Three-Dimensional Histology of Whole Zebrafish by Sub-Micron Synchrotron X-ray Micro-Tomography

Histological studies providing cellular insights into tissue architecture have been central to biological discovery and remain clinically invaluable today. Extending histology to three dimensions would be transformational for research and diagnostics. However, three-dimensional histology is impractical using current techniques. We have customized sample preparation, synchrotron X-ray tomographic parameters, and three-dimensional image analysis to allow for complete histological phenotyping using whole larval and juvenile zebrafish. The resulting digital zebrafish can be virtually sectioned and visualized in any plane. Whole-animal reconstructions at subcellular resolution also enable computational characterization of the zebrafish nervous system by region-specific detection of cell nuclei and quantitative assessment of individual phenotypic variation. Three-dimensional histological phenotyping has potential use in genetic and chemical screens, and in clinical and toxicological tissue diagnostics. One Sentence Summary Synchrotron X-ray micro-tomography can be used to rapidly create 3-dimensional images of fixed and stained specimens without sectioning, enabling computational histological phenotyping at cellular resolution. Abbreviations : 3D : three-dimensional H&E : hematoxylin & eosin 2D : two-dimensional micro-CT : X-ray micro-tomography dpf : days post-fertilization PTA : phosphotungstic acid RBC : red blood cell