Phase Contrast Mode Research Articles

Nature of laminations and mineralization in rhinoceros enamel using histology and X-ray synchrotron microtomography: Potential implications for palaeoenvironmental isotopic studies

Incremental features of rhinoceros molar enamel were studied quantitatively on histological slices. The different kinds of incremental features of enamel were related to one another and counted to determine the rate and duration of crown formation and to clarify the status of the laminations. The mineralization and the microstructure of the enamel were examined in a rhinoceros molar germ using X-ray synchrotron microtomography respectively using absorption and phase contrast mode at different levels of resolution, from 45.71 μm for the largest scale up to 0.28 μm for the finest one. The different microtomographic data were combined to obtain a multiscale integrated overview of the microstructure and mineralization in order to understand better the relationship between these two aspects. Additional human and herbivorous mammal teeth were examined qualitatively using histological sectioning to obtain a better understanding of laminations in mammal tooth enamel. Laminations, regularly spaced features parallel to the Retzius lines, represent regular isochrons of enamel deposition. This study shows that laminations and cross-striations are equivalent daily features in enamel of rhinoceros, humans, and probably of middle sized and large herbivorous mammals in general. Laminations are shown to be three-dimensional alignments of cross-striations. In addition to the fact that cross-striations are visible only in prismatic enamel, observation of histological slices, and high resolution phase contrast X-ray synchrotron microtomography demonstrate three main parameters influencing the dominant aspect of daily features in rhinoceros and human enamel: the angle between prisms and Retzius lines, the enamel deposition rate and the thickness of the histological slices. Given their daily nature, laminations can be used to determine the crown formation time of middle sized and big herbivorous mammals. Quantitative study of the enamel mineralization in the rhinoceros germ fragment using X-ray synchrotron microtomography suggests that the innermost 20 μm of the enamel near the enamel–dentin junction are highly mineralised (77–90% of the final enamel mineralization) during, or just after the deposition of the corresponding enamel matrix. Integrated microtomographic study shows that this area corresponds to the thin aprismatic enamel layer at the EDJ level plus a thin layer of radial enamel directly adjacent. It appears that, in some parts of the enamel, there may be a considerable time lag between matrix deposition and full maturation, which could have important implications for geochemical studies. This study reinforces the previously suggested hypothesis that isotopic measurements derived from the innermost enamel may represent the period most closely related to matrix secretion. By using information derived from incremental features (laminations and Retzius lines) and restricting the isotopic measurements to the innermost part of the enamel, precise temporal calibration of isotopic signals should be possible, and the complication of time averaging due to enamel maturation could be avoided or strongly reduced.

The determination of firing distance applying a microscopic quantitative method and confocal laser scanning microscopy for detection of gunshot residue particles

In this study, we applied a microscopic quantitative method based on the use of sodium rhodizonate to verify the presence of residues and their distribution on the cutis of gunshot wounds. A total of 250 skin samples were selected from cases in which the manner of death (accidental, suicide, and homicide) and the shooting distance could be reliably determined. The samples were examined under a light microscope, in transmitted bright field illumination and phase contrast mode, and with confocal laser scanning microscopy. In all skin specimens the area of each histological section was directly measured by an image analysis system. Both the number and the size of powder particles were measured. The distribution of gunshot residues (GSR) in the epidermal and subepidermal layers was also analyzed. The evaluation of the microscopic entrance wounds demonstrated different findings related to the range of fire. The data derived from the evaluation of both macroscopic and microscopic features demonstrated that the amount and the spatial distribution of GSR deposits in the skin surrounding entrance wounds strictly correlate with shooting distance.

