Table 1. List of animals undergoing CT, mCT, and MRI scanning and the use of anaesthetics. Animal Whiteknee tarantula (Acanthoscurria geniculate) Rice field eel (Monopterus albus) African lungfish (Protopterus annectens) Cane toad (Rhinella marina) Anaesthesia Anaesthesia with 100% CO2 Anaesthesia and termination with 3 g/kg ethyl p-aminobenzoat in water Anaesthesia with 3 g/kg ethyl p-aminobenzoat in water Anaesthesia and termination with 1% ethyl p-aminobenzoat in water Monitor lizard (Varanus exanthematicus) Termination with 100 mg/kg pentobarbital American alligator (Alligator mississippiensis) Termination with 100 mg/kg pentobarbital Ball python (Python regius) Yellow anaconda (Eunectes notaeus) Termination with 100 mg/kg pentobarbital Termination with 100 mg/kg pentobarbital Red-eared slider (Trachemys scripta) Termination with 100 mg/kg pentobarbital Domestic pig (Sus scrofa domesticus) Giraffe (Giraffa camelopardalis) doi:10.1371/journal.pone.0017879.t001 Premedication with ketamin and midazolam followed by ventilation with 1% isoflurane and termination with pentobarbital Heart delivered perfusion fixed from an expedition to Africa MRI mCT CT CT CT CT CT CT CT CT and mCT CT and MRI Modern Imaging Techniques to Reveal Animal Anatomy Image modality Contrast enhancement Gastrointestinal structures enhanced by ingestion of Dotarem-filled cockroach Vascular filling with Microfil No contrast enhancement applied Vascular filling with Mixobar in gelatinous solution No contrast enhancement applied Vascular filling with Mixobar in gelatinous solution No contrast enhancement applied Vascular filling with Mixobar in gelatinous solution Vascular filling with Mixobar in gelatinous solution Vascular filling with Mixobar in gelatinous solution Vascular filling with Mixobar in gelatinous solution in this study are presented in Table 1, including anaesthetics used. For ex vivo angiography, the vascular beds of the rice field eel were perfused with a commercially available CT contrast agent (Microfil; Flow Tech Inc., Carver, MA). Vascular fillings of cane toad, anaconda, red-eared slider, pig, and giraffe heart were accomplished with a mixture of gelatine and MRI (Dotarem; Guerbet, Paris, France) and CT (Mixobar Colon; Astra Tech, Mo¨ lndal, Sweden) contrast agent as described by Rasmussen et al. [7]. Arterial and venous injections were performed on deeply anesthetised animals, terminated in anaesthesia, with a pressure- controlled pump after initial flushing with heparinised saline. Physiological pressure was used as the intentional injection pressure. After filling of the entire vasculature, the animal was cooled on ice to rapidly solidify the injected contrast agents. Computed Tomography (CT) CT provides radiography-based thin cross sectional images without disturbing internal structures. The Ro¨ ntgen tube, detectors and associated electronics are contained within the gantry that rotates around the animal, which is positioned in the centre of the scanner. During CT scanning, electromagnetic radiation (x-rays) penetrates the study object from 360u. Because some radiation is absorbed by the tissues, the initial data processed by the CT computer system is in fact shadow projections of the absorption from various angles. The pre-defined field of view is divided into volume elements (voxels) and the absorption coefficients, measured in Houndsfield units (HU), of these voxels are computed into elements (pixels), creating thin cross-sectional images of the study object that are displayed as individual grey shades on a screen. These grey shades finally allow differentiation between tissues and structures according to their radiodensity. In this study, CT scans were performed using a 64-slice Siemens Somatom Definition (Siemens Medical Solutions, Germany). Acquisition parameters included a slice collimation of 4 mm; a pitch of 2u; 32 rotations, a spatial resolution of 0.216 mm3/voxel (reconstructed to 0.027 mm3/voxel) and a scan duration of 10– 30 s (depending on the size of the object). Additional mCT scans were performed using a Scanco Medical mCT 40 scanner (Scanco Medical, Z u¨rich, Switzerland) containing an x-ray source at 80 kV and 160 mA. The mCT images were reconstructed three- dimensionally with an isotropic voxel size of 646106 mm3/voxel. Magnetic resonance Imaging (MRI) MRI provides detailed images of the body in any directional plane by aligning the spin of hydrogen nuclei in the study object by aid of a strong external magnetic field. The study object is divided into voxels by coding field strength, spin frequency and spin phase in three-dimensional space. Hydrogen nuclei situated in various environments, i.e. tissue types, differ by intrinsic magnetic relaxation times, and this physical characteristic is used to produce contrast between different soft tissues. The ability to translate these magnetic relaxation times to grey-white signal intensities makes MRI particularly sensitive to delineate all anatomical structures other than air-filled regions and calcified compounds (skeleton and bones) [29]. In this study, MRI was performed with two clinically available 1.5 Tesla systems (Philips Medical Systems, Netherlands; and Siemens Medical Solutions, Germany). The animal was positioned in a quadrature radiofrequency receiver-coil. A fast localizer scan was followed by a high-resolution 3D gradient–echo sequence with the following parameters: field-of-view depending on animal size; thickness 0.5 mm; TR 23.1 ms; TE 1.6 ms and excitation flip angle 30u. A stack of multiple slices (with no gaps) was acquired, covering the entire specimen of interest with scan durations of 10–60 min. Images were isotropically acquired with a spatial resolution of 0.125 mm3/voxel. Image analysis Data acquired both by MRI and CT were exported in DICOM format and 3D reconstructions were generated using the free PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17879