Synchrotron X-ray micro-computed tomography for the observation of the 3D structure of the zebrafish brain with cellular resolution.

X-ray micro-computed tomography (micro-CT) analysis has been utilized to visualize the three-dimensional (3D) structure of a vertebrate brain. In this study, X-ray micro-CT with young adult zebrafish brains was performed at a synchrotron radiation facility, SPring-8. Single neuronal cell bodies were assessed in virtual cross-sectional tomographic images of the retina and brains, stained with phosphotungstic acid solution (PTA). From these sequential cross-sectional images, 3D brain images were made to observe different sectional planes from the same sample. We could discriminate brain nuclei, layered structures and a pair of identifiable neurons, Mauthner cells, from virtual sectional images. Finally, we also could observe single cell like structures in the tomographic images of zebrafish brains without any metal staining. In tomographic images of the telencephalon and diencephalon from the unstained sample, some brain nuclei could be discriminated, as seen in images of the PTA-stained samples. Therefore, the datasets generated by these techniques will allow us to generate a 3D brain atlas at cellular resolution. This methodology of X-ray micro-CT for the central nervous system will advance the more detailed understanding of the patterns of the 3D structure of the vertebrate brain at cellular resolution in developmental, genetic and comparative neurobiology.