MicroCT for developmental biology: A versatile tool for high‐contrast 3D imaging at histological resolutions

Abstract

Understanding developmental processes requires accurate visualization and parameterization of threedimensional embryos. Tomographic imaging methods offer automatically aligned and calibrated volumetric images, but the usefulness of X-ray CT imaging for developmental biology has been limited by the low inherent contrast of embryonic tissues. Here, I demonstrate simple staining methods that allow high-contrast imaging of embryonic tissues at histological resolutions using a commercial microCT system. Quantitative comparisons of images of chick embryos treated with different contrast agents show that three very simple methods using inorganic iodine and phosphotungstic acid produce overall contrast and differential tissue contrast for X-ray imaging at least as high as that obtained with osmium. The stains can be used after any common fixation and after storage in aqueous or alcoholic media, and on a wide variety of species. These methods establish microCT imaging as a useful tool for comparative developmental studies, embryo phenotyping, and quantitative modeling of development. Developmental Dynamics 238: 632-640, 2009.

X‐ray computed tomography and its potential in ecological research: A review of studies and optimization of specimen preparation

“…Samples are certainly more easily prepared for MRI than X‐ray CT, in particular for those studies which require metal‐based staining for soft tissue imaging (Metscher, 2009a, 2013). Nevertheless, magnetic resonance scanning systems are limited in resolution (Metscher, 2013; Metzner et al., 2015) and often have prohibitive costs of operation (Ziegler et al., 2011).…”

“…For small vertebrates (e.g., laboratory mice, zebra fish, and embryos), it is also common to store specimen in formalin (Cnudde et al., 2008; Hoshino et al., 2012; Kim et al., 2012; Metscher, 2009a,b; Seo et al., 2015). Sombke et al.…”

“…Although several combinations of methods for fixation, staining, and drying have previously been assessed and compared (Metscher, 2009a,b; Sombke et al., 2015), the authors generally used several species (vertebrate and invertebrate) and target tissues, and have carried out tomography without any phase retrieval. Here, we systematically compare different staining and fixation approaches, focusing on only one species, and consider in particular the more recent phase‐contrast modality.…”

“…PTA failed to stain the complete sample (neither head nor abdomen), and the few stained regions had very strong contrast, precluding adequate tissue recognition (Figure 4a‐d). The unstained parts of the samples exposed to PTA had minor contrast, resembling tissues that had not been exposed to any stain (Degenhardt et al., 2010; Metscher, 2009a), thus indicating that the tissue penetration was insufficient and samples would have to be exposed for more days to attain the desired stain.…”

“…(2016) and also this study—it might be considered when the sample tissue is thin or removal of parts of the exoskeleton is possible (to facilitate stain perfusion). While osmium tetroxide (OsO 4 ) has been a popular staining agent in previous X‐ray CT studies (Jahn et al., 2018; Kim et al., 2012; Metscher, 2009a,b; Ribi, Senden, Sakellariou, Limaye, & Zhang, 2008), we did not consider it because of its undesirable toxicity, high costs, limited tissue penetration, and failure to work properly in tissues preserved in alcohol (Hayat, 1970; Metscher, 2009a; Smith, Carson, & Ferguson, 1974; Smith et al., 2016). …”