A high-resolution MACROscope with differential phase contrast, transmitted light, confocal fluorescence, and hyperspectral capabilities for large-area tissue imaging

Recent advances in imaging technology have contributed greatly to biological science. Confocal fluorescence microscopes can acquire two-dimensional and three-dimensional images of biological samples such as live or fixed cells and tissues. Specimens that are large (e.g., a 10 mm/spl times/10 mm tissue section) and overfill the field of view (FOV) of typical microscope objectives require the use of image tiling to cover the entire specimen. This can be time consuming and cause artifacts in the composite image. The MACROscope system (Biomedical Photometrics Inc., Waterloo, ON, Canada) is a confocal device with a 22 mm/spl times/70 mm FOV designed for imaging large tissue sections in a single frame. The prototype demonstrated here can obtain images in reflected, transmitted, fluorescence, phase contrast, and hyperspectral modes. The new spectral imaging mode is characterized with a series of test targets, and sampled spectra are compared to a commercial spectrometer. Fluorescence images of human SiHa tumor xenografts stained with CD31-Cy3, showing blood vessel location, and EF5-Cy5, showing areas of tissue hypoxia, were collected. Differential phase contrast images of the same section, as well as human epithelial cells, were recorded to assess the phase contrast mode. Additionally, fluorescence images of Cytokeratin-Cy3 stained squamous cell carcinoma tissue sections were captured. Finally, red, green, blue transmitted light images of human tongue were obtained. This new device avoids the need for image tiling and provides simultaneous imaging of multiple fluorescently labeled tissue-specific markers in large biological samples. This enables time- and cost-efficient imaging of (immuno)histopathological samples. This device may also serve in the imaging of high-throughput DNA and tissue arrays.

Evaluation of Al/SiC Wetting Characteristics in Functionally Graded Metal-Matrix Composites by Synchrotron Radiation Microtomography

The concept of functionally graded material (FGM), may be considered as a model particularly interesting to be applied in components for the automotive industry, if reliability and cost can be controlled in an advantageous way. In fact, coupling superior superficial wear resistance with a significant bulk toughness, without compromising important weight savings, by using Al-Si metal matrix composites (MMC’s) selectively reinforced at the surface with SiC particles, is likely to be considered as a innovative advance to that industrial field, if adequate production techniques are developed. Casting under a centrifugal force may well be considered as one of the most effective methods for processing Al-based FGM s. A primary problem to be faced when producing MMC s reinforced with ceramic particles is related with the imperfect wetting of the ceramic particles by the molten matrix alloy. A first consequence of defective wetting may be the formation of ceramic-ceramic, ceramic-gas and/or metal-gas interfaces, instead of the desired metal-ceramic interface. Secondly, wetting phenomena play an essential role regarding the physical, chemical and mechanical characteristics of the metal/ceramic interface. A general consequence of these aspects may be related with the degradation of the material properties, be it mechanical, chemical, or thermal in nature. The present work refers to an X-ray microtomography experiment aiming at the elucidation of some aspects regarding particle distribution in SiCp-reinforced functionally graded aluminium composites. Precursor composites were produced by rheocasting. These were then molten and centrifugally cast in order to produce the FGM composites. From these, small cylindrical samples were extracted and observed by X-ray microtomography at the European Synchrotron Radiation Facility (ESRF). The 3D tomographic images were obtained in edge-detection mode (phase-contrast mode), and an adequate segmentation procedure was employed to isolate the pores and SiC particles from the Al matrix. This has allowed a study of the relations between the matrix, the SiC particles, and locally intervening porosities of varying shapes, aiming at a better understanding of the mechanisms involved.

X-ray microscopy in Zernike phase contrast mode at 4 keV photon energy with 60 nm resolution

We report on x-ray microscopy of advanced microelectronic devices imaged in Zernike-type phase contrast mode at 4 keV photon energy. Fresnel zone plates were used as high resolution x-ray objectives providing 60 nm spatial resolution. Integrated circuit copper interconnect structures were imaged in positive as well as negative phase contrast. In both cases the phase contrast in the x-ray images is about five times higher than the pure absorption contrast.

Electrorelease of Escherichia coli nucleoids.

