Noise-induced hearing loss can be caused by sudden or prolonged exposure to loud noise. Noise exposure is known to contribute to the degeneration of sensory cells, disrupting the conversion of mechanical sound waves into electrical impulses and further their transmission to the brain. To determine the pathophysiological condition of the inner ear cells in animal models, the measurement of the animals' hearing is essential. The follow-up examination of the cochlea is particularly important as it provides information on the cellular morphology changes. Our aim was therefore to investigate the hair cell survival in the inner ear of mice exposed to two high noise levels using synchrotron radiation-based microtomography. Its spatial resolution allows for the reconstruction of three-dimensional images of unstained cochlea at the cellular level. We segmented the basilar membrane via automatic cell segmentation and fast manual cell removal, and determined its length using a one-dimensional Isomap embedding. After extracting its middle region and image slices aligned with it, surviving inner and outer hair cell locations were semi-automatically determined and then manually corrected using the ImageJ plugin PointPicker. These results were compared with the confocal microscopy data. The data collected provides meaningful information about healthy and damaged hair cells in the adult cochlea.
Alzheimer's disease (AD) remains one of the foremost public health challenges of our time. Recently, attention has turned to the gut-brain axis, a complex network of communication between the gastrointestinal tract and the brain, as a potential player in the pathogenesis of AD. Here we exploited x-ray Phase Contrast Tomography to provide an in-depth analysis of the link between the gut condition and AD, exploring gut anatomy and structure in murine models. We conducted a comprehensive analysis by comparing the outcomes in various mouse models of cognitive impairment, including AD, frail mice, and frontotemporal dementia affected mice. We discovered an association between substantial changes in the gut structure and the presence of amyloid-beta (Aβ) in the brain. We found that the most important gut alterations are related to Aβ occurrence in the brain. In particular, we investigated the gut morphology, the distribution of enteric micro-processes and neurons in the ileum.
Caries affects billions of individuals worldwide, thus pointing out the importance of advancements in restorative dentistry. Dental resin composites yield restorations with satisfying mechanical properties, therefore the focus of development has shifted to accelerated treatments and esthetic aspects. Challenges in matching tooth color arise due to limited options, application changes, and color variations over time. Single-shade composites with the 'chameleon effect' adapt their color to the surrounding enamel by closely matching the tooth's optical spectrum, enhancing color blending. Structural color, based on light interference, contributes to this effect. The study investigates the submicron filler particles' impact on optical properties and the chameleon effect. Four single-shade dental resin composite materials were investigated. Needle-like samples about 100 μm in diameter were prepared and imaged in a scanning electron microscope. Light transmission through the materials for wavelengths between 200 and 900 nm was measured using a spectrophotometer. Three-dimensional nanotomography data were obtained through transmission X-ray microscopy at the ANATOMIX beamline, Synchrotron SOLEIL, France in both absorption and Zernike phase contrast mode with 23 nm voxel size. The real space information was complemented with small-angle X-ray scattering. These experiments revealed substantial differences in the microscopic structure of the materials. In the case of Omnichroma, the filler consists of almost identical spheres with a diameter of 260 nm while Filtek Universal exhibits polydisperse, irregularly shaped fillers. Additionally, Venus Pearl One’s fillers have a polyhedral shape and a wide size distribution. Finally, the setups used did not reveal any clearly identified microstructure of the Chroma Fill composite. Although all investigated materials are known to exhibit the chameleon effect, their differences in micro- and nanostructure call into question previous hypotheses on the chameleon effect’s origin from structural color. While we have now a reasonable understanding of filler morphology, size distribution and spatial arrangement, more information is needed on the exact chemical composition of filler and matrix and their interaction with electromagnetic waves, including possible nonlinear effects.
Joint tissues consist of trabecular and cortical bone as well as calcified and hyaline cartilage, which presents a challenge for hard X-ray-based visualization on the sub-cellular level due to the wide range of local X-ray absorption values. The density of the calcified tissues requires rather high photon energy, which often leads to insufficient contrast within the cartilage and impedes the visualization of individual biological cells. Decalcification of the tissues reduces the total and local X-ray absorption values and allows for selecting a lower photon energy. Further contrast enhancement can be achieved by ethanol fixation and paraffin tissue embedding. In this study, we (i) searched for an appropriate visualization method to investigate lesions generated by a laser osteotome and (ii) visualized a decalcified porcine joint after ethanol fixation and subsequent paraffin embedding using laboratory- and synchrotron radiation-based microtomography. The experiments at the ANATOMIX beamline of Synchrotron SOLEIL were performed in off-axis scan mode with a pixel size of 1.3 µm. Individual cells in all layers of the joint could be made visible and the effect of ethanol fixation and paraffin embedding demonstrated.
