High-resolution Phase Contrast Research Articles

Immuno-surveillance and protection of the human cochlea.

Despite its location near infection-prone areas, the human inner ear demonstrates remarkable resilience. This suggests that there are inherent instruments deterring the invasion and spread of pathogens into the inner ear. Here, we combined high-resolution light microscopy, super-resolution immunohistochemistry (SR-SIM) and synchrotron phase contrast imaging (SR-PCI) to identify the protection and barrier systems in the various parts of the human inner ear, focusing on the lateral wall, spiral ganglion, and endolymphatic sac. Light microscopy was conducted on mid-modiolar, semi-thin sections, after direct glutaraldehyde/osmium tetroxide fixation. The tonotopic locations were estimated using SR-PCI and 3D reconstruction in cadaveric specimens. The sections were analyzed for leucocyte and macrophage activity, and the results were correlated with immunohistochemistry using confocal microscopy and SR-SIM. Light microscopy revealed unprecedented preservation of cell anatomy and several macrophage-like cells that were localized in the cochlea. Immunohistochemistry demonstrated IBA1 cells frequently co-expressing MHC II in the spiral ganglion, nerve fibers, lateral wall, spiral limbus, and tympanic covering layer at all cochlear turns as well as in the endolymphatic sac. RNAscope assays revealed extensive expression of fractalkine gene transcripts in type I spiral ganglion cells. CD4 and CD8 cells occasionally surrounded blood vessels in the modiolus and lateral wall. TMEM119 and P2Y12 were not expressed, indicating that the cells labeled with IBA1 were not microglia. The round window niche, compact basilar membrane, and secondary spiral lamina may form protective shields in the cochlear base. The results suggest that the human cochlea is surveilled by dwelling and circulating immune cells. Resident and blood-borne macrophages may initiate protective immune responses via chemokine signaling in the lateral wall, spiral lamina, and spiral ganglion at different frequency locations. Synchrotron imaging revealed intriguing protective barriers in the base of the cochlea. The role of the endolymphatic sac in human inner ear innate and adaptive immunity is discussed.

Automated human induced pluripotent stem cell colony segmentation for use in cell culture automation applications

Human induced pluripotent stem cells (hiPSCs) have demonstrated great promise for a variety of applications that include cell therapy and regenerative medicine. Production of clinical grade hiPSCs requires reproducible manufacturing methods with stringent quality-controls such as those provided by image-controlled robotic processing systems. In this paper we present an automated image analysis method for identifying and picking hiPSC colonies for clonal expansion using the CellXTM robotic cell processing system. This method couples a light weight deep learning segmentation approach based on the U-Net architecture to automatically segment the hiPSC colonies in full field of view (FOV) high resolution phase contrast images with a standardized approach for suggesting pick locations. The utility of this method is demonstrated using images and data obtained from the CellXTM system where clinical grade hiPSCs were reprogrammed, clonally expanded, and differentiated into retinal organoids for use in treatment of patients with inherited retinal degenerative blindness.

Heterotopic mineral deposits in intact rat Achilles tendons are characterized by a unique fiber-like structure

Heterotopic mineralization entails pathological mineral formation inside soft tissues. In human tendons mineralization is often associated with tendinopathies, tendon weakness and pain. In Achilles tendons, mineralization is considered to occur through heterotopic ossification (HO) primarily in response to tendon pathologies. However, refined details regarding HO deposition and microstructure are unknown. In this study, we characterize HO in intact rat Achilles tendons through high-resolution phase contrast enhanced synchrotron X-ray tomography. Furthermore, we test the potential of studying local tissue injury by needling intact Achilles tendons and the relation between tissue microdamage and HO. The results show that HO occurs in all intact Achilles tendons at 16 weeks of age. HO deposits are characterized by an elongated ellipsoidal shape and by a fiber-like internal structure which suggests that some collagen fibers have mineralized. The data indicates that deposition along fibers initiates in the pericellular area, and propagates into the intercellular area. Within HO deposits cells are larger and more rounded compared to tenocytes between unmineralized fibers, which are fewer and elongated. The results also indicate that multiple HO deposits may merge into bigger structures with time by accession along unmineralized fibers. Furthermore, the presence of unmineralized regions within the deposits may indicate that HOs are not only growing, but mineral resorption may also occur. Additionally, phase contrast synchrotron X-ray tomography allowed to distinguish microdamage at the fiber level in response to needling. The needle injury protocol could in the future enable to elucidate the relation between local inflammation, microdamage, and HO deposition.

