Registration of phase-contrast images in propagation-based X-ray phase tomography.

X-ray phase tomography aims at reconstructing the 3D electron density distribution of an object. It offers enhanced sensitivity compared to attenuation-based X-ray absorption tomography. In propagation-based methods, phase contrast is achieved by letting the beam propagate after interaction with the object. The phase shift is then retrieved at each projection angle, and subsequently used in tomographic reconstruction to obtain the refractive index decrement distribution, which is proportional to the electron density. Accurate phase retrieval is achieved by combining images at different propagation distances. For reconstructions of good quality, the phase-contrast images recorded at different distances need to be accurately aligned. In this work, we characterise the artefacts related to misalignment of the phase-contrast images, and investigate the use of different registration algorithms for aligning in-line phase-contrast images. The characterisation of artefacts is done by a simulation study and comparison with experimental data. Loss in resolution due to vibrations is found to be comparable to attenuation-based computed tomography. Further, it is shown that registration of phase-contrast images is nontrivial due to the difference in contrast between the different images, and the often periodical artefacts present in the phase-contrast images if multilayer X-ray optics are used. To address this, we compared two registration algorithms for aligning phase-contrast images acquired by magnified X-ray nanotomography: one based on cross-correlation and one based on mutual information. We found that the mutual information-based registration algorithm was more robust than a correlation-based method.

Cortical bone elasticity measured by resonant ultrasound spectroscopy is not altered by defatting and synchrotron X-ray imaging.

In the study of mechanical properties of human bone, specimens may be defatted before experiments to prevent contamination and the risk of infections. High energy synchrotron radiation micro-computed tomography (SR-µCT) is a popular technique to study bone microstructure. However, little is known about the effects of defatting or irradiation during SR-µCT imaging on different elastic coefficients including shear and longitudinal moduli in different anatomical directions. In this work, these effects are evaluated on a set of 24 samples using resonant ultrasound spectroscopy (RUS), which allows one to accurately measure the complete set of elastic coefficients of cortical bone non destructively. The results show that defatting with diethylether and methanol and irradiation up to 2.5kGy has no detectable effect on any of the elastic coefficients of human cortical bone.

Multiscale and multimodality computed tomography for cortical bone analysis.

In clinical studies, high resolution peripheral quantitative computed tomography (HR-pQCT) is used to separately evaluate cortical bone and trabecular bone with an isotropic voxel of 82 µm3, and typical cortical parameters are cortical density (D.comp), thickness (Ct.Th), and porosity (Ct.Po). In vitro, micro-computed tomography (micro-CT) is used to explore the internal cortical bone micro-structure with isotropic voxels and high resolution synchrotron radiation (SR); micro-CT is considered the 'gold standard'. In 16 tibias and 8 femurs, HR-pQCT measurements were compared to conventional micro-CT measurements. To test modality effects, conventional micro-CT measurements were compared to SR micro-CT measurements at 7.5 µm3; SR micro-CT measurements were also tested at different voxel sizes for the femurs, specifically, 7.5 µm3 versus 2.8 µm3. D.comp (r = -0.88, p < 10-3) was the parameter best correlated with porosity (Po.V/TV). The correlation was not affected by the removal of pores under 130 µm. Ct.Th was also significantly highly correlated (r = -0.89 p < 10-3), while Ct.Po was correlated with its counterpart Po.V/TV (r = 0.74, p < 10-3). From SR micro-CT and conventional micro-CT at 7.5 µm3 in matching areas, Po.V/TV and pore diameter were underestimated in conventional micro-CT with mean ± standard deviation (SD) biases of -2.5 ± 1.9% and -0.08 ± 0.08 mm, respectively. In contrast, pore number (Po.N) and pore separation (Po.Sp) were overestimated with mean ± SD biases of +0.03 ± 0.04 mm-1 and +0.02 ± 0.04 mm, respectively. The results from the tibia and femur were similar when the results of SR micro-CT at 7.5 µm3 and 2.8 µm3 were compared. Po.V/TV, specific surface of pores (Po.S/Po.V), and Po.N were underestimated with mean biases of -1.7 ± 0.9%, -4.6 ± 4.4 mm-1, and -0.26 ± 0.15 mm-1, respectively. In contrast, pore spacing was overestimated at 7.5 µm3 compared to 2.8 µm3 with mean biases of 0.05 ± 0.03 mm. Cortical bone measurements from HR-pQCT images provided consistent results compared to those obtained using conventional micro-CT at the distal tibia. D.comp was highly correlated to Po.V/TV because it considers both the micro-porosity (Haversian systems) and macro-porosity (resorption lacunae) of cortical bone. The complexity of canal organization, (including shape, connectivity, and surface) are not fully considered in conventional micro-CT in relation to beam hardening and cone beam reconstruction artifacts. With the exception of Po.V/TV measurements, morphological and topological measurements depend on the characteristics of the x-ray beam, and to a lesser extent, on image resolution.

