Mouse embryo phenotyping using X-ray microCT

Abstract

Microscopic X-ray computed tomography (microCT) is a structural ex vivo imaging technique providing genuine isotropic 3D images from biological samples at micron resolution. MicroCT imaging is non-destructive and combines well with other modalities such as light and electron microscopy in correlative imaging workflows. Protocols for staining embryos with X-ray dense contrast agents enable the acquisition of high-contrast and high-resolution datasets of whole embryos and specific organ systems. High sample throughput is achieved with dedicated setups. Consequently, microCT has gained enormous importance for both qualitative and quantitative phenotyping of mouse development. We here summarize state-of-the-art protocols of sample preparation and imaging procedures, showcase contemporary applications, and discuss possible pitfalls and sources for artefacts. In addition, we give an outlook on phenotyping workflows using microscopic dual energy CT (microDECT) and tissue-specific contrast agents.

Enhanced contrast synchrotron X-ray microtomography for describing skeleton-associated soft tissue defects in zebrafish mutants

“…Our histopathological findings in bone-associated soft tissues obtained by applying DICE-PPC-SRµCT demonstrates a great potential of this technique for the investigation of small fish skeletal disease models involving gene mutants and regulatory sequence mutants that often display subtle phenotypes ( 66 ), and also for skeleton-associated regeneration experiments and general developmental studies of, for example, mineralization progression. For some questions, particularly those involving adult fish and large soft tissues, conventional µCT combined with contrasting agents may be sufficient, as has been already shown for mouse ( 67 ) and in a limited number of zebrafish studies (e.g. 22 , 68 ).…”

OCT Meets micro-CT: A Subject-Specific Correlative Multimodal Imaging Workflow for Early Chick Heart Development Modeling

“…OCT delivers dynamic in vivo anatomical and morphological information at acquisition speeds of up to multiple volumes per second [73], and also functional information if Doppler OCT is applied to detect axial blood flow velocity at the same spatiotemporal resolution as structural OCT [21]. Micro-CT data provides high-detail isotropic 3D data that is calibrated for both geometry and intensity and that can be acquired over a large number of developmental stages [29]. The combination of these modalities, however, not only overcome the limitations of isolated standard imaging approaches, but also provide unique and complementary information only achievable through the correlation of these data.…”

“…As micro-CT data is calibrated in geometry, it could, in theory, serve as an excellent tool for validating the geometry obtained from OCT imaging. However, tissue preparation necessary for micro-CT imaging can lead to significant tissue shrinkage, caused by the fixation and staining method used [29], among other things. However, tissue shrinkage can be minimized either by polymer perfusion [70] or by the combination of formaldehyde-fixation, hydrogel-embedding [71], and neutral-buffered iodine infiltration [72].…”

“…This is because OCT allows for non-invasive, label-free, and cross-sectional (B-scan) imaging of tissues in vivo with micrometer-scale spatial resolution [9,10]. So far, OCT is routinely used as a diagnostic tool how altered hemodynamic loads caused by certain treatments or interventions can lead to congenital heart defects: OCT imaging can inform about subject-specific changes to hemodynamics and overall heart geometry, while micro-CT can provide subsequent subjectspecific information needed to quantitatively describe (micro-)structural alterations to the heart [21,29].…”

Visualisation of gene expression within the context of tissues: an X-ray computed tomography-based multimodal approach

“…Due to its high penetration depth, X-ray computed tomography is particularly suitable for anatomical imaging of whole organisms with high-throughput (Matula et al 2021; Weinhardt et al 2018; dos Santos Rolo et al 2014; van de Kamp et al 2018). Based on absorption contrast agents, pan-resolution histology-like, also called histotomography volumes, are obtained in diverse specimens (Ding et al 2019; Busse et al 2018; Holme et al 2014), enabling phenotypic screens (Handschuh and Glösmann 2022; Schoborg et al 2019) and analysis of pathologies (Kavkova et al 2021; Eckermann et al 2020). We therefore decided to combine X-ray tomography and light-sheet microscopy that would achieve simultaneous localisation of fluorescence markers and tissue architecture with pan-resolution in hundreds of micrometre thick specimens.…”