Bacterial chromosome is assembled and folded into one or several nucleoids, depending on the metabolic status of the cell. Development of reliable nucleoid isolation protocols has always been an objective for researchers. A rapid and reproducible procedure for isolation of E. coli nucleoids is described here, while the cell envelope is maintained. Membrane dispersions and vesicles were prepared by lysozyme-EDTA treatment with subsequent rupture of the spheroplasts by electric field. Under these conditions the yield of electroreleased nucleoids was around 90%. The extent of DNA-envelope contacts was determined by light microscopy employing phase contrast and fluorescence modes.

Surface topography, corrosion and microhardness of nitrogen-diffusion-hardened titanium alloy.

Mechanical-electrochemical interactions accelerate corrosion in mixed-metal modular hip prostheses. These interactions can be reduced by improving the modular component machining tolerances or by improving the resistance of the components to scratch or fretting damage. Wrought cobalt-alloy (CoCrMo) is known to have better tribological properties compared to the titanium alloy (Ti64). Thus, improving the tribological properties of this mixed-metal interface should center around improving the tribological properties of the Ti64 alloy. This study used scanning probe microscopy (contact, tapping and phase contrast mode), scanning electron microscopy, corrosion testing, and microhardness testing to determine the effect of a nitrogen-diffusion hardening process on the surface morphology, electrochemistry and surface hardness of the Ti64 alloy. The nitrogen-diffusion-hardened titanium alloy samples (N-Ti64) had a more pronounced grain structure, more nodular surface, and significantly (P<0.01) higher mean roughness values than the control-Ti64 samples. The N-Ti64 samples also exhibited at least equivalent corrosion behavior and a definite increase in surface hardness compared to the control Ti64 samples. The equivalent corrosion behavior and improved surface hardness indicate the potential for N-Ti64 samples to resist similar and mixed-metal scratch and fretting damage. The use of N-Ti64 as opposed to control-Ti64 may therefore reduce the occurrence of mechanical-electrochemical degradation in mixed-metal modular total hip prostheses.

Lorentz And Interference Electron Microscopy On A Scanning Tem

Magnetic imaging can be performed both on conventional (projection) and scanning TEM (CTEM/STEM). The CTEM-based magnetic imaging, classified into Fresnel, Foucault, holography, and other modes, has proven successful by now in observing various magnetic objects, and has accordingly been well documented elsewhere. The STEMbased one has emerged relatively recently prompted by the rapid growth of current STEM technology.The most ubiquitous implementation of Lorentz microscopy on a STEM is the differential phase contrast (DPC) mode. Each scanning signal obtained in the DPC mode is linear to Lorentz deflection, thus reflects the magnitude of an in-plane component of magnetic induction integrated along the optical axis(Fig.l). The signals therefore suffice to generate integrated in-plane magnetic induction maps(Fig.2). Furthermore, by making use of the fact that the Lorentz deflection distribution across the image plane forms an irrotational (vortex-free) 2D vector field, a pertinent numerical image processing yields a function equivalent to the electron phase function representing magnetically distorted electron wavefront.

Numerical removal of ring artifacts in microtomography

Defective and insufficient calibrated detector elements introduce ring or half-circle artifacts in microtomographic image reconstructions. A computationally efficient numerical filter is presented which suppresses these defects by taking advantage of the particular appearance of ring artifacts in the Fourier transforms of the recorded sinograms. The performance of the filter is demonstrated on experimental data taken with high-energy synchrotron radiation in phase-contrast (outline) mode.

