ray detection in a new analytical electron microscope
Authors: LYMAN C. E. , GOLDSTEIN J. I. , WILLIAMS D. B. , ACKLAND D. W. , VON HARRACH S. , NICHOLLS A. W. , STATHAM P. J.
Published: August 2011

X‐ray detection by energy‐dispersive spectrometry in the analytical electron microscope (AEM) is often limited by low collected X‐ray intensity (P), modest peak‐to‐background (P/B) ratios, and limitations on total counting time (τ) due to specimen drift and contamination. A new AEM has been designed with maximization of P, P/B, and τ as the primary considerations. Maximization of P has been accomplished by employing a field‐emission electron gun, X‐ray detectors with high collection angles, high‐speed beam blanking to allow only one photon into the detector at a time, and simultaneous collection from two detectors. P/B has been maximized by reducing extraneous background signals generated at the specimen holder, the polepieces and the detector collimator. The maximum practical τ has been increased by reducing specimen contamination and employing electronic drift correction. Performance improvements have been measured using the NIST standard Cr thin film. The 0·3 steradian solid angle of X‐ray collection is the highest value available. The beam blanking scheme for X‐ray detection provides 3–4 times greater throughput of X‐rays at high count rates into a recorded spectrum than normal systems employing pulsepileup rejection circuits. Simultaneous X‐ray collection from two detectors allows the highest X‐ray intensity yet recorded to be collected from the NIST Cr thin film. The measured P/B of 6300 is the highest level recorded for an AEM. In addition to collected X‐ray intensity (cps/nA) and P/B measured on the standard Cr film, the product of these can be used as a figure‐of‐merit to evaluate instruments. Estimated minimum mass fraction (MMF) for Cr measured on the standard NIST Cr thin film is also proposed as a figure‐of‐merit for comparing X‐ray detection in AEMs. Determinations here of the MMF of Cr detectable show at least a threefold improvement over previous instruments.
The preparation of cryosections from plant tissue: An alternative method appropriate for secondary ion mass spectrometry studies of nutrient tracers and trace metals
Authors: LAZOF D. B. , GOLDSMITH J. K. G. , RUFTY T. W. , SUGGS C. , LINTON R. W.
Published: August 2011

A method involving cryostat sectioning (10 μm thickness) and freeze‐drying is presented for the preparation of plant tissue for microanalytical studies. The method is well suited for semi‐quantitative imaging by secondary ion mass spectrometry (SIMS) and offers significant advantages over bulk freeze‐dried or freeze‐substitution preparations. Segments of corn or soybean root (5 mm) are quench‐frozen, embedded externally, sectioned in a cryostat (10 μm), pressed onto ultrapure Si and slowly freeze‐dried. Images of these sections with secondary electron microscopy and SIMS indicated good morphological preservation. It was possible to section tissues of a wide developmental range, as well as roots varying sixfold in diameter. SIMS images are presented which demonstrate the ability to detect and localize nutrient tracers, such as Rb+, following brief exposures (10 min) to the intact plant. Likewise, a toxic metal (Al) was localized in root tissue after brief exposure (<1 day) of the intact plant root to micromolar external concentrations. Elemental redistribution during processing was minimal, as demonstrated most explicitly by the lack of movement of loosely bound Ca from the outer cell walls into the adjacent embedding material. Preservation of compositional differences between cellular content and cell wall was supported by a semi‐quantitative treatment of SIMS images.
In situ analysis of microbial consortia in activated sludge using fluorescently labelled, rRNA‐targeted oligonucleotide probes and confocal scanning laser microscopy
Authors: WAGNER M. , AßMUS B. , HARTMANN A. , HUTZLER P. , AMANN R.
Published: August 2011