Characterizing microcrack orientation distribution functions in osteonal bone samples.

Prefailure microdamage in bone tissue is considered to be the most detrimental factor in defining its strength and toughness with respect to age and disease. To understand the influence of microcracks on bone mechanics it is necessary to assess their morphology and three-dimensional distribution. This requirement reaches beyond classic histology and stereology, and methods to obtain such information are currently missing. Therefore, the aim of the study was to develop a methodology that allows to characterize three-dimensional microcrack distributions in bulk bone samples. Four dumbbell-shaped specimens of human cortical bone of a 77-year-old female donor were loaded beyond yield in either tension, compression or torsion (one control). Subsequently, synchrotron radiation micro-computed tomography (SRµCT) was used to obtain phase-contrast images of the damaged samples. A microcrack segmentation algorithm was developed and used to segment microcrack families for which microcrack orientation distribution functions were determined. Distinct microcrack families were observed for each load case that resulted in distinct orientation distribution functions. Microcracks had median areas of approximately 4.7 µm2 , 33.3 µm2 and 64.0 µm2 for tension, compression and torsion. Verifying the segmentation algorithm against a manually segmented ground truth showed good results when comparing the microcrack orientation distribution functions. A size dependence was noted when investigating the orientation distribution functions with respect to the size of the volume of interest used for their determination. Furthermore, a scale separation between tensile, compressive and torsional microcracks was noticeable. Visual comparison to classic histology indicated that microcrack families were successfully distinguished. We propose a methodology to analyse three-dimensional microcrack distributions in overloaded cortical bone. Such information could improve our understanding of bone microdamage and its impact on bone failure in relation to tissue age and disease.

Quantitative evaluation of regularized phase retrieval algorithms on bone scaffolds seeded with bone cells.

In the field of regenerative medicine, there has been a growing interest in studying the combination of bone scaffolds and cells that can maximize newly formed bone. In-line phase-contrast x-ray tomography was used to image porous bone scaffolds (Skelite(©)), seeded with bone forming cells. This technique allows the quantification of both mineralized and soft tissue, unlike with classical x-ray micro-computed tomography. Phase contrast images were acquired at four distances. The reconstruction is typically performed in two successive steps: phase retrieval and tomographic reconstruction. In this work, different regularization methods were applied to the phase retrieval process. The application of a priori terms for heterogeneous objects enables quantitative 3D imaging of not only bone morphology, mineralization, and soft tissue formation, but also cells trapped in the pre-bone matrix. A statistical study was performed to derive statistically significant information on the different culture conditions.

Binary tomography reconstruction from few projections with Total Variation regularization for bone microstructure studies.

Discrete tomography refers to a class of reconstruction methods adapted to discrete-valued images. Many different approaches have been investigated to address the binary case, when a two-phase object is considered. This reconstruction problem is very important in medical or material applications where it is crucial to reduce the number of projections. In this paper, we address the problem of binary image reconstruction for X-ray CT imaging from a small number of projections. We propose a TV (Total Variation) regularization approach and compare the results obtained with or without an additional box convex constraint. The schemes are applied to a simple disk image and to more complex bone cross-sections for various noise levels. The minimization of the regularization functional is performed with the state-of-the-art ADMM (Alternate Direction Minimization Method) algorithm. The methods perform equally well on a simple disk image. The additional box convex constraints improves the reconstruction results for complex structures with fine details.

