title: A quantitative comparison of micro-CT preparations in Dipteran flies.
abstract: 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.
authors.affiliation: Department of Bioengineering, Imperial College London, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Swart
authors.affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London, UK.
authors.forename: Martina
authors.initials: M
authors.lastname: Wicklein
authors.affiliation: Imaging and Analysis Centre, Natural History Museum, Cromwell Road, London, UK.|Henry Moseley X-ray Imaging Facility, University of Manchester, Manchester, UK.
authors.forename: Dan
authors.initials: D
authors.lastname: Sykes
authors.affiliation: Imaging and Analysis Centre, Natural History Museum, Cromwell Road, London, UK.
authors.forename: Farah
authors.initials: F
authors.lastname: Ahmed
authors.affiliation: Department of Bioengineering, Imperial College London, UK.
authors.forename: Holger G
authors.initials: HG
authors.lastname: Krapp
mesh_terms: D000818:Animals; D003287:Contrast Media / Q000737:chemistry; D004175:Diptera / Q000033:anatomy & histology*; D005557:Formaldehyde; D021621:Imaging, Three-Dimensional / Q000379:methods; D007455:Iodine / Q000737:chemistry; D011108:Polymers; D013194:Staining and Labeling / Q000379:methods; D055114:X-Ray Microtomography / Q000379:methods*
chemical_list: D003287:Contrast Media; D011108:Polymers; D005557:Formaldehyde; D007455:Iodine; C003043:paraform