Volumetric High-Resolution X-Ray Phase-Contrast Virtual Histology of Breast Specimens With a Compact Laboratory System.

The assessment of margin involvement is a fundamental task in breast conserving surgery to prevent recurrences and reoperations. It is usually performed through histology, which makes the process time consuming and can prevent the complete volumetric analysis of large specimens. X-ray phase contrast tomography combines high resolution, sufficient penetration depth and high soft tissue contrast, and can therefore provide a potential solution to this problem. In this work, we used a high-resolution implementation of the edge illumination X-ray phase contrast tomography based on “pixel-skipping” X-ray masks and sample dithering, to provide high definition virtual slices of breast specimens. The scanner was originally designed for intra-operative applications in which short scanning times were prioritised over spatial resolution; however, thanks to the versatility of edge illumination, high-resolution capabilities can be obtained with the same system simply by swapping x-ray masks without this imposing a reduction in the available field of view. This makes possible an improved visibility of fine tissue strands, enabling a direct comparison of selected CT slices with histology, and providing a tool to identify suspect features in large specimens before slicing. Combined with our previous results on fast specimen scanning, this works paves the way for the design of a multi-resolution EI scanner providing intra-operative capabilities as well as serving as a digital pathology system.

Phase contrast imaging with inelastically scattered electrons from any layer of a thick specimen

A controversy exists as to whether the signal in a high resolution phase contrast electron micrograph of a particle in a thick specimen is the same irrespective of the particle’s position along the beam axis. Different conceptions of inelastic scattering and its effects on wave interference have led to radically different expectations about the degree of phase contrast vs. depth. Here we examine the information available from bright field phase contrast images of small crystalline particles on the top or bottom of a thick support. The support is an aluminium foil which has strong plasmon resonances that cause a large proportion of the electron beam to lose energy in transit. Phase contrast micrographs of the atomic lattice of two ensembles of platinum particles were measured in an energy loss window corresponding to the first plasmon resonance. The signal measured for particles on top was equal to that for particles on the bottom of the foil to within a 99% confidence interval, and the measurements exclude other models of depth dependent phase contrast in the literature to >5σ. These observations are consistent with quantum theory which considers dynamical effects as independent of event sequence and is distinct from the “top-bottom effect” observed in amplitude contrast. We thus confirm that phase contrast using inelastically scattered electrons can be obtained equally well from particles within any layer of a thick specimen.

Imaging biological macromolecules in thick specimens: The role of inelastic scattering in cryoEM

We investigate potential improvements in using electron cryomicroscopy to image thick specimens with high-resolution phase contrast imaging. In particular, using model experiments, electron scattering theory, Monte Carlo and multislice simulations, we determine the potential for improving electron cryomicrographs of proteins within a cell using chromatic aberration (Cc) correction. We show that inelastically scattered electrons lose a quantifiable amount of spatial coherence as they transit the specimen, yet can be used to enhance the signal from thick biological specimens (in the 1000 to 5000 Å range) provided they are imaged close to focus with an achromatic lens. This loss of information quantified here, which we call “specimen induced decoherence”, is a fundamental limit on imaging biological molecules in situ. We further show that with foreseeable advances in transmission electron microscope technology, it should be possible to directly locate and uniquely identify sub-100 kDa proteins without the need for labels, in a vitrified specimen taken from a cell.

High-Resolution X-ray Phase-Contrast Imaging and Sensory and Rheometer Tests in Cooked Edamame.

Although several reports exist on the use of X-ray analysis in vegetables and fruits to examine internal disorders, cavities, and porosity, information on X-ray analysis of qualities, such as texture, is lacking as well as information on X-ray analysis of legumes. Therefore, this study aimed to perform X-ray analysis with sensory and rheometer tests in cooked vegetable soybean (edamame). Edamame is popular worldwide due to its deliciousness and nutritional value. Vascular structures and cracks around them were clearly visualized using X-ray phase-contrast computed tomography (CT) imaging. In addition, we observed the fine structure of the seed coat, which could be important for seed development, germination, and processing. The density in the edamame beans declined as the boiling time increased, promoting a reduction in hardness described in sensory and rheometer tests. The reduction in density proceeded from the gap between cotyledons, the opposite side of the hypocotyl, and the crack. Collectively, the findings show that the high-resolution X-ray phase-contrast CT imaging conducted in a nondestructive manner may help in effectively evaluating the quality of vegetables and in observing the internal structures related to plant development.

