A method for investigating spatiotemporal growth patterns at cell and tissue levels during C-looping in the embryonic chick heart.

We developed a workflow using multi-scale and multi-disciplinary experimental and computational approaches to analyze C-looping (the first phase of cardiac looping) of the chick across four developing hearts. We provide the first 3D datasets for the C-looping heart with cell to organism level information, including datasets of heart images and segmented myocardial cells within the heart. We used these datasets to investigate, as a proof-of-concept, the differential spatiotemporal patterns of growth at both the cellular and tissue levels, and demonstrate how geometrical changes of C-looping at the tissue level are linked to growth features at the cellular level. Our methodological pipeline provides preliminary results for qualitative and quantitative evidence of various cellular and tissue features as potential candidates regarding the mechanism of C-looping. This pipeline can be used and extended in future studies to include larger specimen samples for detailed analyses of, and potentially new insights into, cardiac C-looping.

Micro-CT guided illustration of the head anatomy of penguins (Aves: Sphenisciformes: Spheniscidae).

The illustration is an important tool to aid in the description and understanding of anatomy, and penguins (Aves: Sphenisciformes: Spheniscidae) are an important clade in environmental monitoring, paleontology, and other research fields. Traditionally, anatomic illustration has been informed by dissection. More recently, micro-computed tomography (micro-CT) has proven to be a powerful tool for three-dimensional anatomic imaging, although larger specimens are more challenging to image due to increased X-ray attenuation. Here, we used traditional dissection and micro-CT to illustrate the skulls of Aptenodytes patagonicus, Eudyptula minor, and Pygoscelis papua, and the extracranial soft tissue of E. minor. Micro-CT prevented the loss of orientation, disarticulation, and distortion of bones that might result from cleaning and drying skulls, while immobilization was achieved by freezing the specimens before imaging. All bony elements in the head were accurately depicted. Fixing, dehydrating, and diffusion staining with iodine (diceCT) enabled the identification of muscles and other large nonmineralized structures, but specimen preparation precluded the ability to show smaller nerves and vessels. The results presented here provide a guide for anatomic studies of penguins and our summary of sample preparation and imaging techniques are applicable for studies of other similarly sized biological specimens.

Skeletal elements of the penguin eye and their functional and phylogenetic implications (Aves: Sphenisciformes: Spheniscidae).

Scleral ossicles and other bony elements are present in the eyes of many vertebrates, including birds. In this study, the skeletal elements present in the penguin eye and orbit were imaged using macro photographs and micro-computed tomography (micro-CT), to help elucidate their function and significance. A total of 36 scleral rings and three whole skulls were imaged. King (Aptenodytes patagonicus), Fiordland crested (Eudyptes pachyrhynchus), Snares crested (Eudyptes robustus), royal (Eudyptes schlegeli) and yellow-eyed (Megadyptes antipodes) penguins had between 12 and 14 elements in their scleral ring while the gentoo (Pygoscelis papua) had 14 and 17; little penguins (Eudyptula sp.) consistently had between 10 and 12 elements. All had at least two elements that overlapped, usually totally, each neighbour, and two that were overlapped by each neighbour. The interior structure of all ossicles revealed a lattice-like arrangement of struts typical of cancellous bone, the whole being surrounded by thick cortical bone. The scleral ring of a 10 week gentoo chick was not completely ossified but rather had multiple small holes within it on micro-CT. A large os opticus was present in one king penguin but in another bird of the same age and gender there was no such bone. Much smaller accessory bones were found in the posterior pole of one Snares crested and one little penguin. We conclude that the penguin scleral ring not only maintains the shape of the eye but also provides protection and a site of insertion for rectus muscles. However, the extreme variability in the os opticus suggests that it is not essential to normal function.

Surface imaging microscopy using an ultramiller for large volume 3D reconstruction of wax- and resin-embedded tissues.

Three-dimensional reconstruction of large tissue volumes using histological thin sections poses difficulties because of registration of sections, section distortion, and the possibility of incomplete data set collection due to section loss. We have constructed an integrated surface imaging system that successfully addresses these problems. Embedded tissue is mounted on a high precision XYZ stage and the upper surface is iteratively: (i) stained to provide an effective optical section, (ii) imaged using a digital camera, and (iii) removed with an ultramiller. This approach provides for the reconstruction of high-quality 3D images by inherently preserving image registration, eliminates section distortion, thus removing the need for complex realignment and correction, and also ensures full capture of all image planes. The system has the capacity to acquire images of tissue structure with voxel sizes from 0.5 to 50 mum over dimensions ranging from micrometers to tens of millimeters. The ultramiller enables large samples to be imaged by reliably removing tissue over their full extent. The ability to visualize key features of 3D tissue structure across such a range of scale and resolution will facilitate the development of a greater understanding of the relationship between structure and function. This understanding is essential for better analyses of the structural changes associated with different disease states, and the development of structure-based computer models of biological function.

Automated imaging of extended tissue volumes using confocal microscopy.

Confocal microscopy enables constitutive elements of cells and tissues to be viewed at high resolution and reconstructed in three dimensions, but is constrained by the limited extent of the volumes that can be imaged. We have developed an automated technique that enables serial confocal images to be acquired over large tissue areas and volumes. The computer-controlled system, which integrates a confocal microscope and an ultramill using a high-precision translation stage, inherently preserves specimen registration, and the user control interface enables flexible specification of imaging protocols over a wide range of scales and resolutions. With this system it is possible to reconstruct specified morphological features in three dimensions and locate them accurately throughout a tissue sample. We have successfully imaged various samples at 1-mum voxel resolution on volumes up to 4 mm3 and on areas up to 75 mm2. Used in conjunction with appropriate embedding media and immuno-histochemical probes, the techniques described in this paper make it possible to routinely map the distributions of key intracellular structures over much larger tissue domains than has been easily achievable in the past.