Staining and resin embedding of whole Daphnia samples for micro-CT imaging enabling 3D visualization of cells, tissues, and organs of various thicknesses

Micro-CT imaging is a powerful tool for generating high resolution, isotropic three-dimensional datasets of whole, centimeter-scale model organisms that can be used for qualitative and quantitative analysis. The small size, global freshwater distribution, wide range of cell size and structures of micron scale, and common use of D. magna in toxicological and environmental studies make it an ideal model for demonstrating the potential power of micro-CT-enabled whole-organism phenotyping. This protocol details the steps involved in D. magna samples preparation for micro-CT: euthanasia, fixation, staining, and resin embedding. Micro-CT reconstructions of samples imaged using synchrotron micro-CT reveal histological (microanatomic) features of organ systems, tissues, and cells in the context of the entire organism at sub-micron resolution, and in 3 dimensionality. The enabled "3D histology" and 3D renderings can be used towards morphometric analyses across cells, tissues, and organ systems for both descriptive and hypothesis testing studies.