3D X-ray ultra-microscopy of bone tissue.

We review the current X-ray techniques with 3D imaging capability at the nano-scale: transmission X-ray microscopy, ptychography and in-line phase nano-tomography. We further review the different ultra-structural features that have so far been resolved: the lacuno-canalicular network, collagen orientation, nano-scale mineralization and their use as basis for mechanical simulations. X-ray computed tomography at the micro-metric scale is increasingly considered as the reference technique in imaging of bone micro-structure. The trend has been to push towards increasingly higher resolution. Due to the difficulty of realizing optics in the hard X-ray regime, the magnification has mainly been due to the use of visible light optics and indirect detection of the X-rays, which limits the attainable resolution with respect to the wavelength of the visible light used in detection. Recent developments in X-ray optics and instrumentation have allowed to implement several types of methods that achieve imaging that is limited in resolution by the X-ray wavelength, thus enabling computed tomography at the nano-scale. We review here the X-ray techniques with 3D imaging capability at the nano-scale: transmission X-ray microscopy, ptychography and in-line phase nano-tomography. Further, we review the different ultra-structural features that have so far been resolved and the applications that have been reported: imaging of the lacuno-canalicular network, direct analysis of collagen orientation, analysis of mineralization on the nano-scale and use of 3D images at the nano-scale to drive mechanical simulations. Finally, we discuss the issue of going beyond qualitative description to quantification of ultra-structural features.

Computer vision tools to optimize reconstruction parameters in x-ray in-line phase tomography.

In this article, a set of three computer vision tools, including scale invariant feature transform (SIFT), a measure of focus, and a measure based on tractography are demonstrated to be useful in replacing the eye of the expert in the optimization of the reconstruction parameters in x-ray in-line phase tomography. We demonstrate how these computer vision tools can be used to inject priors on the shape and scale of the object to be reconstructed. This is illustrated with the Paganin single intensity image phase retrieval algorithm in heterogeneous soft tissues of biomedical interest, where the selection of the reconstruction parameters was previously made from visual inspection or physical assumptions on the composition of the sample.

Synchrotron radiation X-ray phase micro-computed tomography as a new method to detect iron oxide nanoparticles in the brain.

PURPOSE: The purpose of this study was to introduce synchrotron radiation X-ray phase computed tomography (SR-PCT) as a new method of visualizing ultrasmall superparamagnetic particles of iron oxide (USPIO) distribution into the brains of mice with neuroinflammation. PROCEDURES: The sensitivity of the technique was assessed by performing back-to-back SR-PCT and magnetic resonance imaging (MRI) in mice stereotaxically injected with a range of USPIO concentrations. Eight mice with cerebral ischemia were then intravenously injected with USPIOs and imaged back-to-back with MRI and SR-PCT. RESULTS: SR-PCT proved sensitive enough to detect iron in nanomolar quantities. In stroke-induced animals, SR-PCT showed hyperintense areas in the regions of MR signal loss and immunostaining for macrophages. SR-PCT, moreover, identified brain anatomy as clearly as histology, without the need for sectioning or staining, with an examination time of 44 min per brain at an isotropic spatial resolution of 8 µm. CONCLUSION: SR-PCT has potential for cellular imaging in intact brain, with unequaled neuroanatomy.

3D characterization of pores in the cortical bone of human femur in the elderly at different locations as determined by synchrotron micro-computed tomography images.