Activated sludge flocs are complex consortia of various micro‐organisms. The community structures of samples taken from municipal sewage treatment plants were characterized using fluorescently labelled, 16S and 23S rRNA‐targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM). In comparison with conventional epifluorescence microscopy, CSLM considerably improved the capability to visualize directly the spatial distribution of defined bacterial populations inside the sludge flocs. Analyses could be performed at high resolution undisturbed by problems such as autofluorescence or limited spatial resolution in thick samples. In addition, CSLM was used to analyse some structural properties of paraformaldehyde‐fixed activated sludge flocs, such as floc size and homogeneity. Typical floc sizes were found to be in the range between 5 and 50 μm. Whereas most of the flocs were completely colonized by bacteria, there were also examples of flocs containing gas bubbles or particles in the interior.
Scanning interference and confocal microscopy
Authors: JUŠKAITIS R. , WILSON T.
Published: August 2011

The form of the interference term image in scanning confocal and scanning conventional interference microscopes is identical in all respects including optical sectioning. This observation is used to obtain confocal images and surface profiles from conventional scanning interference microscope images.
Time‐ and wavelength‐resolved spectroscopy in two‐photon excited fluorescence microscopy
Authors: ANDERSSON‐ENGELS S. , ROKAHR I. , CARLSSON J.
Published: August 2011

Two‐photon excited fluorescence spectroscopy has been performed at a microscopic scale in combination with normal, white‐light microscopy. This gave simultaneously a spectral resolution of 20 nm and a temporal resolution of 20 ps, from a volume element less than 5 μm in all three dimensions. The sample was excited with the light from a continuously mode‐locked Ti: sapphire laser that was focused on the sample in a fluorescence microscope. A polychromator and a streak‐camera were used for detection. The method has been used on tissue, plant and paper samples. It has also been demonstrated how substances naturally occurring in the samples can be identified from their spectroscopic properties and the spatial distribution of these substances can be observed.
A versatile 2π‐tilting device for fluorescence microscopes
Authors: BRADL J. , HAUSMANN M. , SCHNEIDER B. , RINKE B. , CREMER C.
Published: August 2011

A tilting device for biological specimens (rotation angle up to 2π), especially fluorescence‐labelled cell nuclei, was developed. It consists of a quartz glass capillary and a mounting adapter for the microscope stage. The applicability of the device was tested for several epifluorescence and confocal scanning laser fluorescence microscopes. The axis of rotation is perpendicular to the optical axis of the microscope. The capillary can be tilted around its axis at any desired angle or in equiangular steps. This can be done manually or by remote control using a stepping motor.

The three‐dimensional (3‐D) image‐forming properties of the capillary system were experimentally examined using an inverse confocal scanning laser microscope. The results were compared with measurements obtained from the same microscope with the standard stage for plane slides with cover glasses. The measured point spread function suggested that in spite of aberration effects, the optical arrangement used allows a gain in the 3‐D resolution by tilting the object.

A low‐cost, fully automated 3‐D imaging system was built on the basis of a conventional epifluorescence microscope with a cooled black‐and‐white CCD camera. The system was operated by a personal computer. The online visualization (‘movie’) of rotating objects indicates the feasibility of the system.
Continuous wave excitation two‐photon fluorescence microscopy
Authors: HÄNNINEN P. E. , SOINI E. , HELL S. W.
Published: August 2011

Two‐photon excitation fluorescence imaging is feasible with continuous wave lasers. Images of biological specimens are obtained by employing photon counting in conjunction with an increased recording time. The approach allows two‐photon three‐dimensional imaging of fluorescently labelled specimens with inexpensive lasers.
Studies of porphyrin‐containing specimens using an optical spectrometer connected to a confocal scanning laser microscope
Authors: TREPTE O. , ROKAHR I. , ANDERSSON‐ENGELS S. , CARLSSON K.
Published: August 2011

A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user‐defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277 K, thereby allowing integration times of up to 60 s. The spectral resolving power, λ/Δλ, ranges from 350 at λ = 400 nm to 100 at λ = 700 nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three‐dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings.