The famous Swiss mummy, the "Lady of the Franciscan Church", is located in Basel. Subsequent to discovery in 1975, many investigations on her origin were performed without success. Four decades later, however, the mummy’s origin has been identified: Anna Catharina Gernler-Bischoff (1719-1787), who was a direct ancestor of Boris Johnson, the former Prime Minister of the United Kingdom. Further details about her life have been collected. For this purpose, teeth were removed and physically sliced for imaging using optical microscopy. One single tooth remained and became available for this cementochronology study. We have applied microtomography to visualize the annual deposits in tooth cementum, termed incremental layers, without the need of physical slicing. The applicability of synchrotron radiation-based tomography to cementochronology has previously been investigated and showed promise. It is, however, unclear how far tomography will work for the mummy tooth. Laboratory-based microtomography enabled us to discriminate between enamel, dentin, and cementum, but the annual layers remained invisible. The improved density and spatial resolution of the tomography setup at the beamline ANATOMIX, Synchrotron SOLEIL, France, however, brought the incremental layers to light. We have counted 30 layers on average. Their thickness corresponds to (4.6 ± 1.4) µm. The available tomography data of the entire tooth should be further analyzed to correlate the life history of Anna Catharina Gernler-Bischoff with thickness and contrast of the incremental layers. The pipeline developed and the gained knowledge will be beneficial for cementochronology of humans dead and alive.
Mammalian brains are extremely complex: a mouse brain contains one hundred million neurons. Mapping an entire brain’s three-dimensional cytoarchitecture from the nano- to centimeter-scale is a monumental challenge. For standard microtomography with sub-µm pixel size, reconstructed volume is limited to about a few mm3 . For full brain mapping, even for the mouse brain with volume of 450 mm3 , the field-of-view must be significantly increased in all three dimensions. We demonstrate mosaic tiling to extend imaged volume by 400× and a dedicated pipeline to process these tera-voxel sized datasets. Here, an entire mouse brain was imaged with 0.65 µm-wide voxels. The datasets, which are 6 TB in size at 16-bit depth, contain a wealth of microanatomical information but present challenges for registration and segmentation.
Dental restorations should match the color of the surrounding enamel. Carefully selecting the appropriate shade for the filling material is a challenge for dentists. Moreover, tooth color can change over time due to habits such as smoking or drinking coffee. In the last few years, single-shade dental composites have come to the market. They rely on a chameleon effect to provide acceptable to good color matching regardless of the tooth color. The chameleon effect refers to a dental filling’s ability to guide light in such a way that its color blends in with that of the tooth. Structural color is a contributing factor to the chameleon effect and an active area of research where structures at the submicron scale play a critical role. We investigated the size, shape, and three-dimensional spatial arrangement of filler particles in single-shade dental resin composites. Cylindrical samples of dental composites were prepared and imaged with the transmission X-ray microscope at the ANATOMIX beamline, Synchrotron SOLEIL, France. The centers of the filler particles were determined from the tomography data. Combined with shape information from scanning electron microscopy, a Monte Carlo approach was used to model the transmittance for light at wavelengths from the visible to the ultraviolet. The results were compared to optical transmission measurements. The combination of nanotomography and simulation can thus help to understand the influence of the size and distribution of filler particles on the chameleon effect.
Cementum deposits on mammalian teeth contain layered microstructures associated with the chronological age of an animal and other details of their life history. Hard X-ray tomography data captured this record contained within the cementum deposits from whole teeth without sectioning. We investigated three teeth of African bovids, namely gemsbok (Oryx gazella), eland (Taurotragus oryx), and African or Cape buffalo (Syncerus caffer) using the laboratorybased system nanotom m for measuring each complete tooth to identify relevant regions, which were scanned at the ANATOMIX beamline of Synchrotron SOLEIL, France. Using microtomography in archaeological materials such as teeth, eliminates the need for tooth sectioning, making it a desirable alternative for archaeologists and museum curators. Synchrotron measurements enabled the application of pixel sizes as low as 0.65µm, which generated around 40 TB of data. The three adult bovids investigated here, have a known day of death and season of death, and come from regions with distinct seasonal patterns in temperature and/or rainfall. They also have an estimated age at death based on occlusal wear. The known information serves as a control to determine the applicability of microtomography on whole teeth of large bovids. Preliminary results show that microtomography can successfully replace the need of sectioning in cementum dental analysis. Our future goal is to develop a protocol to standardize procedures of tooth cementum analysis in bovids using microtomography.
Comparable to annual rings present in a tree trunk, human tooth cementum contains yearly deposited incremental layers often termed incremental lines, which are generally visualized from tooth slides with optical microscopy in two dimensions. These micrometer-thin incremental lines are used to decode age-at-death and stress periods over the lifetime of an individuum. One can also visualize these layers without physical slicing by means of hard X rays because of density modulations. Within this project, two optically almost transparent tooth slides were used to record optical data in two dimensions with submicron pixel sizes. These data were registered with projections of available synchrotron radiation-based tomography data of the slides. Such data were also acquired for an entire tooth to determine thickness variations in each layer, the intra-layer thickness, and variations between the layers, the inter-layer thickness, automatically.
Purpose: Synchrotron radiation-based tomography yields microanatomical features in human and animal tissues without physical slicing. Recent advances in instrumentation have made laboratory-based phase tomography feasible. We compared the performance of three cutting-edge laboratory systems benchmarked by synchrotron radiation-based tomography for three specimens. As an additional criterion, the user-friendliness of the three microtomography systems was considered.
