Phosphotungstic acid‐enhanced microCT: Optimized protocols for embryonic and early postnatal mice

Abstract

Background Given the need for descriptive and increasingly mechanistic morphological analyses, contrast‐enhanced microcomputed tomography (microCT) represents perhaps the best method for visualizing 3D biological soft tissues in situ. Although staining protocols using phosphotungstic acid (PTA) have been published with beautiful visualizations of soft tissue structures, these protocols are often aimed at highly specific research questions and are applicable to a limited set of model organisms, specimen ages, or tissue types. We provide detailed protocols for micro‐level visualization of soft tissue structures in mice at several embryonic and early postnatal ages using PTA‐enhanced microCT. Results Our protocols produce microCT scans that enable visualization and quantitative analyses of whole organisms, individual tissues, and organ systems while preserving 3D morphology and relationships with surrounding structures, with minimal soft tissue shrinkage. Of particular note, both internal and external features of the murine heart, lungs, and liver, as well as embryonic cartilage, are captured at high resolution. Conclusion These protocols have broad applicability to mouse models for a variety of diseases and conditions. Minor experimentation in the staining duration can expand this protocol to additional age groups, permitting ontogenetic studies of internal organs and soft tissue structures within their 3D in situ position.

Multimodal Atlas of the Murine Inner Ear: From Embryo to Adult

“…Using these ex vivo imaging modalities, we were able to evaluate the inner ear within the intact whole head in two and three dimensions at these developmental stages. In the embryo, we performed Micro-CT following immersion in phosphotungstic acid (PTA), a radio dense contrast agent (11), allowing distinction of tissues through variable X-ray attenuation that would otherwise appear homogenous on Micro-CT or MRI.…”

ViceCT and whiceCT for simultaneous high-resolution visualization of craniofacial, brain and ventricular anatomy from micro-computed tomography

“…A drawback of whiceCT may be that the incubation time is prolonged to 18 days, as compared to diceCT protocols for mouse brain imaging that report only 1–2 days incubation to reach homogeneous contrast enhancement 30 – 32 . However, this incubation time is within an acceptable range, since more recent techniques employing PTA-enhanced µCT optimized for embryonic and early postnatal mice require even longer incubation periods in adult mice 34 . The advantage of the whiceCT protocol that outweighs the relatively long incubation time is that the craniofacial structure is conserved.…”

“…Alternative protocols have explored the use of selectively perfusable iodine-based contrast-enhanced computed tomography (spiceCT) to administer contrast to the entire mouse body leaving the entire anatomy intact including the craniofacial structure, but this strategy does not manage to provide contrast into the brain 33 . Imaging protocols using other contrast agents such as phosphotungstic acid (PTA) have enabled the visualization of bone and soft tissue from µCT scans with minimal shrinkage 34 , but have not yet been optimized for adult mice, are more costly and require more elaborated sample processing. Finally, the brain ventricles are not visible in any of these approaches, which implies an extra limitation for the neurodegenerative and neurodevelopmental disorders research fields, where the ventricular anatomy or volume are important biomarkers 35 – 38 .…”

CloudReg: Automatic Terabyte-Scale Cross-Modal Brain Volume Registration

“…16 We specifically developed algorithms for distributed local intensity correction and cross-modal registration while leveraging existing state-of-the-art, open-source tools. 12,13 We applied CloudReg to various datasets including in vivo human brain MRI, 17 ex vivo macaque brain MRI, 18,19 ex vivo in situ mouse brain micro-CT, 20,21 and LSM-imaged cleared mouse and rat brains. 3,4,5 Figure 1 shows an overview of the CloudReg pipeline.…”