Synchrotron Phase-contrast Imaging Research Articles

A novel therapeutic pathway to the human cochlear nerve.

Traditional approaches to the human cochlear nerve have been impeded by its bony encasement deep inside the skull base. We present an innovative, minimally invasive, therapeutic pathway for direct access to the nerve to deliver novel regenerative therapies. Neuroanatomical studies on 10 cadaveric human temporal bones were undertaken to identify a potentially safe therapeutic pathway to the cochlear nerve. Simulations based on three-dimensional delineation of anatomical structures obtained from synchrotron phase-contrast imaging were analyzed. This enabled the identification of an approach to the nerve in the fundus of the internal auditory meatus by trephining the medial modiolar wall of the cochlea via the round window for a median depth of 1.48mm (range 1.21-1.91mm). The anatomical access was validated on 9 additional human temporal bones using radio-opaque markers and contrast injection with micro-computed tomography surveillance. We thus created an effective conduit for the delivery of therapeutic agents to the cochlear nerve.

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.

Unlocking the human inner ear for therapeutic intervention

The human inner ear contains minute three-dimensional neurosensory structures that are deeply embedded within the skull base, rendering them relatively inaccessible to regenerative therapies for hearing loss. Here we provide a detailed characterisation of the functional architecture of the space that hosts the cell bodies of the auditory nerve to make them safely accessible for the first time for therapeutic intervention. We used synchrotron phase-contrast imaging which offers the required microscopic soft-tissue contrast definition while simultaneously displaying precise bony anatomic detail. Using volume-rendering software we constructed highly accurate 3-dimensional representations of the inner ear. The cell bodies are arranged in a bony helical canal that spirals from the base of the cochlea to its apex; the canal volume is 1.6 μL but with a diffusion potential of 15 μL. Modelling data from 10 temporal bones enabled definition of a safe trajectory for therapeutic access while preserving the cochlea’s internal architecture. We validated the approach through surgical simulation, anatomical dissection and micro-radiographic analysis. These findings will facilitate future clinical trials of novel therapeutic interventions to restore hearing.

Synchrotron phase-contrast imaging applied to the anatomical study of the hand and its vascularization.

Microscopic anatomical study of the hand requires difficult or destructive dissection techniques for each anatomical structure. Synchrotron phase-contrast imaging (sPCI) allows us to study precisely, at a microscopic resolution and in a nondestructive approach, the soft tissues and bone structures within a single 3D image. Therefore, we aimed to assess the capacity of sPCI to study the arterial anatomy of the hand and digits in human cadavers for anatomical purposes. A non-injected hand from an embalmed body was imaged using sPCI at 21-µm pixel size. The vascularization and innervation of the hands were virtually reconstructed at 84-µm resolution, and the medial neurovascular bundle of the third digit at 21µm. The thinner-most distal structures were observed and reported. The diameter and thickness of the vascular and neural structures were defined on 2D computed tomographic axial projections, and using a granulometry method coupled to the 3D reconstructions. The vascularization of the hand was visible from the radial and ulnar arteries to the distal digital transverse anastomoses. The thinnest structure observed was the anastomotic arterial network around the proper palmar digital nerve. The latter emerged from the proper palmar digital artery and vascularized the nerve around its whole length and circumference. The perineural arterioles individualizable at this resolution had a diameter of 66-309µm. In conclusion, sPCI allows both the arterial and neural anatomy of the hand to be studied at the same time, as well as the anatomical interactions between both networks. It facilitates the study of structures that have different sizes, diameters, thickness, and histological origin with great precision, in a noninvasive way, and using a single technique.