Approach: The three tomography systems—SkyScan 2214 (Bruker-microCT, Kontich, Belgium), Exciscope prototype (Stockholm, Sweden), and Xradia 620 Versa (Zeiss, Oberkochen, Germany)—were given 36 h to measure three medically relevant specimens, namely, zebrafish larva, archaeological human tooth, and porcine nerve. The obtained datasets were registered to the benchmark synchrotron radiation-based tomography from the same specimens and selected ones to the SkyScan 1275 and phoenix nanotom m® laboratory systems to characterize development over the last decade.
Results: Next-generation laboratory-based microtomography almost reached the quality achieved by synchrotron-radiation facilities with respect to spatial and density resolution, as indicated by the visualization of the medically relevant microanatomical features. The SkyScan 2214 system and the Exciscope prototype demonstrated the complementarity of phase information by imaging the eyes of the zebrafish larva. The 3-μm thin annual layers in the tooth cementum were identified using Xradia 620 Versa.
Conclusions: SkyScan 2214 was the simplest system and was well-suited to visualizing the wealth of anatomical features in the zebrafish larva. Data from the Exciscope prototype with the high photon flux from the liquid metal source showed the spiral nature of the myelin sheaths in the porcine nerve. Xradia 620 Versa, with detector optics as typically installed for synchrotron tomography beamlines, enabled the three-dimensional visualization of the zebrafish larva with comparable quality to the synchrotron data and the annual layers in the tooth cementum.
Tooth cementum annulation (TCA) is used for determining age-at-death and stress periods based on yearly deposited lines in the root cementum of human teeth. Traditionally, TCA analysis employs optical microscopy, which requires cutting sections of the root and provides only sparse sampling in the third dimension. Ancient teeth are unique specimens that should not be sliced. In this imaging study, we show that extended field of view synchrotron radiation-based tomography provides true micrometer resolution and coverage for non-destructively surveying for incremental lines. To rapidly review the root cementum layer of four teeth from early 19th century cemetery with historical records of life events, we employed machine learning for semi-automatic detection and analysis of incremental lines. Surveying large regions of the root cementum enables detection of incremental lines and hence improves TCA analysis as an alternative to slicing of the unique teeth.
Inline phase tomography using synchrotron radiation with sub-micrometer voxel sizes is nowadays the gold standard for investigation of soft and hard tissues with micron resolution. Recent developments on detectors and X-ray sources allow the transfer of the technique into laboratory environment. For the comparison of three manufacturers, we performed microtomography with advanced laboratory microtomography devices with micron resolution on a porcine nerve, a zebrafish embryo and a historic human tooth. These data sets were also compared with data acquired at the ANATOMIX beamline at Synchrotron Soleil and the TOMCAT beamline at SLS. For the lab-based experiments following scanners were chosen: Skyscan 2214 (Bruker-microCT, Kontich, Belgium), Xradia 620 Versa (Zeiss, Oberkochen, Germany) and a prototype with a MetalJet X-ray source from Exillum from the company Exciscope (Stockholm, Sweden). All devices contained detectors including X-ray optics.
Formalin fixation and paraffin embedding of post mortem tissue specimens is widely used for high-resolution neuroimaging with both conventional and X-ray virtual histology. Exchange of embedding solutions generates non-uniform brain shrinkage and changes relative tissue densities. We used synchrotron radiation-based X-ray micro computed tomography to visualize the embedding process for a single mouse brain. Non-rigid registration was employed to determine the volumetric strain fields and to track the X-ray absorption changes of corresponding features. This allows for a correction of the observed microanatomy to improve the anatomical context. Through embedding, the entire brain shrinks to around 40% of its volume in formalin. Shrinkage is non-uniform and varies over anatomical regions and the distance to external surfaces.
Biological matter may change shape via water absorption or loss. For example, brain tissue shows non-uniform shrinkage during formalin fixation and paraffin embedding, which is the most common tissue preparation for conventional histological analysis. Local deformations can be analyzed with non-rigid registration of non-destructive three-dimensional imaging datasets. We utilized synchrotron radiation microtomography at the ANATOMIX beamline of Synchrotron SOLEIL to image a mouse brain with 3 micron voxel length after formalin fixation, immersion in ascending alcohol series and xylene, and after paraffin embedding. We created a pipeline for non- rigid registration to align the volumes and extract volumetric strain fields. In this way, we could visualize the swelling/shrinkage of anatomical features. This method avoids time-consuming segmentation of brain regions, however it is sensitive to the registration parameters. In this proceedings paper, we discuss the selection of registration parameters in order to generate plausible volumetric strain fields. This protocol can be deployed to any type of shape change of biological matter and allows for the quantification of the related processes.
X-ray grating interferometry (XGI) is a phase-contrast imaging technique that allows for a quantitative measurement of the refractive index with high density resolution in a model-independent manner—i.e. without a priori knowledge of the specimen composition. However, the retrieval of the X-ray wavefront phase shift relies on the accurate measurement of the interference pattern phase shift, making XGI vulnerable to phase wrapping when the interference pattern phase shift, related to the derivative of the wavefront phase shift, is large. Standard procedure for avoiding phase wrapping involves submerging the specimen in a water bath to reduce the mismatch of the index of refraction at the boundaries, but this requires a top-down rotation stage and is susceptible to gas bubble formation inside the water bath. Our team has presented an algorithm to remove phase wrapping artifacts for cylindrically shaped specimens that is applied to the phase-retrieved sinogram. This algorithm models and replaces phase-wrapped data to prevent the spread of “cupping” artifacts due to the integration of the differential phase during reconstruction. We give a criterion for selecting the modeling parameters so that the resulting measurement of the index of refraction matches the results of measurements without phase wrapping. We also apply this technique to cases where phase wrapping occurs at multiple interfaces. This algorithm allows for XGI measurements without a water bath and top-down rotation stage at synchrotron and laboratory facilities, especially as sensitivity increases.
