Ideal three-dimensional imaging of complex samples made up of micron-scale structures extending over mm to cm, such as biological tissues, requires both wide field of view and high resolution. For existing optics and detectors used for micro-CT (computed tomography) imaging, sub-micron pixel resolution can only be achieved for fields of view of <2 mm. This article presents a unique detector system with a 6 mm field-of-view image circle and 0.5 µm pixel size that can be used in micro-CT units utilizing both synchrotron and commercial X-ray sources. A resolution-test pattern with linear microstructures and whole adult Daphnia magna were imaged at beamline 8.3.2 of the Berkeley Advanced Light Source. Volumes of 10000 × 10000 × 7096 isotropic 0.5 µm voxels were reconstructed over a 5.0 mm × 3.5 mm field of view. Measurements in the projection domain confirmed a 0.90 µm measured spatial resolution that is largely Nyquist-limited. This unprecedented combination of field of view and resolution dramatically reduces the need for sectional scans and computational stitching for large samples, ultimately offering the means to elucidate changes in tissue and cellular morphology in the context of larger, whole, intact model organisms and specimens. This system is also anticipated to benefit micro-CT imaging in materials science, microelectronics, agricultural science and biomedical engineering.

Daphnia, an important model system for the study of evolution, development, phenotypic plasticity, and environmental health, lacks a modern reference atlas for microanatomy. To facilitate the comprehensive assessment of phenotypic effects of genes and environment, we created the histology reference atlas for Daphnia (http://daphnia.io/anatomy/), a tractable, interactive web-based tool that provides insight into normal phenotype through vectorized annotations overlaid onto digital histology sections imaged at 40X magnification. Guided by our expert-curated and multimodal informed hierarchical anatomical ontology, we show that this resource can be used to elucidate sex-specific differences of female and male Daphnia magna in each of 3 orthogonal planes, providing new insight for the study of sex-specific traits. It is our intention that this atlas aids in phenotypic anchoring of large-scale biomolecular (multi-omics) data from comparative toxicological studies. Greater access to high-quality histological data may clarify cross-correlations between microanatomic and multi-omic phenotypes caused by genetic variation, environment, and disease across phylogeny.

Ideal 3-dimensional imaging of many complex samples, such as biological tissues made up of micro-scale structures extending over millimeter- to centimeter-scale tissue samples and organisms, requires both a wide field-of-view and high resolution. With existing optics and detectors used for micro-CT imaging, sub-micron pixel resolution can only be achieved for fields-of-view of <2 mm. This manuscript presents a unique detector system with a 6-mm field-of-view image circle and 0.5 μm pixel size that can be used in both synchrotron facilities and tabletop micro-CT units. A resolution-test pattern with linear microstructures and whole adult Daphnia magna were imaged on Beamline 8.3.2 of the Advanced Light Source. Volumes of 10,000 × 10,000 × 7,096 isotropic 0.5 μm voxels were reconstructed over a 5.0 × 3.5 mm field-of-view. Measurements in the projection domain confirmed a 1.182 μm measured spatial resolution that is largely Nyquist-limited. This unprecedented combination of field-of-view and resolution dramatically reduces the need for sectional scans and computational stitching for large samples, ultimately offering the means to elucidate change in tissue and cellular morphology in the context of larger whole, intact model organisms and specimens. This development is also anticipated to benefit micro-CT imaging in materials science, microelectronics, agricultural science, and biomedical engineering.

Micro-CT imaging is a powerful tool for generating high resolution, isotropic three-dimensional datasets of whole, small model organisms that are useful for qualitative and quantitative analysis. D. magna, one of the most common ecological model organisms used for toxicity testing and evaluation of environmental stressors, could benefit from this imaging method for enhancing whole-organism phenotype assessment. This protocol details the steps involved in Daphnia sample preparation for micro-CT imaging: euthanasia, fixation, staining, and resin embedding. The resulting 3D reconstructions of samples imaged using synchrotron micro-CT revealed histological (microanatomic) features of organ systems, tissues, and cells in the context of the entire organism at sub-micron resolution. 3D histology and renderings enabled by this imaging method could contribute to morphometric analysis of any tissue or organ system for both descriptive and hypothesis testing studies.