3D ring artifacts removal algorithm combined low-rank tensor decomposition with spatial-sequential total variation regularization and its application in phase-contrast microtomography.

High-resolution synchrotron radiation X-ray phase contrast microtomography (PC-μCT) images often suffer from severe ring artifacts, which are mainly caused by undesirable responses of detector elements. In the medical imaging field, the existence of ring artifacts can lead to degraded visual quality and can directly affect diagnosis accuracy. Thus, removing or at least effectively reducing ring artifacts is indispensable. The existing ring artifacts removal algorithms mainly focus on two-dimensional (matrix-based) priors, and these algorithms fail to consider correlations hidden in sequential computed tomography (CT) images. This paper proposed a novel three-dimensional (tensor-based) ring artifacts removal algorithm for synchrotron radiation X-ray PC-μCT images. In the sinogram domain, ring artifacts manifest as vertical stripe artifacts. From an image decomposition perspective, a degraded sinogram can be decomposed into a stripe artifacts component and an underlying clean sinogram component. The proposed algorithm is designed to detect and remove stripe artifacts from a degraded sinogram by fully identifying the characteristics of the two components. Specifically, for the stripe artifacts component, tensor Tucker decomposition is used to describe its low-rank character. For the underlying clean sinogram component, spatial-sequential total variation regularization is adopted to enhance the piecewise smoothness. Moreover, the Frobenius norm term is further used to model Gaussian noise. An efficient augmented Lagrange multiplier method is designed to solve the proposed optimization model. The proposed method is evaluated utilizing both simulations and real data containing different ring artifacts patterns. In the simulations, the human chest CT images are used for evaluating the proposed method. We compare the peak signal-to-noise ratio (PSNR), structural similarity (SSIM), and mean absolute error (MAE) results of our algorithm with the Naghia's method, the RRRTV method, the wavelet-FFT method, and the SDRSD-GIF method. The proposed method was also evaluated on real data from rat liver samples and rat tooth samples. Our proposed method outperforms the competing methods in terms of both qualitative and quantitative evaluation results. Additionally, the 3D visualization results were presented to make the ring artifacts removal effect more intuitive. The experimental results on simulations and real data clearly demonstrated that the proposed algorithm can significantly improve the quality of PC-μCT images compared with the existing popular algorithms, and it has great potential to promote the application of high-resolution imaging for visualizing biological tissues.

High-resolution and sensitivity bi-directional x-ray phase contrast imaging using 2D Talbot array illuminators.

Two-dimensional (2D) Talbot array illuminators (TAIs) were designed, fabricated, and evaluated for high-resolution high-contrast x-ray phase imaging of soft tissue at 10-20 keV. The TAIs create intensity modulations with a high compression ratio on the micrometer scale at short propagation distances. Their performance was compared with various other wavefront markers in terms of period, visibility, flux efficiency, and flexibility to be adapted for limited beam coherence and detector resolution. Differential x-ray phase contrast and dark-field imaging were demonstrated with a one-dimensional, linear phase stepping approach yielding 2D phase sensitivity using unified modulated pattern analysis (UMPA) for phase retrieval. The method was employed for x-ray phase computed tomography reaching a resolution of 3 µm on an unstained murine artery. It opens new possibilities for three-dimensional, non-destructive, and quantitative imaging of soft matter such as virtual histology. The phase modulators can also be used for various other x-ray applications such as dynamic phase imaging, super-resolution structured illumination microscopy, or wavefront sensing.

Assessment of single-vessel cerebral blood velocity by phase contrast fMRI.

