Micro-CT and X-ray diffraction: an ontogeny investigation of T. miliaris tadpole

Micro-CT has been used as an important tool in biological research, in particular for amphibians, due to its is non-invasive method allowing to carry out more accurate studies on the morphology of frogs, such as skeleton, muscle, ocular structure and leaves the specimen intact. A peculiar species of tadpole called Thoropa miliaris, found along the Brazilian Atlantic Rain Forest, reproduces under extreme environmental conditions and lives in water films that run along rock outcrops along the forest, near or isolated from river and rivulets. In early stages post-hatching of the egg, T. miliaris tadpole presents deposits of calcium carbonate crystals in their lymphatic glands, known as endolymphatic calcium deposits (ECD), which are composed of aragonite and likely represent an important source of calcium for bone formation during and after metamorphosis. T. miliaris tadpoles were scanned in the X-ray microtomography beamline (IMX) at Brazilian Synchrotron Light Laboratory (LNLS) to obtaining a detailed anatomical description of soft tissue. Images were acquired with 5x or 2x magnification lenses, which give a virtual pixel size of 4.11 μm and 1.64 μm. Depending of sample size we used 1000 projections over 180° or 2000 projections over 360°. The distance sample-to-detector was 20 cm to increase phase contrast effect. Tomographic images were obtained with and without silicon filter depending on the type of sample (different stages of development, ECD or extracted bones) and the exposure time that it was submitted to optimize image quality. Specimen of T. miliaris has tissues whose densities are similar and IMX beamline afforded to obtain tomographic images, improving excellent edge definition between regions of different tissues. Furthermore, by means of X-ray diffraction (XRD1 beamline, with energy of 12 keV) we evaluated different stages (from 25 to 46) of the specimen T. miliaris in order to verify the mineral composition of the remaining samples, such as crystal structure of the ECD and bones. The new Brazilian synchrotron light source, Sirius, will allow us to carry out quantitative and qualitative analyzes with higher quality and fast acquisition time for biological interest (i.e., morphology, ontology, heterochrony), as well as development of new methodologies for biological samples. (author)