Cardiovascular diseases are the number one cause of death and morbidity in the world. Understanding disease
development in terms of lumen morphology and tissue composition of constricted arteries is essential to improve
treatment and patient outcome. X-ray tomography provides non-destructive three-dimensional data with micrometer-resolution.
However, a common problem is simultaneous visualization of soft and hard tissue-containing specimens,
such as atherosclerotic human coronary arteries. Unlike absorption based techniques, where X-ray absorption strongly
depends on atomic number and tissue density, phase contrast methods such as grating interferometry have significant
advantages as the phase shift is only a linear function of the atomic number. We demonstrate that grating interferometry-based
phase tomography is a powerful method to three-dimensionally visualize a variety of anatomical features in
atherosclerotic human coronary arteries, including plaque, muscle, fat, and connective tissue. Three formalin-fixed,
human coronary arteries were measured using advanced laboratory μCT. While this technique gives information about
plaque morphology, it is impossible to extract the lumen morphology. Therefore, selected regions were measured using
grating based phase tomography, sinograms were treated with a wavelet-Fourier filter to remove ring artifacts, and
reconstructed data were processed to allow extraction of vessel lumen morphology. Phase tomography data in
combination with conventional laboratory μCT data of the same specimen shows potential, through use of a joint
histogram, to identify more tissue types than either technique alone. Such phase tomography data was also rigidly
registered to subsequently decalcified arteries that were histologically sectioned, although the quality of registration was
insufficient for joint histogram analysis.
The complex hierarchical structure of human tooth hard tissues, enamel and dentin, guarantees function for decades. On the micrometer level the dentin morphology is dominated by the tubules, micrometer-narrow channels extending from the dentin-enamel junction to the pulp chamber. Their structure has been extensively studied, mainly with two-dimensional approaches. Dentin tubules are formed during tooth growth and their orientation is linked to the morphology of the nanometer-sized components, which is of interest for example for the development of bio-inspired dental fillings. Therefore, a method has to be identified that can access the three-dimensional organization of the tubules, e.g. density and orientation. Tomographic setups with pixel sizes in the sub-micrometer range allow for the three-dimensional visualization of tooth dentin tubules both in phase and absorption contrast modes. We compare high-resolution tomographic scans reconstructed with propagation based phase retrieval algorithms as well as reconstructions without phase retrieval concerning spatial and density resolution as well as rendering of the dentin microstructure to determine the approach best suited for dentin tubule imaging. Reasonable results were obtained with a single-distance phase retrieval algorithm and a propagation distance of about 75% of the critical distance of d2/λ, where d is the size of the smallest objects identifiable in the specimen and λ is the X-ray wavelength.
Atherosclerosis, the narrowing of vessel diameter and build-up of plaques in coronary arteries, leads to an increase in the shear stresses present, which can be used as a physics-based trigger for targeted drug delivery. In order to develop
appropriate nanometer-size containers, one has to know the morphology of the critical stenoses with isotropic
micrometer resolution. Micro computed tomography in absorption and phase contrast mode provides the necessary
spatial resolution and contrast. The present communication describes the pros and cons of the conventional and
synchrotron radiation-based approaches in the visualization of diseased human and murine arteries. Using registered
datasets, it also demonstrates that multi-modal imaging, including established histology, is even more powerful. The
tomography data were evaluated with respect to cross-section, vessel radius and maximal constriction. The average
cross-section of the diseased human artery (2.31 mm2) was almost an order of magnitude larger than the murine one (0.27 mm2), whereas the minimal radius differs only by a factor of two (0.51 mm versus 0.24 mm). The maximal constriction, however, was much larger for the human specimen (85% versus 49%). We could also show that a plastic model used for recent experiments in targeted drug delivery represents a very similar morphology, which is, for example, characterized by a maximal constriction of 82%. The tomography data build a sound basis for flow simulations, which allows for conclusions on shear stress distributions in stenosed blood vessels.
Basic research is required to develop more powerful approaches to prevent, diagnose, and above all to treat cancer, although the clever combination of surgery, chemical, pharmacological, and radiation therapies is well established. Our research activity concentrates on the quantification of the three-dimensional micro-morphology of vessel trees formed in cancerous and healthy tissues of a mouse model post mortem. While in several cases it is possible to extract the vessel tree from corrosion casts, phase contrast imaging modalities are needed for cancerous tissues with a significant amount of damaged vessel walls. Differences between cancerous and healthy tissues could be identified. The sum-of-angle metrics is found to be constant for vessel segments in cancerous and healthy tissues with lengths between 12 and 220 μm and corresponds to (0.62 ± 0.10) rad/μm.