New applications in studies of waterborne coatings by atomic force microscopy

Atomic force microscopy (AFM) is rapidly emerging as an important tool for coatings characterization. We report several new applications of AFM of particular value to the development of improved waterborne coatings systems. First, an AFM method was developed to quantitatively assess the extent of coalescence and film formation for latex films by measurement of particle number density of protruding (uncoalesced) particles in dried coatings. Second, the use of topographic imaging to evaluate environmental (temperature) effects on film formation was investigated for a waterborne latex system. Finally, specular gloss of waterborne epoxy coatings was studied by AFM and optical measurements, and topographic features analyzed using power spectral density calculations were found to correlate with optical gloss measurements. Mechanisms for gloss reduction over time (particularly in early pot life coatings) were elucidated in the studies. Further applications in coatings studies will be driven by the development of new modes of AFM (friction force, force modulation, and phase contrast) that can be used to map mechanical properties (friction, stiffness, and adhesion) while simultaneously imaging topography. Examples of the use of the phase contrast mode to identify chemically different domains in early pot life waterborne epoxy coatings are presented.

Nanometer-scale structure of hectorite–aniline intercalates

The inclusion of the organic guest aniline into Cu(II)-exchanged hectorite thin films has been investigated. The subsequent polymerization of aniline on the clay surface and in the intergallery regions are studied using scanning force microscopy (SFM), electron paramagnetic resonance (EPR) spectroscopy, powder x-ray diffraction (XRD), and impedance spectroscopy. EPR and XRD data show that in addition to strong polymerization of aniline on the clay surface, polymerization also occurs in the intergallery regions of the clay. Using standard lift-off techniques or razor cleaving, the exposed intergallery polyaniline is successfully imaged for the first time using noncontract SFM in phase-contrast mode. The nearly two-dimensional polymer sheets formed in these regions exhibit none of the nanometer-scale grain or bundle structure commonly associated with polyaniline synthesized using techniques such as electrochemical or vacuum deposition. The electrical response of the resulting clay-conducting polymer composites is also investigated for thin films fabricated on 15 μm interdigitated arrays. Preliminary results indicate that these composite materials may be highly suitable as chemical sensors for a variety of important gases and vapors such as ethanol and hexane.

Regimes of X-ray phase-contrast imaging with perfect crystals

Perfect crystals have recently been used as X-ray wavefront analysers to help produce phase-contrast images of non-periodic objects. Such images are essentially the maps of the phase gradients introduced in a plane X-ray wave upon passage through a weakly absorbing object. We show that the nature of the contrast in the images is determined by the ratio between the local wavefront curvature and the width of the crystal rocking curve. Depending on this ratio being small or large, two quite distinct regimes for image formation can be identified, namely the differential phase-contrast mode and the refractometric mode. We derive simple analytical formulae which can be used for the analysis of X-ray images of phase objects obtained in these two regimes.

Electron Phase Counting Micromagnetometry Using a Differential Phase Contrast/Interference Scanning Transmission Electron Microscope

A new method of micromagnetometry, electron phase counting (EPC), using a scanning transmission electron microscope with differential phase contrast and interference imaging modes has been proposed and tested. By counting the relative phase changes occurring in a pair of mutually coherent fine probes as they are moved from an observation point to a nearby reference point, integrated magnetic flux density at the observation point can be quantified without recourse to instrumental signal intensity calibration. Its feasibility for practical micromagnetometry has been confirmed by the successful application of two different EPC procedures to a NiFe alloy film.

Domain structures in epitaxially grown cobalt thin films

Following the epitaxial growth and characterization of single crystal c-axis oriented hcp cobalt thin films (with thicknesses in the range of 200-600 AA), we have investigated their micromagnetic domain properties by Lorentz electron microscopy (LEM). In particular we have used the differential phase contrast mode of LEM because this imaging technique allows a wide range of quantitative analysis to be carried out directly. The domain structures were studied in the as-grown, ac-demagnetized and remanent states. Large domains with regular magnetization ripple and cross ties were observed in the thinner films, whilst stripe domains were seen in films with thicknesses >or=400 AA. Detailed analysis of the DPC images led to information pertaining to the domain wall width and its associated energy density being attained.