With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photo‐dynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and δ‐amino levulinic acid (ALA), a precursor to protoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound Photofrin, or protoporphyrin IX, respectively, is accumulated. For our study Wistar/Furth rats were injected either with Photofrin or with ALA 3–5 h before they were killed. The organs were removed directly after, and snap‐frozen in carbon dioxide ice with isopentane. No further staining or fixation procedures were adopted.
Modelling of inclined and curved surfaces in the reflection scanning acoustic microscope
Authors: WEISE W. , ZININ P. , BOSECK S.
Published: August 2011

An expression is derived for the output signal when an inclined plane surface is imaged by the reflection scanning acoustic microscope, which is modelled as a spherical transducer. This expression is applied to model non‐planar surfaces. The accuracy of this approach is tested for perfectly reflecting spherical surfaces. The influence of inclination on V(z) curves is considered when Rayleigh waves occur.
Imaging in the far‐red with electronic light microscopy: Requirements and limitations
Authors: CULLANDER C.
Published: August 2011

The acquisition of simultaneous dual confocal images with red and far‐red light has both advantages (e.g. lower autofluorescence) and limitations. An understanding of these requisites is necessary to acquire high‐quality images and to avoid the misinterpretation of experimental data. The poor detection of far‐red light mandates a high optical transfer efficiency for the system, thus the transmittance of the objective lens and its axial and lateral chromatic aberration in the far‐red are important factors for consideration. This technical note is an attempt to ‘demystify’ the process of filter set design for confocal microscopy by discussing the considerations that went into the construction of a filter set for use with the reagents cyanine 3.18 (Cy3) and cyanine 5.18 (Cy5), and thus to encourage users to look beyond the multi‐purpose designs available commercially. The 568‐nm laser line exciting Cy3 is at its emission maximum, which limits the collectable Cy3 fluorescence. High‐transmission optical filters with sharp band pass cutoffs are thus desirable for maximum light throughput. Light path mirror efficiency rapidly degrades above 700 nm, but the loss of this portion of the Cy5 emission spectrum is acceptable since the fluorophore is very bright, and these very long wavelengths are also likely to introduce aberration. While resolution is decreased with far‐red light, there is also greater penetration and less scattering, and it is thus possible to obtain high‐quality images from deeper within the specimen. Although only one make and model of confocal microscope (the Bio‐Rad MRC–600) is considered, similar considerations pertain to the design of filter sets for any confocal microscope that accommodates user‐installed filters.
Atomic force microscopy in the photochemistry of chalcones
Authors: KAUPP G.
Published: August 2011

The application of atomic force microscopy (AFM) to photodimerization of crystalline chalcones provides new insights into the detailed mechanisms of solid‐state reactions on the molecular level. Well‐directed long‐range transport phenomena are found which reach far beyond the crystal lattice distances. Reactions occur in the surface region where the light is absorbed. Characteristic features are built up that depend on crystal structure and crystal face. This could not be foreseen by previous theories based solely on a topochemical postulate/principle. There is now a much more intimate correlation of crystal structure with solid‐state reactivity and this is directly studied and proven experimentally by AFM. Even solid‐state reactions which are in opposition to topochemistry can be studied and understood on a molecular basis. The three‐dimensional resolution of undisturbed insulating surfaces which is obtained by AFM is not available by any other technique.
Enhanced retention of magnetic particles (e.g. microtomed sections) in a TEM
Authors: FURDANOWICZ W. A. , DOWNEY K. E.
Published: August 2011

An electron‐transparent layer of adhesive is used to restrain magnetic particles from being stripped off the support film by the magnetic field of an objective lens in a TEM.
In vivo determination of fibril orientation in plant cell walls with polarization CSLM
Authors: VERBELEN J.‐P. , STICKENS D.
Published: August 2011

Congo Red fluorescence is used to detect cellulose in the wall of plant cells. The orientation of the cellulose fibrils is determined by using polarized light for excitation. The absorption characteristics of Congo Red make this approach a method of choice for applications with any standard confocal scanning laser microscope (CSLM). The semiquantitative character of CSLM observations combined with the non‐toxicity of the stain allow a very fast and reliable assessment of cellulose orientation in the wall of living plant cells.
Confocal microscopy in the analysis of the etched nuclear particle tracks in polymers
Authors: JAKES J. , GAIS P. , SCHRAUBE H.
Published: August 2011