The brain has an outstanding functional importance in the human organism. Therefore, there is a strong need for three-dimensional brain imaging modalities. Magnetic resonance imaging provides deep insights but its spatial resolution is insufficient to study the structure on the cellular level. X-ray absorption microtomography yields the necessary spatial resolution, but shows only marginal contrast between the different types of brain tissue. Alternatively, differential X-ray phase contrast obtained with grating interferometry, which is known for much better differentiations between soft tissues can be used for the visualization of the human brain. As important structures of the human brain such as the human thalamus have dimensions of several centimeters, a large field of view is required. In the present communication, we report an evaluation of grating-based X-ray phase contrast microtomography in the off-axis modus which allows to expand the field of view up to a factor of two but may reduce the image quality. We demonstrate that tomograms with comparable contrast-to-noise values, about 10%, and 50% inferior spatial resolution can be generated with off-axis measurements. As one can reduce the effective pixel size up to a factor of two, the choice of an asymmetrical rotation axis can give rise to an improvement of the spatial resolution by 20%.
Fabrication of double-side-polished single-crystal beryllium foils for X-ray window applications
were reported in an earlier paper. It was stipulated that unlike the conventional windows, polished
single-crystal windows - nearly free from granular boundaries, voids, impurities, and inclusions - would essentially transmit an incident X-ray beam unaltered, except for a uniform attenuation.
This paper reports on further X-ray characterization of these windows. Near- and far-field
transmission images of the windows have been obtained, and the impact of the windows on the wave
quality and coherence properties of the transmitted X-ray beam is demonstrated. Compared with
conventional X-ray windows, single-crystal beryllium windows are shown to introduce significantly
less artifacts in the transmitted beam but largely preserve coherence. The cause of sporadic and faint
features in the transmitted images is discussed and wave optics computation is used to simulate
some features.
Minimally invasive deep brain neurosurgical interventions require a profound knowledge of the morphology of the
human brain. Generic brain atlases are based on histology including multiple preparation steps during the sectioning and
staining. In order to correct the distortions induced in the anisotropic, inhomogeneous soft matter and therefore improve
the accuracy of brain atlases, a non-destructive 3D imaging technique with the required spatial and density resolution is
of great significance. Micro computed tomography provides true micrometer resolution. The application to post mortem
human brain, however, is questionable because the differences of the components concerning X-ray absorption are weak.
Therefore, magnetic resonance tomography has become the method of choice for three-dimensional imaging of human
brain. Because the spatial resolution of this method is limited, an alternative has to be found for the three-dimensional
imaging of cellular microstructures within the brain. Therefore, the present study relies on the synchrotron radiationbased
micro computed tomography in the recently developed grating-based phase contrast mode. Using data acquired at
the beamline ID 19 (ESRF, Grenoble, France) we demonstrate that grating-based tomography yields premium images of
human thalamus, which can be used for the correction of histological distortions by 3D non-rigid registration.
In this paper we describe the design of different X-ray Talbot interferometers that have been built at the tomography
beamline ID19 of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France, and give a short review of
performance characteristics, of current developments, and of the results obtained with these instruments so far. Among the
applications so far, soft-tissue imaging has been a particular focus, as demonstrated in a recent paper by Schulz et al. (J.
Roy. Soc. Interface, in press).
Phase contrast imaging with conventional X-ray tubes as e.g. in computer tomography scanners (CTscanners)
requires a setup of three different types of optical gratings. One grating is used to obtain
a spatially coherent radiation, the second grating defines a periodic phase shift and the third is used
as a periodic absorption grating. In order to absorb high energy radiation, absorption gratings with
periods of a few microns only and extreme aspect ratios (>80) are fabricated, employing a modified
LIGA process. However, above a critical structural height, structures collapse due to e.g. capillary
effects. To overcome this limitation a new variant of the LIGA process has been developed. It is
characterized by structuring of a resist on both sides of a membrane, resulting in a moderate aspect
ratio on both sides of the membrane instead of an extreme aspect ratio on one side. To get a perfect
overlay of both structures the grating structure on the front side of a membrane patterned by the
standard LIGA-process is used as the mask for structuring the second resist layer on the backside of
the membrane. A second electroforming step fills the gaps on the backside.
We present a systematic study in which multilayers of different composition (W/Si, Mo/Si, Pd/B4C), periodicity (from
2.5 to 5.5 nm), and numbers of layers have been characterised. Particularly, we investigated the intrinsic quality
(roughness and reflectivity) as well as the performance (flatness and coherence of the outgoing beam) as a
monochromator for synchrotron radiation hard X-ray micro-imaging. The results indicate that the material composition
is the dominating factor for the performance. This is of high importance for synchrotron-based hard X-ray imaging
which has become a widely applied tool for probing the microstructure of bulk samples. The high spatial resolution and
different contrast modalities available here strongly depend on using coherent beams from highly brilliant sources. In
order to satisfy the demand for a high flux of quasi-monochromatic photons, multilayer-coated mirrors are commonly
used as monochromators. Their properties present a good tradeoff between spectral bandwidth and photon flux density.
