Imaging anatomical features of the human brain at cellular resolution currently relies on series of physical sections with related slicing artefacts. So far, microtomography has been employed to image an entire human brain at a voxel size of 20µm and selected regions using 6µm. This study aims to demonstrate the feasibility of imaging the entire human brain with cellular resolution without the need for physical sectioning using hard x-ray computed tomography. 1.2mm high sections of two human brains, one embedded in ethanol, the other in paraffin, were imaged using microtomography at the P07 beamline at DESY, Hamburg, Germany with a monochromatic beam at 67keV. The extended field of view necessary to cover the ca. 10 cm wide specimens at 2.54µm voxel size was realized by projection tiling with eight to ten rings. The resulting reconstructed slices measured 39,000×39,000 voxels. This synchrotron radiation-based study shows the feasibility of employing x-ray tomography to image the entire human brain with isotropic voxels of 2.54µm resolution. Next, we need to tackle the vertical stitching of several 10,000 slices of 6GB each, posing the challenge of processing the big data of an entire PB-sized human brain and making it accessible to the research community.
Transport of immune cells, nutrients and waste products via the cerebrospinal fluid (CSF) has been implicated in the development of neurological disorders. Using time-resolved in vivo microtomography, we investigated pulsatile motion of CSF spaces in the mouse brain as a potential driver of CSF flow. Here we present a method for detecting motion captured in murine brain images acquired in vivo at the European Synchrotron Radiation Facility. Anesthetized mice were placed in a heated holder that was designed to minimize head motion and maintain physiological body temperature. Contrast agent was infused into the ventricle to improve visibility of the CSF spaces. Projections were retrospectively sorted based on the ECG recording. Cardiac phase images were reconstructed in 10ms intervals from the ECG peak and automatically analyzed for decorrelation. Motion was automatically quantified by non-rigid registration. Regions with high intensity structures, large motion magnitudes, large improvements in image similarity due to registration, or at the contrast-enhanced ventricles were visually inspected for structures with motion artifacts prior to registration. We detected mainly motion in the nasopharynx, skin, ear channels, and bones in the range of 2.3 to 14.8µm magnitude. Small motion artifacts were detectable only for high-contrast structures. No misalignments were visible for the contrast-enhanced ventricles at a voxel resolution of 6.30 to 6.45µm. In the future, dedicated active gating to ensure regular sampling and local scans with improved spatial resolution will be used to investigate the limits to the detection of in vivo ventricular motion in mice.
The acquisition of large tomography volumes, exceeding the typical detector field-of-view, requires advanced acquisition techniques. Current approaches are the tiling of local reconstructed volumes or the tiling in projection space, also known as mosaic tomography. Reconstruction tiling has the advantage that standard reconstruction software can be used and acquisition can be interrupted and resumed relatively easily. The disadvantage is that there is the need for volume registration and transformation. Projection tiling is faster and more dose efficient, however a custom reconstruction pipeline is required, registration in projection space is challenging due to lower contrast, and there is a high sensitivity to mechanical instabilities. In this work we propose a third, hybrid approach, to profit from the advantages of projection tiling, but limit the risks. The volume to be imaged is covered by overlapping cylinders, each corresponding to the reconstructed volume of one mosaic tomogram. The number of rings per cylinder and the total number of cylinders can be tuned to the specimen at hand. We demonstrate this approach for a 2cm-wide section of a human brain stem, imaged at the Anatomix beamline of Synchrotron Soleil, France with 0.65µm voxel size, resulting in reconstructed slices 29,650 voxels wide. For mosaic reconstruction we used our team’s existing pipeline. For stitching of volumes, image registration was performed in the overlap regions. As pairwise displacements between cylinders are not independent, we modified the registration approach to force a consistent solution. The results of the hybrid acquisition in seven tiles with four rings were compared to a pure projection tiling approach with eight rings and to local regions representing reconstruction tiling. In conclusion, we propose an extended field of view acquisition scheme building on the speed and dose efficiency of mosaic acquisition, but relaxing the requirements for mechanical and beam stability.
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.
Caries is detected visually, often supported by palpation and conventional radiographs, and is characterized by reduced Xray absorption. The fringe regions of carious lesions, however, are hardly distinguished. Therefore, the treatment is usually suboptimal, because either a substantial part of healthy crown is removed or the carious lesion is still partially present. This in vitro study applied conventional micro computed tomography to quantify remaining carious tissue after treatment with respect to the volume of tissue mechanically removed. For this purpose, 16 teeth with 23 lesions were treated by an experienced dentist. The teeth were imaged before and after caries removal using a SKYSCAN 1275 system, Bruker, Belgium. Pre- and post-treatment datasets were rigidly registered. Manual segmentation gave rise to the volume of the remaining carious tissue. Twenty of the 23 lesions had a residual carious lesion that amounted to less than 3% of the mechanically erased tissue. Still, residual caries with volumes between 0.8 and 3.4 mm3 was found in three cases. Thus, more detailed microtomography studies are to be performed to give the desired feedback for caries therapy to clinicians.
