Alexander Ziegler; Cornelius Faber; Susanne Mueller; Thomas Bartolomaeus

“ Whereas μCT is mainly used to display hard tissue such as bone or calcite structures, MRI provides images depicting primarily soft tissue anatomy. MRI is based on the principles of nuclear magnetic resonance (NMR) and is predominantly used to measure the distribution of hydrogen protons within a sample. Image contrast is achieved due to the different physical properties of tissues. Both μCT and MRI produce standardized digital data, principally permitting an interactive access through virtual collections or voxel libraries as envisaged previously [3] , [4] . ”

“ For example, in case of sea urchins (Echinoidea, Echinodermata) , some species are deep-sea dwellers or confined to remote regions of the world. It is therefore a considerable problem to obtain these animals in a freshly fixed state. Understandably, the desirable use of specimens from museum collections is usually not permitted for invasive and destructive analyses.
The application of non-invasive imaging techniques such as micro-computed tomography (μCT; see, for example, [1] - [2] ) or magnetic resonance imaging (MRI; see below) has substantially increased in basic morphological research. ”

“ Although several specimens of the echinoid taxon missing in our study, the Echinothurioida (or leather urchins) , were found in museum collections, their shape prevented successful soft tissue imaging. These sea urchins possess a flexible calcite endoskeleton as well as specialized internal muscles that contract when the animal is fixed, resulting in a pancake-like shape of the organism. ”

“ Extensive morphological or developmental studies can thus be carried out, although anaesthetic substances may have to be applied to reduce movement artefacts during the long scanning times that are needed for high-resolution datasets and 3D reconstructions. Another very important advantage of non-invasive imaging techniques in general is the fact that the digitally acquired image stacks do not have to be manually aligned, as is the case with digital pictures from histological or electronmicroscopical sections. ”

“ Sea urchins are spherical, oval or flattened free-moving echinoderms covered with spines. Their soft tissue anatomy is characterized by a dominant digestive tract, a varying number of gonads, and the highly specialized water vascular system with its external appendages, the tube or ambulacral feet. ”

“ Although these datasets can be used to generate highly resolved digitized 3D models of invertebrates as well (see, for example, [34] , [35] ) , these analyses are extremely demanding in terms of specimen preparation, slice alignment and 3D reconstruction. In comparison, 3D reconstruction of MRI data is simple and highly accurate, but the segmentation process carried out for the present work is still subjective and our primary hypotheses regarding organ designation had to be evaluated against the information available from classical histological literature. ”

“ The space between the major organ systems is composed of the main body cavities (the so-called perivisceral coeloms) that are filled with coelomic fluid [24] . Therefore, echinoids possess several of the internal structures encountered in most other taxa, although a number of highly specialized organs are present as well. This taxon can thus serve as a model for analyses using non-invasive imaging techniques on a broad systematic scale. ”

“ For a reliable comparison of internal structures it would be necessary to scan specimens prior to contraction of these powerful muscles.
3D reconstruction of selected internal organs
The 3D reconstructions presented here are limited to the major soft tissue structures identifiable in all datasets, if present in the respective species (Figure 3C–E and 4C–E) : digestive tract, Stewart's organs, axial complex, siphon, gonads, buccal sacs (or 'gills') and gastric caecum. ”

“ Artefacts, for example deformation or compression of histological slices, and the problematic alignment of sections make 3D reconstructions a laborious and often highly subjective procedure. The problems encountered are further aggravated by the logistic challenges that arise when comparative studies on selected species of a whole taxon are to be carried out. ”

“ Their state of preservation was surprisingly good and seemed mainly to depend on the mode of collection, the fixation mode, and the subsequent preservation in alcohol. Following the dissections, MRI experiments using freshly fixed and museum specimens of P. miliariswere carried out. Structures visible in the freshly fixed specimen (Figure 1E) were recognizable also in the 135-year-old museum specimen (Figure 1F) . The size and location of the anatomical features varied slightly, but this had been observed among different specimens of freshly fixed animals as well. ”

“ Moving down from the aboral to the oral side, the following internal and external structures can be identified, amongst others (Additional files 3, 4, 5, 6) : madreporic plate, gonads, spines, intestine, compass elevator muscles, Aristotle's lantern with corresponding muscles, and the stomach. Some parts of the gut of P. miliariswere filled with paramagnetic sediment of unknown composition (possibly iron or manganese residues) that caused artefacts by altering the homogeneity of the magnetic field inside the MRI instrument. ”

“ However, some authors have greatly extended our knowledge about the internal anatomy of selected, readily available sea urchin species such as Cidaris cidaris (Linnaeus, 1758) [42] , Arbacia punctulata (Lamarck, 1816) [43] , Paracentrotus lividus (Lamarck, 1816) [44] , [45] , Echinus esculentusLinnaeus, 1758 [46] , Sphaerechinus granularis (Lamarck, 1816) [45] , [47] , Spatangus purpureusMüller, 1776 [45] or Echinocardium cordatum (Pennant, 1777) [25] , not to mention the extensive works of Alexander E Agassiz, Hubert L Clark and Theodor J Mortensen. ”

“ CLSM: confocal laser scanning microscopy; μCT: micro-computed tomography; FOV: field of view; MRI: magnetic resonance imaging; NHM: Natural History Museum, London, UK; NMR: nuclear magnetic resonance; USNM: National Museum of Natural History, Washington DC, USA; ZMB: Systematische Zoologie am Museum für Naturkunde, Berlin, Germany.
Authors' contributions
AZ designed and coordinated the experiments, carried out the dissections, prepared specimens and carried out scanning and 3D modelling. CF and SM prepared and scanned specimens. TB supervised the experiments. ”