Since the photon flux density at the sample position is higher than with standard crystal monochromators, better spatial
resolution can be reached. This comes at the cost of reduced energy resolution and stronger non-uniformities in the
incoming beam profile. By helping scientists and engineers specify the design parameters of multilayer monochromators,
our results can contribute to a better exploitation of the advantages of multilayer monochromators over crystal-based
devices; i.e., larger spectral bandwidth and high photon flux density for X-ray imaging.
Cancer belongs to the primary diseases these days. Although different successful treatments including surgery, chemical, pharmacological, and radiation therapies are established, the aggressive proliferation of cancerous cells and the related formation of blood vessels has to be better understood to develop more powerful strategies against the different kinds of cancer. Angiogenesis is one of the crucial steps for the survival and metastasis formation of malignant tumors. Although therapeutic strategies attempting to inhibit these processes are being developed, the biological regulation is still unclear.
This study concentrates on the three-dimensional morphology of vessels formed in a mouse tumor xenograft model post mortem. Synchrotron radiation-based micro computed tomography (SRμCT) could provide the necessary information that is essential for validating the simulations. Using mouse and human brain tissue, the different approaches to extract the vessel tree from SRμCT data are discussed. These approaches include corrosion casting, the application of contrast agents such as barium sulfate, tissue embedding, all of them regarded as materials science based. Alternatively, phase contrast tomography was used, which gave rise to promising results but still not reaches the spatial resolution to uncover the smallest capillaries.
Alexander Rack, Heinrich Riesemeier, Simon Zabler, Timm Weitkamp, Bernd Müller, Gerd Weidemann, Peter Modregger, John Banhart, Lukas Helfen, Andreas Danilewsky, Hans Gräber, Richard Heldele, Boaz Mayzel, Jürgen Goebbels, Tilo Baumbach
The BAMline at the BESSY light source in Berlin and the TopoTomo beamline at the ANKA synchrotron facility in Karlsruhe (both Germany) operate in the hard X-ray regime (above 6 keV) with similiar photon flux density. For typical imaging applications, a double multilayer monochromator or a filtered white beam is used. In order to optimise the field of view and the resolution of the available indirect pixel detectors, different optical systems have been installed, adapted, respectively, to a large field of view (macroscope) and to high spatial resolution (microscope). They can be combined with different camera systems, ranging from 16-bit dynamic range slow-scan CCDs to fast CMOS cameras. The spatial resolution can be brought substantially beyond the micrometer limit by using a Bragg magnifier. The moderate flux of both beamlines compared to other 3rd generation light sources is compensated by a dedicated scintillator concept. For selected applications, X-ray beam collimation has proven to be a reliable approach to increase the available photon flux density. Absorption contrast, phase contrast, holotomography and refraction-enhanced imaging are used depending on the application. Additionally, at the TopoTomo beamline digital white beam synchrotron topography is performed, using the digital X-ray pixel detectors installed.
The coherence requirements for efficient operation of an X-ray grating interferometer are discussed. It is shown how a
Talbot-Lau geometry, in which an array of equidistant secondary sources is used, can be used to decouple fringe visibility
in the interferometer (and thus, its efficiency) from the total size of the X-ray source. This principle can be used for
phase-contrast radiography and tomography with sources of low brilliance, such as X-ray tubes.
In the framework of TWINMIC, a project for the development of a
multipurpose compact X-ray microscopy station capable of both
scanning and full-field imaging, fabrication methods for tantalum
zone plates are being developed at the Laboratory for Micro- and
Nanotechnology of the Paul Scherrer Institut. Tantalum is deposited
on supporting silicon or silicon nitride membranes by magnetron
sputtering. The zone-plate patterns are transferred into the
tantalum layer by reactive ion etching. Electron-beam lithography
with continuous path control using a Leica LION LV1 e-beam machine
has been used to make zone plates with diameters between 250 and
500 μm and thicknesses between 200 and 300 nm, all with an
outermost zone width of 80 nm.
In high-resolution microtomography, the alignment of the axis of
rotation with respect to the optical axis and to the rows of the
detector pixel array is an important issue, misalignment being a
critical source of reconstruction artifacts. A common calibration
method is based on the use of small fiducial markers on a
sample. However, the automatic detection and identification of such
markers is difficult. Moreover, the exact determination of their
positions in the radiographs is prone to errors in the presence of
noise, beam-profile fluctuations or nonuniform detector response. This is largely due to the fact that the markers cover only a small number of pixels in the image, which results in poor signal statistics. We have developed a new method that overcomes this limitation. It is based on the use of a purpose-built reference sample with periodic grid structures that cover large regions of the
radiographs. Straightforward Fourier analysis techniques are used to
determine from the images not only the tilt angles of the rotation
axis, but also the lateral position of the axis and the exact pixel
size.
We have developed a two-grating interferometer for hard X rays that
can be used for phase imaging and tomography. A silicon phase
grating positioned just downstream of the object under study splits
the distorted wavefront into essentially a positive and a negative
first-order beam. At a given distance from this beam-splitter grating, where the two beams still mostly overlap, they form a
pattern of interference fringes that is distorted according to the
wavefront distortions. The fringes may be finer than the resolution
of an area detector used to record the signal, but an absorption
grating with suitable pitch, put in front of the detection plane,
allows the detection of intensity variations that correspond to the
derivative of the wavefront phase taken along the direction perpendicular to the grating lines. A combination of this technique
with the phase-stepping method, in which several exposures are made
which differ in the phase of the fringe pattern, allows to eliminate
effects of non-uniform intensity due to inhomogeneous illumination
and edge-enhancing inline phase contrast. Several examples of
tomograms taken under different experimental conditions are shown,
including a polychromatic "pink-beam" setup.