The possibility of the morphometric analysis of etched tracks, induced by protons and alpha particles in the organic polymer allyl diglycol carbonate (CR‐39), using the confocal scanning laser microscope (CSLM), was studied. The detectors were investigated in two groups of irradiation experiments, namely: (a) irradiated with mono‐energetic neutrons of energy 1–2 MeV, (b) exposed to the alpha radiation from 222Rn and its progeny. Both groups were irradiated at normal incidence. Radiation‐induced latent tracks were electrochemically etched, and their morphometric parameters were investigated in the reflection mode by using the 488‐nm spectral line of an argon ion laser. A constant number of up to 200 optical sections in Z‐scan mode was taken through each selected etched track at vertical spacings of 0·642 μm. Successive reconstructions of Z‐sections were used to determine the following parameters: the mean radius of the opening channel, the maximum diameter and the length of the track, and the angle of the track wall to the surface of the sample. The results show that tracks produced by alpha particles differ from those induced by protons. The radius of the opening channel of alpha‐particle‐induced tracks ranges from 7·9 to 11 μm, whereas for protons the same parameter ranges between 2·0 and 3·8 μm for a specific electrochemical etch procedure.
A method to compensate for light attenuation with depth in three‐dimensional DNA image cytometry using a confocal scanning laser microscope
Authors: LILJEBORG A. , CZADER M. , PORWIT A.
Published: August 2011

A method to compensate for attenuation of detected light with increased depth of the collected optical section, and its application in three‐dimensional (3‐D) DNA image cytometry is described. The method is based on studying the stack of 2‐D histograms that can be formed from each consecutive pair of sections in a stack of optical serial sections. An attenuation factor is calculated interactively and a new compensated section series is computed. Formalin‐fixed paraffin‐embedded rat tissue was stained with propidium iodide. Each cell nucleus is extracted by thresholding and its total intensity is calculated. The coefficient of variation (CV) of the total intensity of all cells in each stack is computed. For comparison the CV of the same cells is computed in the uncompensated stacks. This study shows a significantly lower CV for the compensated data, thus contributing to the accuracy of DNA quantification in 3‐D DNA image cytometry.
Comparison of three‐dimensional imaging properties between two‐photon and single‐photon fluorescence microscopy
Authors: GU MIN , SHEPPARD C. J. R.
Published: August 2011

The imaging performance in single‐photon (1‐p) and two‐photon (2‐p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three‐dimensional (3‐D) point spread function and the 3‐D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2‐p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3‐D optical transfer function has the strongest response along the axial direction.
Double‐pulse fluorescence lifetime imaging in confocal microscopy
Authors: MÜLLER M. , GHAUHARALI R. , VISSCHER K. , BRAKENHOFF G.
Published: August 2011

A theoretical analysis of a new technique for fluorescence lifetime measurement, relying on (near steady state) excitation with short optical pulses, is presented. Application of the technique to confocal microscopy enables local determination of the fluorescence lifetime, which is a parameter sensitive to the local environment of fluorescent probe molecules in biological samples. The novel technique provides high time resolution, since it relies on the laser pulse duration, rather than on electronic gating techniques, and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. The principle of the technique is discussed within a theoretical framework. The sensitivity of the technique is analysed, taking into account: photodegradation, the effect of the laser repetition rate and the effect of non‐steady‐state excitation. The features of the technique are compared to more conventional methods for fluorescence lifetime determination.
Influence of matrix precursors on the microstructure and mechanical properties of C/C composites
Authors: FIGUEIRAS A. , FERNÁNDEZ J. J. , GRANDA M. , BERMEJO J. , CASAL E. , MENÉNDEZ R.
Published: August 2011

The microstructure and properties of uni‐directional pitch‐based carbon‐carbon composites are explained in terms of the chemical composition of pitch precursors. Pitches are characterized by standard procedures (elemental analysis, softening point and solubility tests), extrography which is a simple and rapid silica gel adsorption chromatographic technique, Fourier transform infra‐red and gas chromatography of the toluene‐soluble fraction. Pitch pyrolysis behaviour is monitored by hot‐stage microscopy. The main microstructural features of uni‐directional composites from pitches and commercial PAN‐based carbon fibres are determined by light microscopy and scanning electron microscopy.
Processing and properties of Al2O3/SiC nanocomposites
Authors: BORSA C. E. , JIAO S. , TODD R. I. , BROOK R. J.
Published: August 2011