Current approaches to high-field functional MRI (fMRI) provide 2 means to map hemodynamics at the level of single vessels in the brain. One is through changes in deoxyhemoglobin in venules, i.e., blood oxygenation level-dependent (BOLD) fMRI, while the second is through changes in arteriole diameter, i.e., cerebral blood volume (CBV) fMRI. Here, we introduce cerebral blood flow-related velocity-based fMRI, denoted CBFv-fMRI, which uses high-resolution phase contrast (PC) MRI to form velocity measurements of flow. We use CBFv-fMRI in measure changes in blood velocity in single penetrating microvessels across rat parietal cortex. In contrast to the venule-dominated BOLD and arteriole-dominated CBV fMRI signals, CBFv-fMRI is comparable from both arterioles and venules. A single fMRI platform is used to map changes in blood pO2 (BOLD), volume (CBV), and velocity (CBFv). This combined high-resolution single-vessel fMRI mapping scheme enables vessel-specific hemodynamic mapping in animal models of normal and diseased states and further has translational potential to map vascular dementia in diseased or injured human brains with ultra-high-field fMRI.

A Synchrotron and Micro-CT Study of the Human Endolymphatic Duct System: Is Meniere's Disease Caused by an Acute Endolymph Backflow?

Background: The etiology of Meniere's disease (MD) and endolymphatic hydrops believed to underlie its symptoms remain unknown. One reason may be the exceptional complexity of the human inner ear, its vulnerability, and surrounding hard bone. The vestibular organ contains an endolymphatic duct system (EDS) bridging the different fluid reservoirs. It may be essential for monitoring hydraulic equilibrium, and a dysregulation may result in distension of the fluid spaces or endolymphatic hydrops.Material and Methods: We studied the EDS using high-resolution synchrotron phase contrast non-invasive imaging (SR-PCI), and micro-computed tomography (micro-CT). Ten fresh human temporal bones underwent SR-PCI. One bone underwent micro-CT after fixation and staining with Lugol's iodine solution (I2KI) to increase tissue resolution. Data were processed using volume-rendering software to create 3D reconstructions allowing orthogonal sectioning, cropping, and tissue segmentation.Results: Combined imaging techniques with segmentation and tissue modeling demonstrated the 3D anatomy of the human saccule, utricle, endolymphatic duct, and sac together with connecting pathways. The utricular duct (UD) and utriculo-endolymphatic valve (UEV or Bast's valve) were demonstrated three-dimensionally for the first time. The reunion duct was displayed with micro-CT. It may serve as a safety valve to maintain cochlear endolymph homeostasis under certain conditions.Discussion: The thin reunion duct seems to play a minor role in the exchange of endolymph between the cochlea and vestibule under normal conditions. The saccule wall appears highly flexible, which may explain occult hydrops occasionally preceding symptoms in MD on magnetic resonance imaging (MRI). The design of the UEV and connecting ducts suggests that there is a reciprocal exchange of fluid among the utricle, semicircular canals, and the EDS. Based on the anatomic framework and previous experimental data, we speculate that precipitous vestibular symptoms in MD arise from a sudden increase in endolymph pressure caused by an uncontrolled endolymphatic sac secretion. A rapid rise in UD pressure, mediated along the fairly wide UEV, may underlie the acute vertigo attack, refuting the rupture/K+-intoxication theory.

X-Ray Ptychography with a Laboratory Source.

X-ray ptychography has revolutionized nanoscale phase contrast imaging at large-scale synchrotron sources in recent years. We present here the first successful demonstration of the technique in a small-scale laboratory setting. An experiment was conducted with a liquid metal-jet x-ray source and a single photon-counting detector with a high spectral resolution. The experiment used a spot size of 5 μm to produce a ptychographic phase image of a Siemens star test pattern with a submicron spatial resolution. The result and methodology presented show how high-resolution phase contrast imaging can now be performed at small-scale laboratory sources worldwide.

Deep learning for high-resolution and high-sensitivity interferometric phase contrast imaging

In Talbot-Lau interferometry, the sample position yielding the highest phase sensitivity suffers from strong geometric blur. This trade-off between phase-sensitivity and spatial resolution is a fundamental challenge in such interferometric imaging applications with either neutron or conventional x-ray sources due to their relatively large beam-defining apertures or focal spots. In this study, a deep learning method is introduced to estimate a high phase-sensitive and high spatial resolution image from a trained neural network to attempt to avoid the trade-off for both high phase-sensitivity and high resolution. To realize this, the training data sets of the differential phase contrast images at a pair of sample positions, one of which is close to the phase grating and the other close to the detector, are numerically generated and are used as the inputs for the training data set of a generative adversarial network. The trained network has been applied to the real experimental data sets from a neutron grating interferometer and we have obtained improved images both in phase-sensitivity and spatial resolution.

