As genomic, functional, and developmental evolutionary studies continue to expand far beyond the few traditional model species, the need for general, direct methods of accurate three-dimensional imaging of animal specimens has never been greater. Any comparative, functional, or ontogenetic analysis of morphology requires calibrated three-dimensional representation of anatomical structures with their natural shapes, orientations, and spatial relationships in as close to their natural state as specimen preparation can allow.
Methods for constructing 3D visualizations of animal specimens fall into two broad categories: those based on reconstruction from serial section images and those based on whole-volume imaging. The former set of methods require sectioning the specimen under study and then aligning images of those sections into a 3D dataset – a laborious process even in its recent computer-assisted and semi-automated forms . Whole-volume imaging methods are generally non-destructive but have tended to be more specialized in their applications. MicroMRI has been used for some time to generate volumetric images of soft tissue morphology (e.g ), but it requires rather costly imaging equipment. Optical projection tomography (OPT) has been developed recently as a method for anatomical and molecular imaging . Based on transmission of visible light, OPT requires more-or-less transparent samples, and it is finding a useful range of applications, such as localization and measurement of structures within a whole organ .
The oldest tomographic imaging method is x-ray computed tomography (CT) [5, 6], now in widespread use for clinical imaging. X-ray microtomography (micro-computed tomography, or microCT) is identical in its basic principles to medical CT scanning and has been increasingly utilized in non-clinical research in the last eight years or so. A sample to be imaged is placed in the path of an x-ray beam so that it forms a projection image on a scintillator or other x-ray-sensitive detector array. The sample is rotated and imaged at a large number of angles, and the sequence of projection images is "back-projected" to reconstruct the x-ray absorption at each point within the scanned volume (see  and  for thorough treatments).
A volume image obtained from microCT (or any tomographic method) consists of a stack of reconstructed cross sections normal to the axis of rotation. Reconstruction programs usually generate voxels (volume pixels) that are isotropic, and the voxel dimensions are automatically calculated with the same accuracy as the imaging system's calibration. The numerical value computed for each voxel is a linear x-ray attenuation coefficient at the corresponding point in the sample volume . Thus, a tomographic volume image is represented as a three-dimensional matrix of brightness values, equal to a stack of aligned two-dimensional digital images. Such datasets are becoming more and more useful and versatile with the growing sophistication and availability of 3D image viewing, manipulation, and analysis software.
MicroCT imaging systems can be divided into two general classes based on their x-ray sources: lab-based scanners and synchrotron systems. Lab-based scanners contain their own commercial or custom x-ray source, and a number of different systems are commercially available from several companies. The image resolutions achievable with these systems extend into the range of light microscopy, down to one or a few microns. Most of the self-contained scanning systems currently cost from around 120,000 to over 400,000 Euro.
Synchrotron-based microCT systems are capable of much finer resolutions because the synchrotron's electron stream can be used to generate high-brilliance x-ray beams that have narrow bandwidths at chosen energies and that can be manipulated with diffractive zone plates, analogous to focusing a light beam with refractive lenses in an optical microscope. Despite the obvious drawback that the system must be connected to a beamline at one of a few synchrotron facilities, synchrotron-based x-ray tomographic microscope systems have been used for non-destructive imaging of microfossils  and for sub-cellular imaging at resolutions of 60 nm and below [8–10]. The narrow-band beam is also more conducive to phase-contrast imaging than a broadband x-ray tube, affording one kind of soft-tissue contrast [11, 12], but the contrasts produced by phase effects are much more pronounced in samples with steep phase gradients (i.e. sharper edges) rather than the softer gradients prevalent in most tissues.
MicroCT is already an established technology for imaging diverse mineralized animal tissues (reviewed in ), but the widespread application of microCT imaging in comparative morphology has been limited by the low intrinsic x-ray contrast of non-mineralized tissues. Although x-ray contrast enhancement agents are used routinely in clinical radiography, only a few techniques have appeared for imaging soft tissues in preserved animal specimens: a clinical radiographic contrast preparation containing organically-bound iodine has been shown to impart differential x-ray contrast to post-mortem mouse and rabbit brains ; osmium staining has been demonstrated successfully several microCT applications, including phenotyping mouse embryos with microCT  and imaging honeybee brains ; a reduced-silver nerve staining method has been used to image Drosophila brains with microCT ; and a contrast resin perfusion method has been used successfully by Wirkner et al. [18, 19] for 3D imaging of arthropod circulatory systems.
With sufficient contrast imparted to soft tissues, linear and volumetric size changes in development can be measured readily, and comparisons of those measurements can be made between species or between control and genetically or experimentally manipulated specimens. Thus microCT offers a source of quantitative data for modeling of developmental and evolutionary changes, and microCT imaging is already being applied to quantitative studies of variation  and of development .
A previous report from this lab  highlights the utility of microCT for imaging vertebrate embryos, and the present report demonstrates the broad applicability of a few very simple and effective contrast stains for imaging various animal tissues using x-ray microtomography. These readily available, low-toxicity contrast agents open a wide range of microCT applications in comparative, developmental, and functional morphological research.