Abstract
The characterization of developmental phenotypes often relies on the accurate linear measurement of structures that are small and require laborious preparation. This is tedious and prone to errors, especially when repeated for the multiple replicates that are required for statistical analysis, or when multiple distinct structures have to be analyzed. To address this issue, we have developed a pipeline for characterization of long-bone length using X-ray microtomography (XMT) scans. The pipeline involves semi-automated algorithms for automatic thresholding and fast interactive isolation and 3D-model generation of the main limb bones, using either the open-source ImageJ plugin BoneJ or the commercial Mimics Innovation Suite package. The tests showed the appropriate combination of scanning conditions and analysis parameters yields fast and comparable length results, highly correlated with the measurements obtained via ex vivo skeletal preparations. Moreover, since XMT is not destructive, the samples can be used afterward for histology or other applications. Our new pipelines will help developmental biologists and evolutionary researchers to achieve fast, reproducible and non-destructive length measurement of bone samples from multiple animal species.