Synthesis of anisotropic Au–Cu alloy nanostructures and its application in SERS for detection of methylene blue

Au–Cu alloy nanostructures have been synthesized in aqueous phase through co-reduction of HAuCl4.3H2O and CuCl2.2H2O by glucose in presence of hexadecylamine at ∼80 °C. By changing the synthesis conditions, nanostructures of various shapes such as nanowires, multiply twinned tripod, tetrapod, etc were observed. Systematic variation of the synthesis condition not only leads to change in size and particle morphology but also develops various other crystallographic characters in the nanoparticles. Alloying behavior of Au–Cu has been examined through transmission electron microscope operating in its conventional and analytical modes coupled with high resolution phase contrast microscopy. These results suggested that nanostructures are composed of homogeneous Au–Cu alloy. Preferential attachment along {111} and {100} crystallographic facets of Au–Cu alloy nanoparticles led to the formation of nanowires. Multiply twinned branched shape Au–Cu (width of branch ∼30 nm) nanostructures exhibit localized surface plasmon resonance maxima in the near-infrared region. The branched shape Au–Cu alloy nanostructures display better surface enhanced Raman scattering response in the detection of methylene blue as compared to spherical Au nanoparticles.

Pneumatic unidirectional cell stretching device for mechanobiological studies of cardiomyocytes

In this paper, we present a transparent mechanical stimulation device capable of uniaxial stimulation, which is compatible with standard bioanalytical methods used in cellular mechanobiology. We validate the functionality of the uniaxial stimulation system using human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). The pneumatically controlled device is fabricated from polydimethylsiloxane (PDMS) and provides uniaxial strain and superior optical performance compatible with standard inverted microscopy techniques used for bioanalytics (e.g., fluorescence microscopy and calcium imaging). Therefore, it allows for a continuous investigation of the cell state during stretching experiments. The paper introduces design and fabrication of the device, characterizes the mechanical performance of the device and demonstrates the compatibility with standard bioanalytical analysis tools. Imaging modalities, such as high-resolution live cell phase contrast imaging and video recordings, fluorescent imaging and calcium imaging are possible to perform in the device. Utilizing the different imaging modalities and proposed stretching device, we demonstrate the capability of the device for extensive further studies of hiPSC-CMs. We also demonstrate that sarcomere structures of hiPSC-CMs organize and orient perpendicular to uniaxial strain axis and thus express more maturated nature of cardiomyocytes.

The application of harmonic techniques to enhance resolution in mesh-based x-ray phase imaging

X-ray phase-contrast imaging produces significantly higher contrast than the conventional attenuation-based imaging. However, phase contrast typically requires small low-power sources or precisely machined and aligned gratings to generate the required spatial coherence. To overcome this limitation, we use a simple wire mesh to produce a periodic intensity pattern in the illumination. Distortions in this pattern upon propagation through an object can be used to produce phase contrast images. Unlike Talbot-grating-based techniques, this directly provides a measure of the phase gradient. Due to periodicity, fast Fourier transforms enable rapid retrieval of the phase contrast image. The high contrast of the mesh pattern allows a significant relaxation of the coherence requirement. However, the resolution of the phase contrast image is normally limited by the mesh period. We demonstrate that employing wider windowing functions during the Fourier processing substantially improves the spatial resolution at the cost of introducing artifacts in the reconstructed images. In order to remove these artifacts, a method is presented to combine images acquired while spatially shifting the mesh. Experimental results demonstrate the successful removal of these artifacts. This offers a method for significant resolution improvement in mesh-based phase-contrast imaging, enabling high resolution phase contrast images with a significant relaxation of source coherence requirements.

Synthesis of rod-shaped Au-Cu intermetallic nanoparticles and SERS detection

Intermetallic phases in Au-Cu nanoparticles has been synthesized chemically in liquid phase. The heat-treatment of these nanoparticles at 180 °C for 1 h in solution phase led to the formation of Au-Cu alloy nanoparticles with nearly spherical shapes and monodispersed size of ∼10 nm. Subsequent heat-treatment at 300 °C for 30 min of these Au-Cu alloy nanoparticles not only revealed structural transformation to intermetallic phases (tetragonal AuCu (tP4) and cubic Cu3Au (cP4)) but also shape change occurs from spherical to rods having average aspect ratio ∼3.0. The structural determination at the particle has been carried out through nano-beam electron diffraction coupled with simulation of electron diffraction patterns and high resolution phase contrast images. These Au–Cu intermetallic nanoparticles exhibit an excellent surface enhanced Raman spectroscopic activity with methylene blue compared to that of Au-Cu alloy nanoparticles.

