Comparative hard x-ray tomography for virtual histology of zebrafish larva, human tooth cementum, and porcine nerve.

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 - µ m 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.

Virtual histology of an entire mouse brain from formalin fixation to paraffin embedding. Part 2: Volumetric strain fields and local contrast changes.

BACKGROUND: Fixation and embedding of post mortem brain tissue is a pre-requisite for both gold-standard conventional histology and X-ray virtual histology. This process alters the morphology and density of the brain microanatomy. NEW METHOD: To quantify these changes, we employed synchrotron radiation-based hard X-ray tomography with 3 µm voxel length to visualize the same mouse brain after fixation in 4% formalin, immersion in ethanol solutions (50%, 70%, 80%, 90%, and 100%), xylene, and finally after embedding in a paraffin block. The volumetric data were non-rigidly registered to the initial formalin-fixed state to align the microanatomy within the entire mouse brain. RESULTS: Volumetric strain fields were used to characterize local shrinkage, which was found to depend on the anatomical region and distance to external surface. X-ray contrast was altered and enhanced by preparation-induced inter-tissue density changes. The preparation step can be selected to highlight specific anatomical features. For example, fiber tract contrast is amplified in 100% ethanol. COMPARISON WITH EXISTING METHODS: Our method provides volumetric strain fields, unlike approaches based on feature-to-feature or volume measurements. Volumetric strain fields are produced by non-rigid registration, which is less labor-intensive and observer-dependent than volume change measurements based on manual segmentations. X-ray microtomography provides spatial resolution at least an order of magnitude higher than magnetic resonance microscopy, allowing for analysis of morphology and density changes within the brain's microanatomy. CONCLUSION: Our approach belongs to three-dimensional virtual histology with isotropic micrometer spatial resolution and therefore complements atlases based on a combination of magnetic resonance microscopy and optical micrographs of serial histological sections.

Virtual histology of an entire mouse brain from formalin fixation to paraffin embedding. Part 1: Data acquisition, anatomical feature segmentation, tracking global volume and density changes.

BACKGROUND: Micrometer-resolution neuroimaging with gold-standard conventional histology requires tissue fixation and embedding. The exchange of solvents for the creation of sectionable paraffin blocks modifies tissue density and generates non-uniform brain shrinkage. NEW METHOD: We employed synchrotron radiation-based X-ray microtomography for slicing- and label-free virtual histology of the mouse brain at different stages of the standard preparation protocol from formalin fixation via ascending ethanol solutions and xylene to paraffin embedding. Segmentation of anatomical regions allowed us to quantify non-uniform tissue shrinkage. Global and local changes in X-ray absorption gave insight into contrast enhancement for virtual histology. RESULTS: The volume of the entire mouse brain was 60%, 56%, and 40% of that in formalin for, respectively, 100% ethanol, xylene, and paraffin. The volume changes of anatomical regions such as the hippocampus, anterior commissure, and ventricles differ from the global volume change. X-ray absorption of the full brain decreased, while local absorption differences increased, resulting in enhanced contrast for virtual histology. These trends were also observed with laboratory microtomography measurements. COMPARISON WITH EXISTING METHODS: Microtomography provided sub-10 µm spatial resolution with sufficient density resolution to resolve anatomical structures at each step of the embedding protocol. The spatial resolution of conventional computed tomography and magnetic resonance microscopy is an order of magnitude lower and both do not match the contrast of microtomography over the entire embedding protocol. Unlike feature-to-feature or total volume measurements, our approach allows for calculation of volume change based on segmentation. CONCLUSION: We present isotropic micrometer-resolution imaging to quantify morphology and composition changes in a mouse brain during the standard histological preparation. The proposed method can be employed to identify the most appropriate embedding medium for anatomical feature visualization, to reveal the basis for the dramatic X-ray contrast enhancement observed in numerous embedded tissues, and to quantify morphological changes during tissue fixation and embedding.