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.
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.
The quantification of appearance and geometrical changes between structures captured across several images relies on the determination of correspondences between these image structures. Automatic methods for establishing sparse and dense spatial correspondences are feature tracking and image registration, respectively. Main registration challenges are weak image features, complex appearance change, large shape changes, and huge images. These challenges can make the problem intractable. We present approaches to overcome these challenges for registration tasks including microtomography images. Applications include assessing tissue changes due to embedding material, comparison of microtomography with optical microscopy images, and optimization of process parameters for materials.
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.
X rays have been used for medical imaging since RÖNTGEN's fascinating discovery 125 years ago. The first radiographs of human hands were made public less than a month after his famous paper. The conventional X-ray sources integrated into the CT-machines of today’s hospitals still rely on the same physical principles. X-ray imaging has traditionally offered high spatial resolution and low contrast for soft tissues such as the brain. Magnetic resonance imaging is therefore the method of choice for brain imaging in a clinical setting, although for cellular resolution studies it suffers from limited spatial resolution. The gold standard in post mortem brain imaging is histology, which involves fixation, embedding, physical sectioning, staining, and optical microscopy. Currently, section thickness limits isotropic voxel sizes to 20 μm. Advanced X-ray sources including synchrotron radiation facilities offer complementary modalities such as phase-contrast imaging and spatially resolved small-angle X-ray scattering. We showed that X-ray phase contrast of the human cerebellum with micrometer resolution yields complementary three-dimensional images to magnetic resonance microscopy with even better contrast and spatial resolution. Grating interferometry enabled us to visualize individual Purkinje cells in the nonstained cerebellum. Taking advantage of well-established paraffin embedding, Purkinje cells were visualized within the human cerebellum even with conventional instrumentation. Hard X-ray nano-holotomography allowed for label-free, three-dimensional neuroimaging beyond the optical limit with a spatial resolution below 100 nm. Spatially resolved smallangle X-ray scattering permitted the localization of periodic nanostructures such as myelin sheaths on square-inch brain slices and included the orientational information on the axons. These developments have contributed to the establishment of virtual histology and extended the conventional histology to the third dimension. Further advances are required to image the entire human brain with an isotropic micrometer resolution and to suitably handle the petabyte datasets.
Hard X-ray micro computed tomography can be used for three-dimensional histological phenotyping of zebrafish embryos down to 1 µm or below without the need for staining or physical slicing. Current advances in ze- brafish embryo imaging, however, mostly rely on synchrotron radiation sources or highly advanced laboratory sources, which despite their evident strengths with regard to their beam properties and the implementation of phase contrast imaging techniques, lack accessibility. Therefore, we evaluated the performance of a conventional SkyScan 1275 laboratory µCT scanner in absorption contrast mode for the visualization of anatomical features in ethanol- and paraffin-embedded zebrafish embryos. We compare our results to readily available synchrotron data where 35 anatomical structures were identified. Despite having a more than ten times larger voxel length, approximately two thirds of the features could also be determined with laboratory microtomography. This could allow to monitor morphological changes during development or disease progression on large sample numbers, enabling the performance of preclinical studies in a local laboratory.
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.
Successful tomographic imaging of soft tissues with micrometer resolution includes preparation, acquisition, re- construction, and data evaluation. Tissue preparation is essential for hard X-ray microtomography, because staining- and embedding materials can substantially alter the biological tissue post mortem. We performed to- mographic imaging of zebrafish embryos in alcohol and after paraffin embedding with a conventional X-ray source and at a synchrotron radiation facility. The resulting multi-modal, three-dimensional data were registered for direct comparison. Single-cell precision was reached for the synchrotron radiation-based approach, which allows for segmentation of full organs such as the embryonic kidneys. While this approach offers an order of magnitude higher spatial resolution, many of the anatomical features can be readily recognized with the more accessible laboratory system. Propagation-based data acquisition enabled us to demonstrate the complementary nature of the edge-enhanced absorption contrast- and the phase contrast-based modality for visualizing multiple microanatomical features. While ethanol and paraffin embeddings allowed for identification of the same anatomical structures, paraffin-embedding, however, led to more artefacts and shrinkage. The presented multi-modal imaging approaches can be further extended to visualize three to four orders of magnitude larger volumes such as adult zebrafish or complete organs of larger animals such as mouse brains. Going towards even larger volumes, such as the human brain, presents further challenges related to paraffin embedding, data acquisition and handling of the peta-byte scale data volumes. This study provided a multi-modal imaging strategy by the combination of X-ray sources and sample embeddings which can play a role in addressing these challenges.
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.
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