A synchrotron X-ray study of the changes occurring in the corneal stroma during processing for electron microscopy

Using a high-intensity synchrotron X-ray source, the structural changes occurring in the corneal stroma were monitored during each stage of several different processing runs for the transmission electron microscope (TEM) and scanning electron microscope (SEM). The parameters studied were interfibrillar spacing, intermolecular spacing, D-periodicity and fibril diameter. The processing schedule that produced the least changes in spacings for TEM specimens involved extended fixation in glutaraldehyde followed by low-temperature embedding in Lowicryl K4M resin. However, interfibrillar material was better preserved after embedding in LR White resin or Nanoplast. Almost every processing stage for electron microscopy produced significant changes in one or more structural parameters in the cornea. Glutaraldehyde fixation significantly increased the intermolecular spacings, while resin infiltration and resin polymerization each resulted in shrinkage of all the spacings monitored. Critical-point drying for SEM specimens resulted in considerable shrinkage in all three spacings, but was still preferable to air drying, which caused reduction in the order of the fibril packing, resulting in loss of the interfibrillar X-ray pattern. Perhaps the most drastic effect was caused by post-fixation in osmium tetroxide, which resulted in loss of the intermolecular pattern, and also increased the amount of shrinkage in the interfibrillar spacings and the D-periodicity which occurred during later stages of processing.     

X-ray microscopy: experimental results with the Göttingen X-ray microscope at the electron storage ring BESSY in Berlin

An X-ray microscope and X-ray microscopy experiments with biological specimens are described. The experiments have been performed with a resolution of about 0.05 μm using the synchrotron radiation of the electron storage ring BESSY.     

Transmission electron microscopy staining methods for the cortex of human hair: a modified osmium method and comparison with other stains

For wool, superior staining of a wide range of ultrastructural components is achieved by en bloc treatment of fibres with a chemical reductant followed by osmium tetroxide. For human scalp hair, although staining quality is similar, the penetration of reagents is poor, resulting in large parts of the fibre cortex remaining unstained. Here we describe a modification to the reduction-osmication method in which reagents penetrate through a cut fibre end, allowing visualization of a wide range of features across the cortex. We compare the staining quality, artefacts and range of structure rendered visible using transmission electron microscopy for en bloc reduction-osmication to other staining alternatives including en bloc silver nitrate and section stains based on uranyl acetate and lead citrate, phosphotungstic acid, potassium permanganate, ammoniacal silver nitrate and some combinations of these stains. The effects of hair-care treatments are briefly examined.     

多层螺旋CT及多维重建技术对隐匿性肋骨骨折的诊断价值探讨

目的 :考察多层螺旋CT(MSCT)联合多维重建技术对隐匿性肋骨骨折的临床诊断价值,为该类疾病临床诊断提供参考。方法 :以我院2012年7月至2014年2月间收治的73例隐匿性肋骨骨折患者为研究对象,使用MSCT联合VR、SSD、CRP等多种重建技术对患者进行检查,统计并分析影像学检查结果。结果:73例患者经MSCT检查确诊隐匿性肋骨骨折107处,骨折多发于肋骨角(59.81%)且以非完全线性骨折为主(91.59%)。所用多维重建技术的诊断精度依次为:CRP(100.0%)>MPR(97.19%)>SSD(85.89%)>VR(78.50%)>MIP(27.88%)。结论 :MSCT联合多维重建技术对隐匿性肋骨骨折具有较为明确的诊断价值,MSCT与CRP/MPR及VR等联用可获得较为理想的诊断率。     

