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

by Mee S. Ngu, Daniel J. Vanselow, Andrew L. Sugarman, Rachelle A. Saint-Fort, Carolyn R. Zaino, Maksim A. Yakovlev, Keith C. Cheng, Khai C. Ang

Micro-CT imaging is a powerful tool for generating high-resolution, isotropic, three-dimensional datasets of whole, centimeter-scale model organisms. At histological resolutions, micro-CT can be used for whole-animal qualitative and quantitative characterization of tissue and organismal structure in health and disease. The small size, global freshwater distribution, wide range of cell size and structures of micron scale, and common use of Daphnia 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, including 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 3D. The enabled “3D histology” and 3D renderings can be used for morphometric analyses across cells, tissues, and organ systems.