Direct observation on supersonic microprojectile penetration of carbon fiber composites with ultrafast synchrotron X-ray phase contrast imaging

High-speed penetration into carbon fiber composites is of fundamental importance to materials science and impact engineering, but research along this line suffers considerably from the lack of direct experimental observations. Here we investigate such penetration dynamics of a unidirectional carbon fiber reinforced epoxy (UCFRE) composite, with a combination of in situ, ultrafast, synchrotron phase contrast imaging and finite element (FE) analysis. The experiments yield the first direct observation on projectile trajectories and fiber-scale deformation and damage in the UCFRE composites during supersonic microprojectile penetration, for different fiber orientations (0∘−90∘ from the impact direction) and projectile velocities 600−850m.s-1, at unprecedented temporal (∼100 ps) and spatial (5 μm) resolutions. The maximum penetration depth decreases with increasing fiber orientation angles, as a result of anisotropic damage evolution in the composite sample. Strain localizations are prone to develop along a direction perpendicular to the fiber orientation, while the damage or cavity region, along the fiber direction. FE modeling with a three-dimensional Hashin criterion yields consistent projectile trajectory and cavity morphology with the experimental results. With increasing fiber orientation angles, damage analyses show a transition in the damage mode from fiber compression to matrix compression damage, in line with the increasing maximum penetration depth.

Vascular Supply of the Human Spiral Ganglion: Novel Three-Dimensional Analysis Using Synchrotron Phase-Contrast Imaging and Histology

Human spiral ganglion (HSG) cell bodies located in the bony cochlea depend on a rich vascular supply to maintain excitability. These neurons are targeted by cochlear implantation (CI) to treat deafness, and their viability is critical to ensure successful clinical outcomes. The blood supply of the HSG is difficult to study due to its helical structure and encasement in hard bone. The objective of this study was to present the first three-dimensional (3D) reconstruction and analysis of the HSG blood supply using synchrotron radiation phase-contrast imaging (SR-PCI) in combination with histological analyses of archival human cochlear sections. Twenty-six human temporal bones underwent SR-PCI. Data were processed using volume-rendering software, and a representative three-dimensional (3D) model was created to allow visualization of the vascular anatomy. Histologic analysis was used to verify the segmentations. Results revealed that the HSG is supplied by radial vascular twigs which are separate from the rest of the inner ear and encased in bone. Unlike with most organs, the arteries and veins in the human cochlea do not follow the same conduits. There is a dual venous outflow and a modiolar arterial supply. This organization may explain why the HSG may endure even in cases of advanced cochlear pathology.

Incus Necrosis and Blood Supply: A Micro-CT and Synchrotron Imaging Study.

Incus necrosis is a common complication following stapes surgery and is associated with impaired microcirculation. The objective of this study was to investigate the vascular anatomy of the human incus by using light microscopy, micro-computed tomography (micro-CT), and synchrotron phase-contrast imaging (SR-PCI) for a novel three-dimensional (3D) analysis of the middle ear, mucosal folds, major vascular pathways, and intraosseous vascular bone channels. One-hundred-and-fifty temporal bones from the Uppsala collection were analyzed under light microscopy. Twenty temporal bones underwent high-resolution micro-CT scanning, and an additional seven specimens underwent SR-PCI at the Canadian Lightsource in Saskatoon, Canada. One of these specimens was from an individual who had undergone stapes surgery. Data were processed with volume-rendering software to create 3D reconstructions using scalar opacity mapping for bone transparency, cropping, and soft tissue analyses. Micro-CT and SR-PCI with 3D rendering revealed the extensive vascular plexus within the un-decalcified incus bone communicating with the exterior surface. The relationship between the vessels, lenticular process, and incudostapedial joint were clearly observed. SR-PCI allowed for histologic-level detail while preserving the specimen and its 3D relationships. SR-PCI with 3D reconstructions confirmed the main vascular supply to the lenticular process along the intraosseous lenticular vessels. This is the first synchrotron analysis of a patient having undergone stapes surgery, and it suggests that incus necrosis associated with stapes surgery may be caused by a disruption of the lenticular blood flow induced by the prosthesis loop, and not by strangulation of mucosal vessels as has been previously described.

Three-dimensional imaging of the human internal acoustic canal and arachnoid cistern: a synchrotron study with clinical implications.

