Background

Transgenic mouse models have been extensively used to study the roles of specific genes in heart development. Historically, to analyze morphological defects on heart formation in gain-of-function and loss-of-function models, the formalin-fixed paraffin-embedded embryos were sectioned and analyzed section-by-section. X-ray computed microtomography (µCT) is a powerful tool for visualizing the 3D morphology of the heart and performing morphometric analysis. µCT uses X-rays to create cross-sections of a physical object that can be used to recreate 3D models, reaching very high resolution (<1 µm). Here, we describe a method to apply this technique to biological samples. Our purpose was to study the cardiovascular system in the developing mouse embryo. This method overcomes the limits of traditional histology, such as the difficulty to obtain comparable sections, especially when working with very small and complex samples.

For µCT analysis of embryos, it is mandatory to increase the contrast of tissues. To this end, different methods and contrast agents are described in the literature, with small differences in protocols. The main agents available to this purpose and tested by the authors are iodine solution or phosphotungstic acid (PTA) (Wong et al., 2012 and 2013; Hsu et al., 2016; Handschuh and Glösmann, 2022). Osmium tetroxide is also used as a contrast agent, but because of its high toxicity it has not been tested by the authors. Both approaches of staining were previously used to study cardiac defects (Degenhardt et al., 2010; Lesciotto et al., 2020; Jamet et al., 2022). In our experience, the use of 2%–5% PTA solution requires methanol (3%–10%) to properly penetrate tissues. The concentration of PTA and methanol can be adjusted depending on the size of sample; for example, for small mouse embryos (E12.5), 3% of methanol should be sufficient; for E15.5 embryos, 5% methanol; and for large mouse embryos (E18.5), 10% of methanol should be necessary. We successfully used this method for the staining of E13.5 mouse embryos (Petrillo et al., 2018). Although the PTA method offers excellent contrast, it may cause artifacts due to sample shrinkage in embryos older than E13.5. The maximum size of the object suitable for the µCT is 50 mm; however, increasing sample size will reduce the resolution.

On the contrary, iodine-based staining offers good balance in terms of sample preservation and soft tissue contrast, especially for larger samples. In our experiment, in E15.5 embryos we used a 20% Lugol’s solution and 0.5% Tween-20. To preserve mineral structures, pH can be adjusted to 7.3.