UNLABELLED: Diaphysis, inferior, and lateral superior regions of the femoral neck are subjected to diverse mechanical loads. Using micro-CT based on synchrotron radiation, three-dimensional morphology and connectivity of the pore network are location dependent, underlying different remodeling mechanisms. INTRODUCTION: The three-dimensional (3D) morphology and connectivity of the pore network at various locations in human femurs subjected to diverse mechanical loads were assessed using micro-CT based on synchrotron radiation. METHODS: The cortex from 20 human femurs (mean age, 78.3 ± 12.4 years) was taken from the diaphysis (D), the inferior (IN), and the lateral superior (LS) regions of the femoral neck. The voxel size of the 3D reconstructed image was 7.5 µm. Cortical thickness and pore volume/tissue volume (Po.V/TV), pore diameter (Po.Dm) and spacing (Po.Sp) were determined. The pore surface/pore volume ratio (Po.S/Po.V), the number of pores (Po.N), the degrees of anisotropy (DA), and the connectivity density (ConnD), the degree of mineralization (DMB) were also determined. RESULTS: The characteristics of the pore network in femoral cortical bone were found to be location dependent. There was greater porosity, Po.Dm, and Po.N, and more large (180-270 µm), extra-large (270-360 µm) and giant pores (>360 µm) in the LS compared to the IN and D. The difference in porosity in between the periosteal and endosteal layers was mostly due to an increase of Po.Dm rather than Po.N. There was a lower DMB of bone in the LS, which is consistent with a higher remodeling rate. CONCLUSION: The results provide evidence for large variations in the structure of the internal pore network in cortical bone. These variations could involve different underlying remodeling mechanisms.

Local topological analysis at the distal radius by HR-pQCT: Application to in vivo bone microarchitecture and fracture assessment in the OFELY study.

High-resolution peripheral quantitative computed tomography (HR-pQCT) is an in-vivo technique used to analyze the distal radius and tibia. It provides a voxel size of 82µm. In addition to providing the usual microarchitecture parameters, local topological analysis (LTA) depicting rod- and plate-like trabeculae may improve prediction of bone fragility. Thirty-three women with prevalent wrist fractures from the OFELY cohort were compared with age-matched controls. Bone microarchitecture, including the structural model index (SMI), was assessed by HR-pQCT, and micro-finite element analysis (µFE) was computed on trabecular bone images of the distal radius (XtremeCT, Scanco Medical AG). A new LTA method was applied to label each bone voxel as a rod, plate or node. Then the bone volume fraction (BV/TV*), the rod, plate and node ratios over bone volume (RV/BV*, PV/BV*, NV/BV*) or total volume (RV/TV*, PV/TV*, NV/TV*) and the rod to plate ratio (RV/PV*) were calculated. Associations between LTA parameters and wrist fractures were computed in a conditional logistic regression model. Multivariate models were tested to predict the µFE-derived trabecular bone stiffness. RV/TV* (OR=4.41 [1.05-18.62]) and BV/TV* (OR=6.45 [1.06-39.3]), were significantly associated with prevalent wrist fracture, after adjustment for ultra distal radius aBMD. Multivariate linear models including PV/TV* or BV/TV*+RV/PV* predicted trabecular stiffness with the same magnitude as those including SMI. Conversion from plates into rods was significantly associated with bone fragility, with a negative correlation between RV/PV* and trabecular bone stiffness (r=-0.63, p<0.0001). We conclude that our local topological analysis is feasible for a voxel size of 82µm. After further validation, it may improve bone fragility description.

Anatomical distribution of the degree of mineralization of bone tissue in human femoral neck: impact on biomechanical properties.

Osteoporotic hip fractures represent a major public health problem associated with high human and economic costs. The anatomical variation of the tissue mineral density (TMD) and of the elastic constants in femoral neck cortical bone specimens is an important determinant of bone fragility. The purpose of this study was to show that a Synchrotron radiation microcomputed tomography system coupled with a multiscale biomechanical model allows the determination of the 3-D anatomical dependence of TMD and of the elastic constants (i.e. the mechanical properties of an anisotropic material) in human femoral neck. Bone specimens from the inferior femoral neck were obtained from 18 patients undergoing standard hemiarthroplasty. The specimens were imaged using 3-D synchrotron micro-computed tomography with a voxel size of 10.13 µm, leading to the determination of the anatomical distributions of porosity and TMD. The elastic properties of bone tissue were computed using a multiscale model. The model uses the experimental data obtained at the scale of several micrometers to estimate the components of the elastic tensor of bone at the scale of the organ. Statistical analysis (ANOVA) revealed a significant effect of the radial position on porosity and TMD and a significant effect of axial position on TMD only. Porosity was found to increase in the radial direction moving from the periosteum inwards (p<10(-5)). At any given distance from the periosteum, porosity does not vary noticeably along the bone axis. TMD was found to be significantly higher (p<10(-5)) in the periosteal region than in other bone locations and decreases from the periosteal to the endosteal region with an average slope of 10.05 g.cm(-3).m(-1), the decrease being faster in the porous part of the samples (average slope equal of 30.04 g.cm(-3).m(-1)) than in dense cortical bone. TMD was found to decrease from the distal to the proximal part of the femur neck (average slope of 6.5 g.cm(-3).m(-1)). Considering TMD variations in the radial direction induces weak changes of bone properties compared to constant TMD. TMD variations in the axial direction are responsible for a significant variation of elastic constants. These results demonstrate that the anatomical variations of TMD affect the bone elastic properties, which could be explained by the complex stress field in bone affecting bone remodeling. TMD spatial variations should be taken into account to properly describe the spatial heterogeneity of elastic coefficients of bone tissue at the organ scale.