A thorough knowledge of the gross and micro‐anatomy of the human internal acoustic canal (IAC) is essential in vestibular schwannoma removal, cochlear implantation (CI) surgery, vestibular nerve section, and decompression procedures. Here, we analyzed the acoustic‐facial cistern of the human IAC, including nerves and anastomoses using synchrotron phase contrast imaging (SR‐PCI). A total of 26 fresh human temporal bones underwent SR‐PCI. Data were processed using volume‐rendering software to create three‐dimensional (3D) reconstructions allowing soft tissue analyses, orthogonal sectioning, and cropping. A scalar opacity mapping tool was used to enhance tissue surface borders, and anatomical structures were color‐labeled for improved 3D comprehension of the soft tissues. SR‐PCI reproduced, for the first time, the variable 3D anatomy of the human IAC, including cranial nerve complexes, anastomoses, and arachnoid membrane invagination (acoustic‐facial cistern; an extension of the cerebellopontine cistern) in unprocessed, un‐decalcified specimens. An unrecognized system of arachnoid pillars and trabeculae was found to extend between the arachnoid and cranial nerves. We confirmed earlier findings that intra‐meatal vestibular schwannoma may grow unseparated from adjacent nerves without duplication of the arachnoid layers. The arachnoid pillars may support and stabilize cranial nerves in the IAC and could also play a role in local fluid hydrodynamics.

Unveiling 3D physicochemical changes of sugarcane bagasse during sequential acid/alkali pretreatments by synchrotron phase-contrast imaging

Several pretreatment strategies focus on the removal of a significant part of lignin from plant cell walls, as it is considered the main barrier to the process of deconstructing biomass to simple sugars by hydrolytic enzymes. The ability to chemically differentiate and spatially locate lignins across cell walls provides an important contribution to the effort to improve these processes. Here, we present a novel approach using synchrotron phase-contrast tomography to probe the physicochemical features of plant cell walls. In addition to the 3D cellular architecture, the distribution of dense packed lignin–carbohydrate complexes (or, simply dense lignin) over larger regions and within cell walls has been successfully provided. We examined in particular the effect of the sequential H2SO4 and NaOH pretreatment on sugacane bagasse. Our results revealed that aggregates of dense lignin form elongated 3D structures in cell corners (CC) following the orientation of the sclerenchyma fibers; a remarkable result in the light of the conventional perception that lignin particles are discrete and roughly spherical. After the acid pretreatment, a considerable fraction of the dense lignin primarily located in CCs was removed; and it was further dissolved with the subsequent alkali treatment. Unexpectedly, the two-step pretreatment did not contribute to the cellulose accessibility in terms of available surface area and porosity. This study provides new insights into the underlying mechanisms of biomass deconstruction by pretreatments; and the approach established here can be extended to other systems relevant to the bioenergy and biotechnology arenas.

Combined use of X-ray fluorescence microscopy, phase contrast imaging for high resolution quantitative iron mapping in inflamed cells

X-ray fluorescence microscopy (XRFM) is a powerful technique to detect and localize elements in cells. To derive information useful for biology and medicine, it is essential not only to localize, but also to map quantitatively the element concentration. Here we applied quantitative XRFM to iron in phagocytic cells. Iron, a primary component of living cells, can become toxic when present in excess. In human fluids, free iron is maintained at 10-18 M concentration thanks to iron binding proteins as lactoferrin (Lf). The iron homeostasis, involving the physiological ratio of iron between tissues/secretions and blood, is strictly regulated by ferroportin, the sole protein able to export iron from cells to blood. Inflammatory processes induced by lipopolysaccharide (LPS) or bacterial pathoge inhibit ferroportin synthesis in epithelial and phagocytic cells thus hindering iron export, increasing intracellular iron and bacterial multiplication. In this respect, Lf is emerging as an important regulator of both iron and inflammatory homeostasis. Here we studied phagocytic cells inflamed by bacterial LPS and untreated or treated with milk derived bovine Lf. Quantitative mapping of iron concentration and mass fraction at high spatial resolution is obtained combining X-ray fluorescence microscopy, atomic force microscopy and synchrotron phase contrast imaging.

Thermal and Lattice Misfit Stress Relaxation in Growing AlN Crystal with Simultaneous Evaporation of SiC Substrate

AlN single crystals were prevented from cracking by simultaneous growth and evaporation of SiC substrates. The freestanding crystals (<1 mm thick) were proved continuous by synchrotron phase contrast imaging and used as a model system to investigate the type of dislocation structure near AlN/SiC interface by x-ray diffraction techniques. We have found that, unlike the situation in GaN films, where predominantly edge-type threading dislocations cross the layer along its normal, the dislocations configure to form mosaic structure. We suggest a theoretical model that describes the misfit stress relaxation in growing AlN crystal.

