Power and challenges of using zebrafish as a model for skeletal tissue imaging.

The zebrafish (Danio rerio) is now a widely used model organism in biomedical research. The species is also increasingly used for studying skeletal development and regeneration and for understanding human skeletal diseases. The small size of this model organism is an advantage and an extreme challenge for visualizing and diagnosing the animals' skeleton. This applies especially to early stages of skeletal development. Similar challenges arise for the analysis of the skeleton of other small fish species, such as medaka (Oryzias latipes). High quality histological preparations and knowledge about the special quality of the zebrafish skeleton remain prerequisites for a correct analysis. In addition, new methods for fast and high-resolution 2D and 3D skeletal tissue screening are required for a maximal understanding of skeletal development. We, in this study, review advantages and limitations of adapting current visualization techniques for zebrafish skeletal research. We discuss the methods for in toto visualization, such as X-raying, micro-CT, Alizarin red staining and optical projection tomography. Techniques for in vivo imaging, such as second harmonic generation microscopy and two-photon excitation fluorescence, are also discussed. Finally, we explore the possibilities of light-sheet microscopy for the analysis of the zebrafish skeleton.