In recent years, hard x-ray full field microscopy and tomography has been developed for synchrotron radiation sources based on parabolic refractive x-ray lenses. These optics are used as objective lens in a hard x-ray microscope that can image objects in transmission free of distortion. Using beryllium as lens material, an optical resolution of about 100nm has been reached in a field of view that is larger than 500 micrometers. In the future, the spatial resolution may be improved to below 50nm. Recording a large number of micrographs from different perspectives allows one to reconstruct non-destructively the 3-dimensional inner structure of an object with resolutions well below one micrometer. Different contrast mechanisms can be exploited, such as absorption and near field phase contrast. The method is demonstrated using a microchip as a test sample.
A suite of computer routines is presented that uses scalar wavefront theory to calculate the propagation of arbitrarily-shaped X-ray wavefronts through a series of objects and drift spaces that represent an experimental setup. The routines are coded in IDL, the Interactive Data Language, and can be included into or extended with any other IDL code. Objects and apertures in the X-ray path are modeled in the thin-object approximation, where they are represented by a two-dimensional complex transmission function. Fresnel propagation through drift spaces is carried out in Fourier space in the paraxial approximation. A variety of predefined, ready-for-use objects is included in the package. Among these are optical elements such as gratings, lenses, slits, or pinholes, as well as reference sample objects such as spheres, cylinders, test grids, etc. The materials and geometrical parameters of these objects can be freely chosen. Optical materials constants are looked up automatically in the DABAX database. The modular structure of the code makes it reasonably easy for users to add support for objects of any degree of complexity, or even other propagation schemes.
We derive mathematical relations for hard X-ray moire wavefront analysis with a grating interferometer. In particular, the first derivative of the wavefront phase profile and the local radius of curvature of the wavefront are related to the position and inclination of the observed moiré fringes.
When used in microimaging, hard x rays from third-generation synchrotron radiation (SR) sources inevitably generate noninterferometric or in-line phase contrast. It is formed by the propagation of a distorted x-ray wavefront after the sample. In this paper, we discuss phase contrast and its properties in two altogether different experimental modes. First, in edge-enhanced microtomography, we show by phase- propagation simulations that local tomography is possible without special effort. The second part of the paper discusses phase contrast and phase artifacts in magnified x- ray imaging and tomography using refractive lenses. Here, the phase effects degrade resolution to a considerable extent. This part of the paper contains experimental results from the ESRF beamline ID 22 in the photon energy range around 20 keV that are compared to simulated images and to experimental results from conventional high-resolution microtomography. The experimental results show that coherence-degrading devices can reduce but not completely eliminate phase effects, and recent microtomography data gathered with an x-ray microscope still cannot beat conventional state-of-the-art high-resolution microtomography with micrometer resolution.
Parabolic compound refractive lenses (PCRLs) are high quality imaging optics for hard x-rays that can be used as an objective lens in a new type of hard x-ray full field microscope. Using an aluminium PCRL, this new type of microscope has been shown to have a resolution of 350 nm. Further improvement of the resolution down to 50 nm can be expected using beryllium as a lens material. The large depth of field (several mm) of the microscope results in sharp projection images for samples that fit into the field of view of about 300 micrometers. This allows to combine magnified imaging with tomographic techniques. First results of magnified microtomography are shown. Contrast formation in the microscope and the consequences for tomographic reconstruction are discussed. An outlook on further developments is given.
The inadequacies of current analytical models for grain growth are thought to arise in part from their mean-field nature: they ignore the presence of correlations in the sizes of neighboring grains induced by the process of grain growth itself. Although grain-size correlations have been identified in microstructures generated by computer simulations of grain growth, no comparable evidence has been obtained from real samples - primarily because of the experimental difficulties associated with evaluating this inherently three-dimensional property. Using absorption- contrast x-ray microtomography, we have attempted to characterize the network of grain boundaries in polycrystalline samples of Al doped with up to 3 at.% Sn. In principle, since the tin atoms segregate to the grain boundaries, it should be possible to determine the size and relative position of each grain from a three-dimensional reconstruction of the Sn distribution, from which the desired correlation function could be calculated directly. However, the grain boundaries in Al-Sn are not uniformly decorated with tin, which presents a formidable challenge to quantifying the microstructural properties of such samples. Significant progress toward overcoming this problem has been achieved by applying a constrained phase-field grain-growth algorithm to an approximate microstructure gleaned from the tomographic contrast data.