Electron beam tomography of magnetic recording head fields

The quantitative evaluation of the 3D form of the magnetic field from a thin film recording head has been achieved by electron beam tomography. The data sets for tomographic reconstruction were obtained using the differential phase contrast mode of Lorentz electron microscopy applied in a 200 kV (scanning) transmission instrument. The high accelerating voltage and a novel method of mounting the head in the microscope offer advantages over previous experiments. The two reconstruction algorithms, which have been used previously, have been implemented and the results of their application are compared for theoretical data derived from a model head field and for data obtained experimentally from a production recording head.

Differential phase contrast in TEM for magnetic micro structure observation

Phase contrast in TEM can only give information in a limited band of spatial frequencies. The differential phase contrast mode in STEM does not have this limitation, since one simply measures the angle over which the electron beam is deflected by the specimen. This is useful, for example, for determining the magnetic field distribution in magnetic thin films. Recently, a form of electron holography was developed, also in STEM, to obtain differential phase contrast images. In this method, the illuminating beam is split by a biprism in such a way that there are two mutually coherent electron source images close to the specimen. On a far away CCD detector, two shadow images are formed which interfere to give a fringe pattern. It is this fringe pattern that contains the differential phase information. Here, we show that differential phase contrast can also be obtained in a TEM.In the TEM, two coherent illuminating beams are created by a beam splitter in the condenser system. These beams can be treated as two plane waves, creating a fringe pattern in the plane of observation,as shown in figure 1.

Far-out-of-focus electron holography in a dedicated FEG STEM

Flexible operation of off-axis holography modes is achieved when a biprism is placed in the illumination system of a scanning transmission electron microscope, equipped with a field emission electron source. The separation of the two virtual sources created by the biprism can be varied by changing the voltage applied to the biprism or by simply changing the excitation of the condensor and/or objective lenses. Two distinct modes of holography are used to study the magnetic microstructure in thin magnetic films. In the absolute mode the phase difference changes linearly in regions of constant magnetization and thickness and the slope determines the magnitude of magnetization. In addition, this mode permits the determination of the mean inner potential of a solid of known geometry. In the differential mode the phase difference is constant in regions of constant magnetization, allowing a simple and straightforward determination of domain wall profiles. The contrast obtained in the holography modes is compared to well known contrast in the Fresnel and differential phase contrast modes of Lorentz microscopy. The combination of several scanning transmission electron microscopy based techniques presents a powerful tool for the investigation of magnetic microstructure.

Phase contrast studies of biological specimens with the x-ray microscope at BESSY (invited)

The transmission x-ray microscope at the BESSY storage ring is being used for investigations in the fields of biology, biophysics, medicine, colloid chemistry, and soil sciences. The system has been instrumented for phase contrast studies with the possibility of quick change between imaging in amplitude and phase contrast mode. Phase contrast has the advantage of considerable higher image contrast and reduced radiation dosage compared to amplitude contrast. A cryogenic object chamber has been implemented on the x-ray microscope. First experiments have shown that at cryogenic temperatures the structural stability of biological specimens is increased by three orders of magnitude in comparison with unfixed wet specimens at room temperature.

Micromagnetic characteristics of single layer permalloy films in the nanometre range

Abstract The micromagnetic properties of thin (5–20 nm) permalloy films, prepared by UHV magnetron sputtering, have been studied by Lorentz microscopy. Using the high resolution of the differential phase contrast mode the generation and growth of domains and the development of magnetisation ripple has been studied as a function of layer thickness, field strength and direction.

Quantitative evaluation of a thin film recording head field using the DPC mode of Lorentz electron microscopy

The quantitative evaluation of the recording head field is an important aspect of the goal of increased areal density of data storage in magnetic recording. The differential phase contrast (DPC) mode of Lorentz electron microscopy has been used to determine the stray field from a thin film recording head and this has been achieved by collection of data sets suitable for tomographic reconstruction. By etching part of the alumina present in the vicinity of the poletips it has also proved possible to identify magnetic flux escaping from regions of the poles, other than the poletips, and to investigate the contribution these make to the head field profile as a function of dc drive current.