Our group is developing the use of synchrotron micro-computed tomography (microCT) to examine whole zebrafish in three-dimensions. The image volumes acquired from synchrotron microCT have sub-micron isotropic resolution, enabling visualization of individual cells in the context of the whole organism. Our lab is currently engaged in the design and construction of higher-throughput instrumentation for microCT imaging, allowing many zebrafish to be imaged and characterized for genetic or chemical screens. In preparation for the analysis of the resultant data, we are engaged in the parallel pursuit of analytical tools for phenotyping imaged zebrafish at the tissue and cellular levels. Due to intrinsically digital nature of the images, automated image analysis tools can be applied to enable quantitative phenotyping of the whole zebrafish. Here, we show an automated segmentation method to detect and segment neuronal cell nuclei in both wild-type and mutant zebrafish. The mutant zebrafish, huli hutu (hht), causes cytological and nuclear atypia in multiple organs. First, wild-type and mutant 3-, 4-, and 5-day post-fertilization zebrafish were fixed, stained, and embedded in resin before being imaged at Beamline 2-BM at Advanced Photon Source in Argonne National Laboratory (Lemont, IL). From the synchrotron microCT images, 100 neuronal cell nuclei were manually segmented and averaged to create a template for typical neuronal cell nuclei. Next, areas in the zebrafish volumes that are most similar to the average neuronal cell nuclei are extracted for further analysis. Multiple templates can be used to detect neuronal cell nuclei of different sizes. Once the cell nuclei are extracted, morphological characteristics (i.e size, shape) of the cell nuclei are compared between wild-type and hht zebrafish. The zebrafish volumes can also be registered to existing digital zebrafish brain atlases, enabling the segmentation of the zebrafish brain into different regions. The morphological features of the cell nuclei are then compared among the different brain regions, in both wild-type and hht zebrafish. Overall, the results show that there are fewer neuronal cell nuclei in the mutant zebrafish. The size distribution of the cell nuclei is also different between hht and wild-type zebrafish, with hht having an increased fraction of smaller objects of nuclear density, consistent with nuclear fragmentation/apoptosis. The work presented here shows that automated tools can be used for quantitative phenotyping based on synchrotron microCT images of whole zebrafish. Ongoing research is focusing on extending this method to segment other cell types, organ systems, and tissue phenotypes.