Non-linear iterative phase retrieval based on Frechet derivative.

Several methods of phase retrieval for in-line phase tomography have already been investigated based on the linearization of the relation between the phase shift induced by the object and the diffracted intensity. In this work, we present a non-linear iterative approach using the Frechet derivative of the intensity recorded at a few number of propagation distances. A Landweber type iterative method with an analytic calculation of the Frechet derivative adjoint is proposed. The inverse problem is regularized with the smoothing L2 norm of the phase gradient and evaluated for several different implementations. The evaluation of the method was performed using a simple phase map, both with and without noise. Our approach outperforms the linear methods on simulated noisy data up to high noise levels and thanks to the proposed analytical calculation is suited to the processing of large experimental image data sets.

The fusion of lipid droplets is involved in fat loss during cooking of duck "foie gras".

Fat loss during cooking of duck "foie gras" is the main quality issue in processing plants. To better understand this phenomenon, a histological and ultrastructural study was conducted. The aim was to characterize changes in lipid droplets of duck "foie gras" related to fat loss during cooking. Ten fatty livers were sampled before and after cooking and prepared for optical and transmission electron microscopy. In raw livers, the lipid droplets were nearly spherical while after cooking, they were larger and lost their spherical shape. We also observed a decrease in the number of droplets after cooking, probably due to droplet fusion caused by the heat treatment. Before cooking, there were fewer lipid droplets and a higher osmium tetroxyde staining intensity in the fatty liver, which later gave a lower technological yield. Fat loss during cooking was higher when there was more fusion of lipid droplets before cooking.

Evaluation of bone scaffolds by micro-CT.

This paper reviews the possibilities offered by X-ray micro-CT in bone tissue engineering. This technique provides a fast, nondestructive, and 3D quantification of bone scaffolds, bone ingrowth, and microvascularization. Synchrotron radiation absorption and phase micro-CT offer additional advantages to image newly formed bone in bioceramic scaffolds and pre-bone matrix.

Regularized phase tomography enables study of mineralized and unmineralized tissue in porous bone scaffold.

Regularized phase tomography was used to image non-calcified fibrous matrix in in vitro cell-cultivated porous bone scaffold samples. 3D micro-architecture of bone and bone scaffold has previously been studied by micro-computed tomography, synchrotron radiation (SR) micro-computed tomography and microdiffraction. However, neither of these techniques can resolve the low-calcified immature pre-bone fibrous structures. Skelite porous scaffold discs were seeded with osteoblasts, a combination of osteoblast and pre-osteoclasts and, as controls, with pre-osteoclasts only, and then cultivated for 8 weeks. They were subsequently imaged using SR propagation-based phase contrast imaging. Reconstructions using a regularized holographic phase tomography approach were compared to standard (absorption) SR micro-computed tomography, which show that quantitative analysis, such as volume and thickness measurements, of both the calcified fraction and the immature bone matrix in the reconstructed volumes is enabled. Indications of the effect of this type of culture on Skelite, such as change in mineralization and deposit of mature bone on the walls of the scaffold, are found. The results are verified with a histological study.

Determination of the heterogeneous anisotropic elastic properties of human femoral bone: from nanoscopic to organ scale.