In-line phase-contrast and grating-based phase-contrast synchrotron imaging study of brain micrometastasis of breast cancer.

Current bio-medical imaging researches aim to detect brain micrometastasis in early stage for its increasing incidence and high mortality rates. Synchrotron phase-contrast imaging techniques, such as in-line phase-contrast (IPC) and grating-based phase-contrast (GPC) imaging, could provide a high spatial and density imaging study of biological specimens' 3D structures. In this study, we demonstrated the detection efficiencies of these two imaging tools on breast cancer micrometastasis in an ex vivo mouse brain. We found that both IPC and GPC can differentiate abnormal brain structures induced by micrometastasis from the surrounding normal tissues. We also found that GPC was more sensitive in detecting the small metastasis as compared to IPC.

High-speed synchrotron X-ray phase contrast imaging for analysis of low-Z composite microstructure

The study of high performance composites such as plastic-bonded explosives under extreme conditions often requires innovative experimental techniques. Here, static synchrotron X-ray phase-contrast imaging (PCI) of simulated explosive materials has been performed at high speed in an effort to determine feasibility of imaging material response to dynamic, high-strain rate events (102–107s−1). The microstructure of pristine materials, idealized composites and simulated explosive composites has been characterized with synchrotron PCI at the Advanced Photon Source. High spatial resolution (2μm) of the microstructure was achieved with 5μs exposures, and features such as interfaces, cracks, voids, and bubbles were clearly observed. The likelihood of obtaining sufficient phase information at even faster exposures (e.g., 0.2–0.5μs) is shown to be high.

Bone vascularization and growth in placoderms (Vertebrata): The example of the premedian plate of Romundina stellina Ørvig, 1975

The Placodermi (armored jawed fishes), which appeared during the Lower Silurian and disappeared without leading any descendants at the end of the Famennian (Latest Devonian), have the highest diversity of known Devonian vertebrate groups. As phylogenetically basal gnathostomes (jawed vertebrates), they are potentially informative about primitive jawed vertebrate anatomy and origins. Until recently, the study of their internal or histological structures has required destructive methods such as sectioning or serial grinding. Recent advances in tomography and imaging technologies, especially through the increasing use of synchrotron phase contrast imaging for the study of fossils, allow us to reveal the inner structures of the fossil nondestructively and with unprecedented three-dimensional level of detail. Here, we present for the first time the prerostral anatomy of the small acanthothoracid Romundina stellina, one of the earliest and most basal placoderms. Phase contrast imaging allows us to reconstruct the vascularization and nerve canals of the premedian plate and adjacent parts of the skeleton three-dimensionally in great detail, providing important clues to the growth modes and biology of the animal.

Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function

BackgroundDespite advances in imaging techniques, real-time visualization of the structure and dynamics of tissues and organs inside small living animals has remained elusive. Recently, we have been using synchrotron x-rays to visualize the internal anatomy of millimeter-sized opaque, living animals. This technique takes advantage of partially-coherent x-rays and diffraction to enable clear visualization of internal soft tissue not viewable via conventional absorption radiography. However, because higher quality images require greater x-ray fluxes, there exists an inherent tradeoff between image quality and tissue damage.ResultsWe evaluated the tradeoff between image quality and harm to the animal by determining the impact of targeted synchrotron x-rays on insect physiology, behavior and survival. Using 25 keV x-rays at a flux density of 80 μW/mm-2, high quality video-rate images can be obtained without major detrimental effects on the insects for multiple minutes, a duration sufficient for many physiological studies. At this setting, insects do not heat up. Additionally, we demonstrate the range of uses of synchrotron phase-contrast imaging by showing high-resolution images of internal anatomy and observations of labeled food movement during ingestion and digestion.ConclusionSynchrotron x-ray phase contrast imaging has the potential to revolutionize the study of physiology and internal biomechanics in small animals. This is the only generally applicable technique that has the necessary spatial and temporal resolutions, penetrating power, and sensitivity to soft tissue that is required to visualize the internal physiology of living animals on the scale from millimeters to microns.