A method for estimating the fibre length in fibre‐PLA composites

Wood pulp fibres are an important component of environmentally sound and renewable fibre‐reinforced composite materials. The high aspect ratio of pulp fibres is an essential property with respect to the mechanical properties a given composite material can achieve. The length of pulp fibres is affected by composite processing operations. This thus emphasizes the importance of assessing the pulp fibre length and how this may be affected by a given process for manufacturing composites. In this work a new method for measuring the length distribution of fibres and fibre fragments has been developed. The method is based on; (i) dissolving the composites, (ii) preparing the fibres for image acquisition and (iii) image analysis of the resulting fibre structures. The image analysis part is relatively simple to implement and is based on images acquired with a desktop scanner and a new ImageJ plugin. The quantification of fibre length has demonstrated the fibre shortening effect because of an extrusion process and subsequent injection moulding. Fibres with original lengths of >1 mm where shortened to fibre fragments with length of <200 μm. The shortening seems to be affected by the number of times the fibres have passed through the extruder, the amount of chain extender and the fraction of fibres in the polymer matrix.     

Automatic image acquisition, calibration and montage assembly for biological X-ray microscopy

We describe a system for the automatic acquisition and processing of digital images in a high-resolution X-ray microscope, including the formation of large-field high-resolution image montages. A computer-controlled sample positioning stage provides approximate coordinates for each high-resolution subimage. Individual subimages are corrected to compensate for time-varying, non-uniform illumination and CCD-related artefacts. They are then automatically assembled into a montage. The montage assembly algorithm is designed to use the overlap between each subimage and multiple neighbours to improve the performance of the registration step and the fidelity of the result. This is accomplished by explicit use of recorded stage positions, optimized ordering of subimage insertion, and registration of subimages to the developing montage. Using this procedure registration errors are below the resolution limit of the microscope (43 nm). The image produced is a seamless, large-field montage at full resolution, assembled automatically without human intervention. Beyond this, it is also an accurate X-ray transmission map that allows the quantitative measurement of anatomical and chemical features of the sample. Applying these tools to a biological problem, we have conducted the largest X-ray microscopical study to date.     

Visualizing soft tissue in the mammalian cochlea with coherent hard X-rays

This paper concerns an important aspect of current developments in medical and biological imaging: the possibility for imaging soft tissue at relatively high resolution in the micrometer range or better, without tedious and/or entirely destructive sample preparation. Structures with low absorption contrast have been visualized using in-line phase contrast imaging. The experiments have been performed at the Advanced Photon Source, a third generation source of synchrotron radiation. The source provides highly coherent X-ray radiation with high photon flux (>10(14) photons/s) at high photon energies (5-70 keV). Thick gerbil cochlear slices have been imaged and were compared with those obtained by light microscopy. Furthermore, intact gerbil cochleae have been imaged to identify the soft tissue structures involved in the hearing process. The present experimental approach was essential for visualizing the inner ear structures involved in the hearing process in an intact cochlea.     

Imaging applications of synchrotron X-ray phase-contrast microtomography in biological morphology and biomaterials science. I. General aspects of the technique and its advantages in the analysis of millimetre-sized arthropod structure

Synchrotron‐generated X‐rays provide scientists with a multitude of investigative techniques well suited for the analysis of the composition and structure of all types of materials and specimens. Here, we describe the properties of synchrotron‐generated X‐rays and the advantages that they provide for qualitative morphological research of millimetre‐sized biological organisms and biomaterials. Case studies of the anatomy of insect heads, of whole microarthropods and of the three‐dimensional reconstruction of the cuticular tendons of jumping beetles, all performed at the beamline ID19 of the European Synchrotron Radiation Facility (ESRF), are presented to illustrate the techniques of phase‐contrast tomography available for anatomical and structural investigations. Various sample preparation techniques are described and compared and experimental settings that we have found to be particularly successful are given. On comparing the strengths and weaknesses of the technique with traditional histological thin sectioning, we conclude that synchrotron radiation microtomography has a great potential in biological microanatomy.     

