Modern Imaging Techniques to Reveal Animal Anatomy Figure 1. CT and MRI of a red-eared slider. CT (a, b, d, f, g) and MRI (c, e) in red-eared slider (Trachemys scripta). CT and MRI have different capacities in visualising vasculature (d, e), soft tissue (c, f) and skeleton (a, g). (b, c): Both of the scanning modalities produce thin cross sectional images of the red-eared slider under study. (a, d, e, f, g): Further processing of the thin cross sectional images leads to a three dimensional digital model of the animal by the aid of volume rendering software. doi:10.1371/journal.pone.0017879.g001 DICOM-viewer OsiriX software (www.osirix-viewer.com) for Macintosh computers. In OsiriX, DICOM-files were developed as both still-images and animations, and more advanced features were applied: segmentation for highlighting of individual struc- tures, representations of objects as 3D-rotations, determination of flythrough routes, and cropping (in multiple depths, planes, and directions). Quantitative measures (angles, lengths, and volumes) can easily be extracted from the digital models, although we have not included those in this study. Images were exported as tagged image file format (TIFF) in 300 dpi. Movies were exported in QuickTime file format (MOV). As no general agreement exists about the visual enhancement of zoological structures, the computed images and movies were adjusted with regards to brightness, contrast and colour according to personal experiences. Results Images of a turtle using MRI and CT imaging, respectively, are shown in Figure 1a–g to highlight the differences between these two modalities. As shown in Figure 1c, MRI allows for detailed visualisation of the various organs at a thin sectional level, whereas CT is particularly useful for harder structures (note for example in Figure 1b the intraabdominal eggs in the thin section with CT) and for radio-density contrasts such as the difference between tissue and air, enabling the lungs to be visualised clearly (Figure 1f). Both MRI and CT allow for visualisations of the vasculature (Figure 1d,1e), however it must be stressed that the vessel and organ structures in the CT image (Figure 1d) are visible only because a CT contrast agent has been injected into the vasculature. Also, an MRI contrast agent was used to enhance the vessel structure in Figure 1e. The subsequent figures provide images of skeletal elements (Figure 2 and Movie S1, S2), organ structures (Figure 3 and Movie S3) and vasculature (Figure 4, 5 and Movie S4, S5). Using volume rendering software, three- dimensional representations of anatomical structures were con- structed from cross-sectional images, thereby creating digital models movable in space (Movie S1–S5). Both CT and MRI were capable to generate visualisations of soft tissues (Figure 3). The use of an ingested MRI contrast agent in the white knee tarantula increased the detectable signal from the gastrointestinal tract (Figure 3c, 3d and Movie S3–S5). Irrespective of the smaller sensibility of the CT modality to soft tissue structures, visualisation of major organ structures was still possible, indeed after vascular filling with a contrast agent, exemplified in Figure 3a–b, where the heart and the closely aligned lungs and liver of the alligator are easily distinguished. In cases where adjacent tissues exhibit big differences in radio-absorbance, e.g. air and tissue in lungs and trachea, high-resolution representations of borderline areas can be produced using CT (Figure 1f, 3a, 3b). The intravenous use of contrast agents, such as gadolinium- containing agents for MRI and lead oxide or barium sulphate for CT in solidifying solutions, made it possible to produce ex vivo angiographies (Figure 4, 5 and Movie S4, S5). Perfusion of the cane toad’s lungs with barium sulphate prior to CT allowed visualisation of the pulmonary vessels (Figure 4b). Post-processing and analysis allow for at digital dissection of the animal, thereby isolating the cane toad’s lungs from the surrounding tissue (Figure 4c). Whole body angiography can be performed with a similar method, exemplified with the yellow anaconda and the domestic pig (Figure 4d, 5a and Movie S4, S5). Additionally, by preparing the contrast agent to a suitable viscosity, making it impenetrable to the capillary barrier, either the arterial or the venous side of the vasculature can be visualised separately as exemplified by the MRI venography of the red-eared slider (Figure 1c). High-resolution mCT imaging allows for detection of minute vessel structures, facilitating a complete representation of the vasculature in the head region of the air-breathing rice field eel (Figure 4a). The use of vascular contrast agents and mCT imaging revealed the microvasculature of the coronary arteries in the heart of the yellow anaconda (Figure 5c). Coronary arteriography of a giraffe heart (Figure 5b), as well as a whole body angiography of the domestic pig (Figure 5a and Movie S5), were conducted to display the mammalian vasculature. Discussion This methodological study demonstrates that MRI and CT with appropriate post-processing methods can provide anatomical descriptions of various structures in animals in quality comparable with traditional dissection techniques [18–20,30–33]. Main skeletal architecture was easily visualised (Figure 2 and Movie S1, S2), and it was possible to identify the structure and position of major visceral organs, such as heart, liver, lungs and gastrointes- tinal tract (Figure 1d, Figure 3 and Movie S3). The vasculature could be visualised using solidifying contrast agents (Figure 1d, 1e, 4, 5 and Movie S4, S5). Further, we demonstrated the ability of mCT to acquire images with very high spatial resolution, exemplified by visualisation of the rice field eel vasculature (Figure 4a) and the coronary microvasculature of the anaconda heart (Figure 5c). Both MRI and CT are non-invasive techniques, leaving the animal intact, except in situations where angiographic procedures require vascular filling with a solidifying contrast agent. In fact, because both modalities can be used on live animals, MRI and CT allow for repeated measures on the same individual, which call for longitudinal investigations of anatomical phenomena, e.g. inves- tigations of tissue regeneration and volumetric changes in the digestive system during digestion [34]. Note, however, that both CT and MRI require that the animal is completely immobilized during the entire scan procedure, which necessitates respiratory or heart-beat triggering to avoid most artefacts. MRI and CT complement each other well [35], where MRI is useful to reveal soft tissue structures with subtle differences in composition and can provide images from various angles, and multi-slice CT can produce excellent images of hard calcified structures. The two techniques differ considerably in the speed at which the images can be obtained. While a high-resolution CT acquisition can be performed in less than a minute, a high- resolution MRI usually requires up to hours depending on inherent sequence parameters, magnet field strength, etc. Yet, this technique is relatively fast compared to traditional preserva- tion and dissection procedures. Modern clinical MRI and CT modalities are able to generate images with a maximum resolution of approximately 0.09–0.25 mm2 with an acceptable signal-to- noise ratio. Introduction of experimental high-field MRI or mCT is a way to increase the spatial resolution, allowing MRI image PLoS ONE | www.plosone.org 4 March 2011 | Volume 6 | Issue 3 | e17879