Alumina/SiC nanocomposites were produced by mechanical mixture of commercial powders. The preparation steps involved the vigorous mixing of the powders and drying under conditions where the homogeneous mixture was kept stable. Pressureless sintering of die‐pressed powders achieved reasonable densities (∼97% theoretical density) for 2·5wt% of SiC on sintering at 2073 K. Higher SiC contents strongly reduced the sintered density. The use of a more reactive alumina (finer matrix powder) gave similar results. Hot pressing at 1973 K/1 h/25 MPa produced high‐density materials for SiC contents as high as 20 wt%. Transmission and scanning electron microscopy analysis showed that the SiC particles were well distributed and were situated both inside the grains and on the grain boundaries of the alumina matrix. The SiC strongly inhibited grain growth in the matrix in keeping with the Zener model. The bend strength increased as the SiC content increased, a result partly explained by the grain size refinement. The strength improvement of 20% over monolithic was explained in terms of the change to an intergranular fracture mode.
Spatially resolved electron energy‐loss studies of metal–ceramic interfaces in transition metal/alumina cermets
Authors: BRYDSON R. , MÜLLEJANS H. , BRULEY J. , TRUSTY P. A. , SUN X. , YEOMANS J. A. , RÜHLE M.
Published: August 2011

Composites consisting of an alumina matrix and 20 vol.% transition metal (Ni or Fe) particles, prepared by hot pressing powder blends, have been studied using spatially resolved transmission electron energy‐loss spectroscopy (EELS), and, to a lesser extent, by high‐resolution electron microscopy (HREM). Particular attention was paid to the elucidation of the chemical bonding mechanisms at the metal‐ceramic interface; EELS spectra from interfacial regions being obtained via a spatial difference technique. From both qualitative and quantitative interpretation of EELS near‐edge structures, as well as observed HREM images, the data appear to be consistent with the presence of an Al‐terminated alumina at the interface and the formation of direct transition metal – aluminium bonds in Al(O3M) (M = Ni or Fe) tetrahedral units, possibly as a result of the dissolution and interfacial reprecipitation of Al during processing. These results correlate well with similar model studies on diffusion‐bonded Nb/Al2O3 interfaces and may, in the light of recent theoretical electronic structure calculations, have implications for the resultant interfacial bond strength in such materials.
Identification and surface structure of crystalline cellulose studied by atomic force microscopy
Authors: KUUTTI L. , PELTONEN J. , PERE J. , TELEMAN O.
Published: August 2011

A combination of molecular modelling and atomic force microscopy (AFM) techniques was used to study the surface structure of crystalline cellulose. Two‐dimensional Fourier analysis of the AFM raw data gave crystal parameters as well as a highly filtered inverse‐transformed image. Molecular modelling was used to generate Connolly surfaces based on electron diffraction data for crystalline cellulose. The modelled surfaces were used to interpret the experimental AFM images. Monoclinic () crystal faces were identified. The method used enables the structural analysis of cellulose surfaces at the molecular level, where all biological processes involving cellulose take place.
A scanning near‐field optical microscope having scanning electron tunnelling microscope capability using a single metallic probe tip
Authors: INOUYE Y. , KAWATA S.
Published: August 2011

A new microscope system that has the combined capabilities of a scanning near‐field optical microscope (SNOM) and a scanning tunnelling microscope (STM) is described. This is achieved with the use of a single metallic probe tip. The distance between the probe tip and the sample surface is regulated by keeping the tunnelling current constant. In this mode of operation, information about the optical properties of the sample, such as its refractive index distribution and absorption characteristics, can be disassociated from the information describing its surface structure. Details of the surface structure can be studied at resolutions smaller than the illumination wavelength. The performance of the microscope is evaluated by analysing a grating sample that was made by coating a glass substrate with gold. The results are then compared with the corresponding SNOM and STM images of the grating.