A quantitative comparison of micro-CT preparations in Dipteran flies.

X-ray-based 3D-imaging techniques have gained fundamental significance in research areas ranging from taxonomy to bioengineering. There is demand for the characterisation of species-specific morphological adaptations, micro-CT (μCT) being the method of choice in small-scale animals. This has driven the development of suitable staining techniques to improve absorption-based tissue contrast. A quantitative account on the limits of current staining protocols for preparing μCT specimen, however, is still missing. Here we present a study that quantifies results obtained by combining a variety of different contrast agents and fixative treatments that provides general guidance for μCT applications, particularly suitable for insect species. Using a blowfly model system (Calliphora), we enhanced effective spatial resolution and, in particular, optimised tissue contrast enabling semi-automated segmentation of soft and hard tissue from μCT data. We introduce a novel probabilistic measure of the contrast between tissues: PTC. Our results show that a strong iodine solution provides the greatest overall increase in tissue contrast, however phosphotungstic acid offers better inter-tissue discriminability. We further show that using paraformaldehyde as a fixative as opposed to ethanol, slows down the uptake of a staining solution by approximately a factor of two.