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. 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. 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. 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. 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.

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. 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. 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. 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. 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.

Orthodontic treatments with optically transparent aligners have seen a constant increase in acceptance in recent years. The low acceptance rates of conventional orthodontic devices in adult populations, due to a perceived lack of attractiveness, as well as workflows becoming easier for practitioners, can explain the rising demand for aligner treatments.^1,2 Orthodontic tooth movement is accomplished by employing prolonged pressure on that tooth. The initial movement is rapid, as it occurs within the dental alveolus. The periodontal ligament (PDL), which connects the tooth to the alveolar bone, is stretched on one side and compressed on the other side of the root so that a tension and a compression side can be differentiated.³ The disruption of the mechanical balance of PDL and bone leads to the recruitment of osteoclasts and osteoblasts in the vicinity of the tooth.⁴ These specialized cells are responsible for bone resorption on the compression side and bone apposition on the tension side, respectively.⁵ This bone remodeling usually occurs within 40 days after the initial force is applied.³

Minimal invasive tumor therapies are getting ever more sophisticated with novel treatment approaches and new devices allowing for improved targeting precision. Applying these effectively requires precise localization of the structures of interest. Vital processes, such as respiration and heartbeat, induce organ motion, which cannot be neglected during therapy. This review focuses on 4D organ models to compensate for respiratory motion during therapy. An overview is given on the effects of motion on the therapeutical outcome, methods required to capture and quantify respiratory motion, range of reported tumor motion, types of surrogates used when tumors are not directly observable, and methods for temporal prediction of surrogate motion. Organ motion models, which predict the location of structures of interest from surrogates measured during therapy, are discussed in detail.