A new sample preparation method for biological soft X-ray microscopy: nitrogen-based contrast and radiation tolerance properties of glycol methacrylate-embedded and sectioned tissue

We describe the preparation of a biological tissue for imaging in a transmission soft X-ray microscope. Sections of exocrine pancreas embedded in glycol methacrylate polymer, an embedding medium widely used in visible light and electron microscopy, were examined. Contrast was based primarily on the nitrogen content of the tissue, and dual-wavelength imaging at the nitrogen K-shell absorption edge was used to map the distribution and provide quantitative densitometry of both the protein and embedding matrix components of the sample. The measurements were calibrated by obtaining the absorption spectrum of protein near the nitrogen edge. The contrast was consistent and reproducible, making possible the first large-scale X-ray microscopic study on sections of plastic-embedded soft tissue. At radiation doses of up to 10⁸ Gray, much more than required for routine imaging, no distortion and little mass loss were observed. This sample preparation method should permit routine imaging of tissues in X-ray microscopes, previously a difficult task, as well as multimodal imaging (using visible light, X-ray, electron, and scanned probe microscopies) on the same sample.     

A computer simulation platform for the estimation of measurement uncertainties in dimensional X-ray computed tomography

The knowledge of measurement uncertainty is of great importance in conformance testing in production. The tolerance limit for production must be reduced by the amounts of measurement uncertainty to ensure that the parts are in fact within the tolerance. Over the last 5 years, industrial X-ray computed tomography (CT) has become an important technology for dimensional quality control. In this paper a computer simulation platform is presented which is able to investigate error sources in dimensional CT measurements. The typical workflow in industrial CT metrology is described and methods for estimating measurement uncertainties are briefly discussed. As we will show, the developed virtual CT (VCT) simulator can be adapted to various scanner systems, providing realistic CT data. Using the Monte Carlo method (MCM), measurement uncertainties for a given measuring task can be estimated, taking into account the main error sources for the measurement. This method has the potential to deal with all kinds of systematic and random errors that influence a dimensional CT measurement. A case study demonstrates the practical application of the VCT simulator using numerically generated CT data and statistical evaluation methods.     

On the Application of Six-Port Technology (SPT) in Ultrasonic Imaging for Medical Diagnosis

In this paper, an introduction of six-port technology to ultrasonic imaging for medical diagnosis is presented for the first time. Calculations are made to explore the possibilities and advantages of this application. Analysis shows that the introduction of six-port technology for medical ultrasonic imaging to detect the phase information of the reflected signal will improve the measurement accuracy and simplify the instrumentation of imaging technologies used in medical diagnosis.     

Validation of terrestrial laser scanning and photogrammetry techniques for the measurement of vertical underclearance and beam geometry in structural inspection of bridges

This research shows a novel methodology based on the application of terrestrial laser scanning and photogrammetry techniques as auxiliary metric tools for bridge inspections procedures. These methodologies are validated throughout a case study where the minimum underclearance and the overall geometry of a prestressed concrete beam are obtained. Data obtained are compared with high accurate measurements provided by a total station. Results show a good agreement for beam geometry with high statistical correlation coefficients. Minimum vertical underclearance also shows a good agreement for all the systems, which appears good enough due the metric tolerances required in these inspection works.     

The application of electron spectroscopic imaging for quantification of the area fractions of calcium-containing precipitates in nervous tissue

Energy-filtering transmission electron microscopy has been applied to the quantification of area fractions of calcium-containing cytochemical reaction products in central nervous tissue and the retina of fish. The method of electron spectroscopic imaging using electrons with an energy loss of 250 eV produces images with a very high, structure-sensitive contrast. This is a suitable imaging condition for the reliable detection of reaction products and structural details in unstained ultrathin sections. The images were recorded with a sensitive TV camera and evaluated with the integrated digital image-analysis system of the Zeiss CEM 902 energy-filtering electron microscope. An empirical procedure was developed which objectively detects reaction products and calculates characteristic values, taking into account different staining intensities. This new and sensitive method enabled an assessment to be made of the influence of temperature and light adaptation on cytochemically detectable calcium in nervous tissue of fish. Higher amounts of calcium-containing reaction product were detected in synaptic clefts of the optic tectum in warm-adapted fish than in cold-adapted fish. In synaptic vesicles of photoreceptor cells in the fish retina, higher amounts of reaction product were found in dark-adapted fish than in light-adapted fish.     

