17.06.2020: By using a new contrast agent in combination with dual-energy CT, researchers at Vetmeduni Vienna achieve a quantum leap in imaging. Due to the new method just published in the renowned journal "Development", the skeletal development in mouse and chicken embryos can be presented and analyzed much better than before by means of 3D-visualization and colored X-ray imaging.

For decades, clearing and staining with alcian blue and alizarin red has been the gold standard for depicting the skeletal development of vertebrates. In their now published study, a research team from  Vetmeduni Vienna presents a new workflow for the visualization and analysis of skeletal development by means of microscopic computed tomography (microCT). This allows real 3D-data of the entire developing skeleton to be collected with a resolution of micrometers. "For the first time, our method offers scientists the opportunity to study the development of cartilage and bone in the mouse embryo simultaneously and effectively in 3D. In addition, we can also include the development of other internal organs such as the brain or digestive tract in the evaluation," explains first author Simone Gabner from the Histology and Embryology working group of the Institute of Pathology at Vetmeduni Vienna. "The new method enables both the visualization and qualitative assessment of skeletal development as well as extensive quantitative analysis, i.e. measurements of the size and shape of individual skeletal elements," Gabner continues.

Ruthenium red as a new contrast agent

The novel protocol is based on ethanol fixation and staining with ruthenium red. This X-ray-dense dye efficiently contrasts the cartilage matrix, as the researchers were able to show on mouse fetuses and chicken embryos.  The bone mineral is well preserved during staining, so that the entire embryonic skeleton can be depicted with high contrast. Also new in this context is the use of dual-energy CT. This technology allows the spectral separation of cartilage and bone, which allows to generate colored X-ray images – in contrast to the well-known black-and-white X-ray images. Stephan Handschuh from the VetCore/Imaging Unit at Vetmeduni Vienna says: "Usually microCT produces black-and-white images, by computing virtual sectional images based on a series of X-ray images. With dual-energy CT, we have been able to generate multi-coloured microCT-data for several years, so that in combination with our new method cartilage and bone are color distinguishable due to their X-ray properties."

Powerful and versatile extension of the currently available toolkit

According to the researchers, the new protocol is simple and reproducible. The cartilage contrast in mouse fetuses is sufficient for rapid visual phenotyping and morphometric skeletal parameters can be easily extracted. Stephan Handschuh sums up: "We consider the presented workflow to be a powerful and versatile extension of the currently available toolkit for the qualitative and quantitative phenotyping of the skeletal development of vertebrates, for example for the study of developmental malformations or in the classification for the developmental toxicity of substances."

Interdisciplinary cooperation as a basis for success

The development of the new method was based on the fact that the researchers from the three participating institutes of Vetmeduni Vienna have contributed their different coreexpertise: The histology and embryology working group of the Institute of Pathology has a broad knowledge through long-standing research projects on cartilage biology and in histological staining techniques for cartilage tissue. Laboratory animal science was able to contribute its many years of experience in mouse breeding and in the generation of mouse models to the research project. And the VetCore/Imaging Unit has many years of experience in developing new X-ray contrast techniques and is one of the world's leading working groups in the field of microscopic dual-energy CT.

The article "The Visible Skeleton 2.0: Phenotyping of cartilage and bone in fixed vertebrate embryos and foetuses based on X-ray microCT" by Simone Gabner, Peter Böck, Dieter Fink, Martin Glösmann and Stephan Handschuh was published in Development.