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.

Keywords: 3D visualization; cartilage; embryonic development; x-ray computed tomography.

Figure 1.

Illustration of PTA staining protocol.

Figure 2.

A PTA-stained E14.5 mouse embryo scanned at a resolution of 6 microns. A) Volume rendering of whole PTA stained embryo. B) 2D parasagittal slice of PTA stained embryo. C) Same slice as in B, with 3D reconstructions of the brain and spinal cord (transparent yellow), ventricular system (pale purple), heart (red), lungs (blue), and liver (deep purple).

Figure 3.

Volume rendering of an E17.5 mouse (top) showing the approximate location of the coronal (left) and parasagittal (right) 2D slices of PTA-stained scanned specimens for four developmental ages (bottom). Voxel sizes for each scan were 0.006 mm for E15.5, 0.007 mm for E17.5 and P0, and 0.011 mm for P7, and all 2D slices are shown in the same scale. Note the close association between the brain and developing skull, indicating reduced tissue shrinkage.

Figure 4.

3D reconstructions of mouse brains shown to scale at E15.5, E17.5, P0, and P7. Reconstructions were created in Avizo 9.4, using the segmentation editor. Brain regions were manual segmented at every 5–10 slices and the interpolated for the intervening slices.

Figure 5.

A) Volume rendering of the PTA-stained head of an E17.5 CD1 mouse (surface in gray) and 3D reconstruction of the chondrocranium and Meckel’s cartilage (teal). Red box indicates the area of enlargement in B and C. Note that no smoothing algorithms were used in order to present the raw data. B) Enlargement of the anterior portion of the chondrocranium (including the nasal capsule and ala orbitalis) and Meckel’s cartilage (teal). C) Addition of bone (nasal bones, premaxilla, maxilla, frontal, above and dentary, below) (magenta) segmented from the same PTA-stained scan, showing the relationship between the chondrocranium and Meckel’s cartilage and forming bone. Incisors are developing within their crypts at this stage.

Figure 6.

Heart (top row), liver (middle row), and lungs (bottom row) of an E14.5 C57BL/6J mouse embryo scanned at a resolution of 0.006 mm. Left column: Anterior views of 3D reconstructions of the organs. Middle column: Anterior views of 3D reconstructions of the organs made transparent to visualize the spaces with the organs. Right column: Individual 2D transverse slices from the microCT scan, with the heart shaded in red, liver shaded in purple, and lungs shaded in blue. White arrows indicate spaces between the organs and body wall, indicating soft tissue shrinkage. All scale bars are 2 mm.

Figure 7.

Process for embedding a specimen in wax for scanning. A) Tools used during the embedding process, including several sizes of tubes and dissection tools. B) A 5 mm layer of the 50:50 wax mix is injected into the bottom of a metal tube using a syringe preheated to 70°C. C) The specimen is gently dipped in the wax mix five times. D) The specimen is placed nose down into the tube, on top of the 5 mm base layer of wax mix. E) Additional wax mix is injected to completely surround the specimen. F) Once the wax mix has fully solidified (approximately 5–7 minutes), the sample is gently pushed out of the metal case. G) The final sample of an E17.5 specimen embedded in wax and ready for scanning.