Cortical bone is a multiscale composite material. Its elastic properties are anisotropic and heterogeneous across its cross-section, due to endosteal bone resorption which might affect bone strength. The aim of this paper was to describe a homogenization method leading to the estimation of the variation of the elastic coefficients across the bone cross-section and along the bone longitudinal axis. The method uses the spatial variations of bone porosity and of the degree of mineralization of the bone matrix (DMB) obtained from the analysis of 3-D synchrotron micro-computed tomography images. For all three scales considered (the foam (100 nm), the ultrastructure (5 microm) and the mesoscale (500 microm)), the elastic coefficients were determined using the Eshelby's inclusion problem. DMB values were used at the scale of the foam. Collagen was introduced at the scale of the ultrastructure and bone porosity was introduced at the mesoscale. The pores were considered as parallel cylinders oriented along the bone axis. Each elastic coefficient was computed for different regions of interest, allowing an estimation of its variations across the bone cross-section and along the bone longitudinal axis. The method was applied to a human femoral neck bone specimen, which is a site of osteoporotic fracture. The computed elastic coefficients for cortical bone were in good agreement with experimental results, but some discrepancies were obtained in the endosteal part (trabecular bone). These results highlight the importance of accounting for the heterogeneity of cortical bone properties across bone cross-section and along bone longitudinal axis.

Biodegradation of porous calcium phosphate scaffolds in an ectopic bone formation model studied by X-ray computed microtomograph.

Three types of ceramic scaffolds with different composition and structure [namely synthetic 100% hydroxyapatite (HA; Engipore), synthetic calcium phosphate multiphase biomaterial containing 67% silicon stabilized tricalcium phosphate (Si-TCP; Skelite) and natural bone mineral derived scaffolds (Bio-oss)] were seeded with mesenchymal stem cells (MSC) and ectopically implanted for 8 and 16 weeks in immunodeficient mice. X-ray synchrotron radiation microtomography was used to derive 3D structural information on the same scaffolds both before and after implantation. Meaningful images and morphometric parameters such as scaffold and bone volume fraction, mean thickness and thickness distribution of the different phases as a function of the implantation time, were obtained. The used imaging algorithms allowed a direct comparison and registration of the 3D structure before and after implantation of the same sub-volume of a given scaffold. In this way it was possible to directly monitor the tissue engineered bone growth and the complete or partial degradation of the scaffold. Further, the detailed kinetics studies on Skelite scaffolds implanted for different length of times from 3 days to 24 weeks, revealed in the X-ray absorption histograms two separate peaks associated to HA and TCP. It was therefore possible to observe that the progressive degradation of the Skelite scaffolds was mainly due to the resorption of TCP. The different saturation times in the tissue engineered bone growth and in the TCP resorption confirmed that the bone growth was not limited the scaffold regions that were resorbed but continued in the inward direction with respect to the pore surface.

Relationship between ultrasonic parameters and apparent trabecular bone elastic modulus: a numerical approach.

The physical principles underlying quantitative ultrasound (QUS) measurements in trabecular bone are not fully understood. The translation of QUS results into bone strength remains elusive. However, ultrasound being mechanical waves, it is likely to assess apparent bone elasticity. The aim of this study is to derive the sensitivity of QUS parameters to variations of apparent bone elasticity, a surrogate for strength. The geometry of 34 human trabecular bone samples cut in the great trochanter was reconstructed using 3-D synchrotron micro-computed tomography. Finite-difference time-domain simulations coupled to 3-D micro-structural models were performed in the three perpendicular directions for each sample and each direction. A voxel-based micro-finite element linear analysis was employed to compute the apparent Young's modulus (E) of each sample for each direction. For the antero-posterior direction, the predictive power of speed of sound and normalized broadband ultrasonic attenuation to assess E was equal to 0.9 and 0.87, respectively, which is better than what is obtained using bone density alone or coupled with micro-architectural parameters and of the same order of what can be achieved with the fabric tensor approach. When the direction of testing is parallel to the main trabecular orientation, the predictive power of QUS parameters decreases and the fabric tensor approach always gives the best results. This decrease can be explained by the presence of two longitudinal wave modes. Our results, which were obtained using two distinct simulation tools applied on the same set of samples, highlight the potential of QUS techniques to assess bone strength.