Evidence for spinodal decomposition in Ti-15Mo quenched alloy using transmission electron microscopy

Electron diffraction effect associated with nano-scale periodic modulations observed in a quenched Ti-15 wt%Mo alloy within ω phase forming regime has been investigated in this study by analyzing selected area diffraction patterns and high-resolution phase contrast images. Crystallographic direction of the side bands in electron diffraction associated with the periodic modulation of wavelength 1.3 nm is established as [001]β which is an elastically soft direction for the body-centered cubic lattice. Characteristics of the side bands in the diffraction patterns, such as the correlation of spacing of satellite spots with real-space modulation wavelength and the constancy in the direction of satellites and modulations are interpreted as the proof of phase separation by spinodal decomposition. The striations associated with spinodal decomposition form only in Mo-enriched regions of dimensions ˜ 20 nm distributed throughout the β matrix. Accordingly, in this study, two varieties of compositional modulations that appear in the matrix are experimentally illustrated: (1) Mo-depleted and Mo-enriched domains of 10–20 nm size, and (2) The anisotropic compositional modulation by spinodal decomposition in [001]β direction with a modulation wavelength of 1.3 nm occurring in Mo-enriched pockets. It is confirmed that the structural modulations and ω phase formation occurs only in the regions without striations (Mo-lean regions). By analyzing the diffraction spots, it is also shown that the ω phase does not undergo any phase separation by spinodal decomposition.

Fractal Dimension Analysis of High-Resolution X-Ray Phase Contrast Micro-Tomography Images at Different Threshold Levels in a Mouse Spinal Cord

Fractal analysis is a powerful method for the morphological study of complex systems that is increasingly applied to biomedical images. Spatial resolution and image segmentation are crucial for the discrimination of tissue structures at the multiscale level. In this work, we have applied fractal analysis to high-resolution X-ray phase contrast micro-tomography (XrPCμT) images in both uninjured and injured tissue of a mouse spinal cord. We estimated the fractal dimension (FD) using the box-counting method on tomographic slices segmented at different threshold levels. We observed an increased FD in the ipsilateral injured hemicord compared with the contralateral uninjured tissue, which was almost independent of the chosen threshold. Moreover, we found that images exhibited the highest fractality close to the global histogram threshold level. Finally, we showed that the FD estimate largely depends on the image histogram regardless of tissue appearance. Our results demonstrate that the pre-processing of XrPCμT images is critical to fractal analysis and the estimation of FD.

Laser Use in Endodontic for Increase the Adhesion of Root Canal Filling. A synchrotron radiation micro tomography study

The aim of the present paper is to explore, through a high-resolution synchrotron radiation-based micro computed tomography experiment, the efficiency of the Er:YAG laser involvement in the debridement of the root canal of pathologic teeth, applied after the chemo-mechanical treatment of the canal. The adhesion quality of root canal filling is the consequence of the root canal cleaning and shaping, thus preventing the leakage. Twelve extracted single-rooted teeth were considered. The first 5 mm of the roots, corresponding to the apical region of the teeth, were initially investigated through high-resolution synchrotron radiation phase-contrast micro computed tomography. Afterwards, a chemo-mechanical endodontic treatment was performed and the same region of the teeth was imaged again. Finally, an Er:YAG laser with PIPS tip treatment was applied on the same teeth and a last tomographic scan was done. The chemo-mechanical treatment properly removed the necrotic tissue only in the upper part of the apical area of root canal, but tissue debris were found in the lower and partially also in the medium and upper part of the apical region, especially when the apical area exhibited a larger curvature. The laser treatment applied afterwards gave good results in terms of removing the debris from the apical area of root canal, mainly when they remained in the main canal. The present study proves the capability of Er:YAG with PIPS tips laser treatment to enhance the removing of tissue debris from the apical region of root canals after a chemo-mechanical treatment. The high-resolution synchrotron radiation-based phase contrast micro computed tomography is the most powerful tool for a non-destructive 3D investigation method of the root canal morphology analysis and a proper characterization technique for the assessment of the endodontic treatment.