Quantitative Geometric Modeling of Blood Cells from X-ray Histotomograms of Whole Zebrafish Larvae.

Tissue phenotyping is foundational to understanding and assessing the cellular aspects of disease in organismal context and an important adjunct to molecular studies in the dissection of gene function, chemical effects, and disease. As a first step toward computational tissue phenotyping, we explore the potential of cellular phenotyping from 3-Dimensional (3D), 0.74 µm isotropic voxel resolution, whole zebrafish larval images derived from X-ray histotomography, a form of micro-CT customized for histopathology. As proof of principle towards computational tissue phenotyping of cells, we created a semi-automated mechanism for the segmentation of blood cells in the vascular spaces of zebrafish larvae, followed by modeling and extraction of quantitative geometric parameters. Manually segmented cells were used to train a random forest classifier for blood cells, enabling the use of a generalized cellular segmentation algorithm for the accurate segmentation of blood cells. These models were used to create an automated data segmentation and analysis pipeline to guide the steps in a 3D workflow including blood cell region prediction, cell boundary extraction, and statistical characterization of 3D geometric and cytological features. We were able to distinguish blood cells at two stages in development (4- and 5-days-post-fertilization) and wild-type vs. polA2 huli hutu ( hht ) mutants. The application of geometric modeling across cell types to and across organisms and sample types may comprise a valuable foundation for computational phenotyping that is more open, informative, rapid, objective, and reproducible.

Neonatal and clinical outcomes after transfer of a mosaic embryo identified by preimplantation genetic testing for aneuploidies.

RESEARCH QUESTION: Do clinical and neonatal outcomes differ between mosaic embryo transfers (MET) and euploid embryo transfers (EET)? DESIGN: This retrospective cohort study compared the implantation rate, live birth rate (LBR) and miscarriage rate between 513 euploid embryos and 118 mosaic embryos (72 whole chromosome mosaic [WCM], 40 segmental mosaic and six complex mosaic). Blastocysts were analysed using preimplantation genetic testing for aneuploidies with next-generation sequencing, followed by a single vitrified-warmed embryo transfer. Trophectoderm biopsies were classified as mosaic if they had 20-80% abnormal cells. RESULTS: Overall, EET resulted in a significantly higher implantation rate (47.0%) and LBR (40.7%) than MET (implantation rate 39.0%, P = 0.005; LBR 28.8%, P = 0.008) and WCM embryos (implantation rate 37.5%, P = 0.01; LBR 22.2%, P = 0.007) after covariate adjustment. Segmental mosaic embryos had an implantation rate (47.5%) and LBR (45.0%) comparable to those of euploid embryos. Mosaic embryos with a high percentage of aneuploid cells (≥60%) showed a significantly lower LBR (10.5% versus 40.7%, P = 0.03) than euploid embryos after covariate adjustment, with three of the five implantations of mosaic embryos resulting in miscarriage. Neonatal outcomes did not differ significantly between the mosaic and euploid groups. Of the 34 women with a live birth after MET, 13 had a prenatal or postnatal genetic testing result, and no abnormalities were found. CONCLUSIONS: Mosaic embryos were associated with a lower LBR, while segmental mosaic embryos had similar clinical outcomes to euploid embryos. Mosaic embryos with a high aneuploidy percentage (≥60%) should be assigned a low transfer priority. Neonatal outcomes did not differ significantly between the euploid and mosaic groups.

A wide-field micro-computed tomography detector: micron resolution at half-centimetre scale.

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.

Whole-organism 3D quantitative characterization of zebrafish melanin by silver deposition micro-CT.

We previously described X-ray histotomography, a high-resolution, non-destructive form of X-ray microtomography (micro-CT) imaging customized for three-dimensional (3D), digital histology, allowing quantitative, volumetric tissue and organismal phenotyping (Ding et al., 2019). Here, we have combined micro-CT with a novel application of ionic silver staining to characterize melanin distribution in whole zebrafish larvae. The resulting images enabled whole-body, computational analyses of regional melanin content and morphology. Normalized micro-CT reconstructions of silver-stained fish consistently reproduced pigment patterns seen by light microscopy, and further allowed direct quantitative comparisons of melanin content across wild-type and mutant samples, including subtle phenotypes not previously noticed. Silver staining of melanin for micro-CT provides proof-of-principle for whole-body, 3D computational phenomic analysis of a specific cell type at cellular resolution, with potential applications in other model organisms and melanocytic neoplasms. Advances such as this in whole-organism, high-resolution phenotyping provide superior context for studying the phenotypic effects of genetic, disease, and environmental variables.

Prospects of plasmapheresis for patients with severe COVID-19.

On February 11, 2020, the World Health Organization officially named the infection caused by the new coronavirus "Coronavirus disease 2019" (COVID-19). On February 11, 2020, the International Committee on Taxonomy of Viruses (ICTV) officially named the infectious matter "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2). Emergence of severe complications with new coronavirus disease is due to the development of hypercytokinaemia, also known as "cytokine storm". These complications comprise acute respiratory distress syndrome (ARDS), respiratory failure and death. Emerging data point to the logic of using extracorporeal haemocorrection to normalise cytokine levels and reduce the severity of organ disorders. The analysis of the literature indicates that to date, a certain positive experience has been accumulated in the world in the application of extracorporeal methods in clinical practice in patients with COVID-19.

Whole-animal connectomes of both Caenorhabditis elegans sexes.

Knowledge of connectivity in the nervous system is essential to understanding its function. Here we describe connectomes for both adult sexes of the nematode Caenorhabditis elegans, an important model organism for neuroscience research. We present quantitative connectivity matrices that encompass all connections from sensory input to end-organ output across the entire animal, information that is necessary to model behaviour. Serial electron microscopy reconstructions that are based on the analysis of both new and previously published electron micrographs update previous results and include data on the male head. The nervous system differs between sexes at multiple levels. Several sex-shared neurons that function in circuits for sexual behaviour are sexually dimorphic in structure and connectivity. Inputs from sex-specific circuitry to central circuitry reveal points at which sexual and non-sexual pathways converge. In sex-shared central pathways, a substantial number of connections differ in strength between the sexes. Quantitative connectomes that include all connections serve as the basis for understanding how complex, adaptive behavior is generated.

Computational 3D histological phenotyping of whole zebrafish by X-ray histotomography.

Organismal phenotypes frequently involve multiple organ systems. Histology is a powerful way to detect cellular and tissue phenotypes, but is largely descriptive and subjective. To determine how synchrotron-based X-ray micro-tomography (micro-CT) can yield 3-dimensional whole-organism images suitable for quantitative histological phenotyping, we scanned whole zebrafish, a small vertebrate model with diverse tissues, at ~1 micron voxel resolutions. Micro-CT optimized for cellular characterization (histotomography) allows brain nuclei to be computationally segmented and assigned to brain regions, and cell shapes and volumes to be computed for motor neurons and red blood cells. Striking individual phenotypic variation was apparent from color maps of computed densities of brain nuclei. Unlike histology, the histotomography also allows the study of 3-dimensional structures of millimeter scale that cross multiple tissue planes. We expect the computational and visual insights into 3D cell and tissue architecture provided by histotomography to be useful for reference atlases, hypothesis generation, comprehensive organismal screens, and diagnostics.

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.