Microtomography based on synchrotron radiation sources is a unique technique for the 3D characterization of different materials with a spatial resolution down to about 1 micrometers . The interface between implant materials (metals, ceramics and polymers) and biological matter is nondestructively accessible, i.e. without preparation artifacts. Since the materials exhibit different x-ray absorption, it can become impossible to visualize implant material and tissue, simultaneously. Here, we show that coating of polymer implants, which are invisible in bone tissue, does not only improve the interfacial properties but also allows the imaging of the interface in detail. Titanium implants, on the other hand, absorb the x-rays stronger than bone tissue. The difference, however, is small enough to quantify the bone formation near interface. Another advantage of microtomography with respect to classical histology is the capability to examine samples in a hydrated state. We demonstrate that ceramic hollow spheres can be imaged before sintering and fibroblasts marked by OsO4 are visible on polymer textiles. Consequently, scaffolds of different materials designed for tissue engineering and implant coatings can be optimized on the basis of the tomograms.
One major goal in x-ray tomography is to increase the resolution in space and time. For the methods with high temporal resolution we will present pink beam imaging and tomography. Experiments were realised at the ESRF undulator beamline ID22 with hard x-rays in the range from 11 keV to 20 keV. For the tomographic scans the exposure time per image was reduced by one to two orders of magnitude to less than 50 ms per image. The obtained image quality was comparable to that done with monochromatic beam. Further time reducing for a tomographic scan is possible with an improved acquiring and control system. The goal in the future is to realise tomographic scans within a minute with micrometer resolution. In order to achieve in the hard x-ray range sub-micrometer resolution we will show first results of x-ray magnified tomography. Different lens systems are available for this purpose. We obtained with aluminium parabolic compound refractive lenses a resolution of 1 micrometers and expect to overcome this limit hand in hand with the improvement of lens technology.
The planar microelectronics technology, involving lithography and highly anisotropic plasma etching techniques, allows manufacturing high quality refractive and diffractive lenses, which may be used in hard X-ray microprobe and microscopy applications. These silicon lenses are mechanically robust and can withstand high beat load of the white X-ray beam at third generation synchrotron radiation sources. For the first time we designed and manufactured a new type of lenses: kinoform lenses and parabolic lenses with scaled reduction of curvature radii. The theoretical background for such type of lens features is presented. Focusing properties in the terms of focus spot and efficiency of all these lenses were tested at the ESRF beamlines. Magnified imaging with planar lense was realized. Some future developments are discussed.
We describe parabolic compound refractive lenses for hard x- rays that are genuine imaging devices similar to glass lenses for visible light. They open considerable possibilities in both full field and scanning x-ray microscopy, microanalysis, and coherent scattering. They can operate in a range from about 2 keV to 100 keV, are robust, and withstand the white beam of a third generation undulator source. Using aluminum lenses in full field microscopy a field of view of about 300 micrometer can be imaged with magnifications between 10 and 50 and a resolution of about 300 nm. With beryllium lenses an improvement of the resolution to below 100 nm is expected. For microbeam applications, the synchrotron source is imaged onto the sample in a strongly demagnifying setup. With focal distances between 0.3 m and 2 m, the source can be demagnified by a factor 20 to 200 producing a beam with lateral extensions in the micron and sub-micron range. For aluminum lenses, monochromatic microbeams with fluxes above 1010 ph/s and a gain above 1000 are routinely produced at third generation undulator sources. Compound refractive lenses will allow to produce microbeams at energies up to at least 100 keV, making for example, microfluorescence experiments at the K-edges of heavy elements possible. The modular setup of compound refractive lenses allows to adjust the focal length to ideally match the experimental requirements. Assembling and aligning the lens take about 15 minutes. No order sorting apertures are required and the straight optical path allows to remove the lens to align other components.
Silicon planar parabolic refractive lenses with relief depth of 100 micrometer are realized by microfabrication technique. A set of 5 planar lenses with simple parabolic profiles and equal apertures and equal focal distances is realized. This set consists of different number (from 1 to 8) of individual lenses. Lenses with minimized absorption as a set of parabolic segments are fabricated too. Focusing and spectral properties of silicon planar parabolic lenses were studied with synchrotron radiation in the x-ray energy range 8 - 25 keV at the ESRF. Linear focus spots of 1.5 micrometer width were recorded for the parabolic lenses and 1.8 micrometer for the lenses with minimized absorption. The intensity transmission of the lens with minimized absorption is two times greater than this value of simple parabolic lenses at 8 keV and in the x-ray energy range over 15 keV overcomes 90%. Spectral properties of the lenses with minimized absorption are discussed in details. Heatload properties of the silicon planar lenses are analyzed and compared with the lenses made of diamond.
At the ESRF micro-fluorescence, imaging and diffraction ((mu) - FID) beamline ID 22, a microtomography setup has been operational for several months. The coherence properties of the high-energy (10 to 70 keV) X-ray undulator beam at ID 22 make the setup especially suited for phase-contrast tomography including possible holographic reconstruction, but it has also provided to be well adapted to absorption tomography. A fast- readout, low-noise CCD camera makes time-resolved imaging possible. Recent developments in magnifying X-ray optics such as Compound Refractive Lenses (CRL) and Fresnel Zone Plates (FZP) open up the field of magnified-X-ray imaging with a resolution of less than 300 nm. Imaging techniques using a 'pink beam,' i.e. a beam with limited monochromaticity obtained by filtering one harmonic from the undulator spectrum, can increase flux in intensity-limited experiments.
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