Integrated methodology for the evaluation of cleaning effectiveness in two Russian icons (16th–17th centuries)

This article covers a methodology for evaluating the effectiveness of cleaning two Russian icons. The icons belong to a group of five from the same iconographic school, dating from the 16th to 17th centuries. An integrated and complementary approach to varnish and overpaint removal involved microscopic techniques (optical and scanning electron microscopy) and colorimetry (CIE L*a*b* system). The materials and techniques used in these icons have been characterized previously. Cleaning revealed extensive overpainting that had not only dramatically changed the original appearance, but also the meaning and attribution of one of the two icons. The analyses carried out were useful in determining the extent of the overpainting and led to a better assessment of the results and effectiveness of the restoration. Microsc. Res. Tech. 73:752–760, 2010. © 2009 Wiley‐Liss, Inc.     

A new analytical method for characterising the bonding environment at rough interfaces in high-k gate stacks using electron energy loss spectroscopy

Determining the bonding environment at a rough interface, using for example the near-edge fine structure in electron energy loss spectroscopy (EELS), is problematic since the measurement contains information from the interface and surrounding matrix phase. Here we present a novel analytical method for determining the interfacial EELS difference spectrum (with respect to the matrix phase) from a rough interface of unknown geometry, which, unlike multiple linear least squares (MLLS) fitting, does not require the use of reference spectra from suitable standards. The method is based on analysing a series of EELS spectra with variable interface to matrix volume fraction and, as an example, is applied to a TiN/poly-Si interface containing oxygen in a HfO₂-based, high-k dielectric gate stack. A silicon oxynitride layer was detected at the interface consistent with previous results based on MLLS fitting.     

Optimizing imaging parameters for the separation of multiple labels in a fluorescence image

A theoretical analysis is presented on how to separate the contributions from individual, simultaneously present fluorophores in a spectrally resolved image. Equations are derived that allow the calculation of the signal‐to‐noise ratio of the estimates for such contributions, given the spectral information on the individual fluorophores, the excitation wavelengths and intensities, and the number and widths of the spectral detection channels. We then ask how such imaging parameters have to be chosen for optimal fluorophore separation. We optimize the signal‐to‐noise ratio or optimize a newly defined ‘figure of merit’, which is a measure of efficiency in the use of emitted photons. The influence of photobleaching on the resolution and on the choice of imaging parameters is discussed, as well as the additional resolution gained by including fluorescence lifetime information. A surprisingly small number of spectral channels are required for an almost optimal resolution, if the borders of these channels are optimally selected. The detailed consideration of photobleaching is found to be essential, whenever there is significant bleaching. Consideration of fluorescence lifetime information (in addition to spectral information) improves results, particularly when lifetimes differ by more than a factor of two.     

Generalization of the polar representation in time domain fluorescence lifetime imaging microscopy for biological applications: practical implementation

The polar representation or phasor, which provides a fast and visual indication on the number of exponentials present in the intensity decay of the fluorescence lifetime images is increasingly used in time domain fluorescence lifetime imaging microscopy experiments. The calculations of the polar coordinates in time domain fluorescence lifetime imaging microscopy experiments involve several experimental parameters (e.g. instrumental response function, background, angular frequency, number of temporal channels) whose role has not been exhaustively investigated. Here, we study theoretically, computationally and experimentally the influence of each parameter on the polar calculations and suggest parameter optimization for minimizing errors. We identify several sources of mistakes that may occur in the calculations of the polar coordinates and propose adapted corrections to compensate for them. For instance, we demonstrate that the numerical integration method employed for integrals calculations may induce errors when the number of temporal channels is low. We report theoretical generalized expressions to compensate for these deviations and conserve the semicircle integrity, facilitating the comparison between fluorescence lifetime imaging microscopy images acquired with distinct channels number. These theoretical generalized expressions were finally corroborated with both Monte Carlo simulations and experiments.