An optimized workflow for microCT imaging of formalin‐fixed and paraffin‐embedded (FFPE) early equine embryos

Abstract

Here, we describe a workflow for high‐detail microCT imaging of formalin‐fixed and paraffin‐embedded (FFPE) equine embryos recovered on Day 34 of pregnancy (E34), a period just before placenta formation. The presented imaging methods are suitable for large animals' embryos with intention to study morphological and developmental aspects, but more generally can be adopted for all kinds of FFPE tissue specimens. Microscopic 3D imaging techniques such as microCT are important tools for detecting and studying normal embryogenesis and developmental disorders. To date, microCT imaging of vertebrate embryos was mostly done on embryos that have been stained with an X‐ray dense contrast agent. Here, we describe an alternative imaging procedure that allows to visualize embryo morphology and organ development in unstained FFPE embryos. Two aspects are critical for high‐quality data acquisition: (i) a proper sample mounting leaving as little as possible paraffin around the sample and (ii) an image filtering pipeline that improves signal‐to‐noise ratio in these inherently low‐contrast data sets. The presented workflow allows overview imaging of the whole embryo proper and can be used for determination of organ volumes and development. Furthermore, we show that high‐resolution interior tomographies can provide virtual histology information from selected regions of interest. In addition, we demonstrate that microCT scanned embryos remain intact during the scanning procedure allowing for a subsequent investigation by routine histology and/or immunohistochemistry. This makes the presented workflow applicable also to archival paraffin‐embedded material.

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

“…As both OCT and micro-CT are non-destructive, they lend themselves to integrating even further imaging modalities [21,29,74,75]. In such a workflow, micro-CT imaging will provide complete, isotropic 3D data sets enabling easy co-registration and co-localizations of ROIs in OCT data with either histology or immunohistochemistry, allowing for subsequent downstream analysis [29,43].…”

“…This allows the visualization and quantitative description of the structure and microstructure of healthy and diseased cardiovascular tissues during development and in adults using micro-CT. General contrast stains have been in use for over a decade now, and many of them already allowed for valuable insights into heart tissue substructures, such as myocardial fiber direction, or localization of collagen and elastin [38][39][40][41][42]; see [24] for an extensive review on different contrast agents already used in heart imaging and their characteristics. For further microscopic analyses, the sample preparation methods and the non-destructive nature of micro-CT allow for subsequent investigation by histology, scanning electron microscopy, immunohistochemistry, and immunofluorescence [43,44]. However, micro-CT imaging itself is moving in the direction of 3D histology with the development of new tissue-specific X-ray contrast enhancement protocols [45][46][47][48][49] and protocols showing that micro-CT can even be utilized for imaging of molecular signals [50,51].…”

Mouse embryo phenotyping using X-ray microCT

“…The size of the plastic container used for mounting should match specimen size, because excess medium such as PBS, agarose, or ethanol decreases the signal-to-noise ratio in projection images, particularly in samples with low intrinsic contrast. The adverse effect of excess paraffin coating on soft tissue contrast of unstained horse embryos has recently been demonstrated (Handschuh et al, 2022). Another important aspect in specimen mounting is the proper sealing of the mounting container.…”

A comprehensive study of micro-CT for 3D virtual histology of FFPE tissue blocks

“…The presence of cassette and wax around the tissue is typically unavoidable on standard FFPE block used in the pathology and they can contribute to artifacts and suboptimal conditions for the imaging. First, the slit-like cassette grid structure, gives rise to pronounced cone-beam artifacts [23] [24], and second, the wax around the tissue reduces contrast for the soft tissue [25]. There exists a basic user guide for soft-tissue X-ray imaging [26], as well as a coarse preliminary study on standard operating procedure for using micro-CT in a broader context in pathology [27].…”