ZeBraInspector, a platform for the automated segmentation and analysis of body and brain volumes in whole 5 dpf zebrafish following simultaneous visualization with identical orientations

Abstract

In recent years, zebrafish have become well established for the study of fundamental biology, toxicology and drug discovery. Here we propose fast, standardized protocols for high-throughput and high-content 3D imaging of 5dpf zebrafish EE using a confocal laser scanning microscope. Using a lipophilic fluorescent dye, we generated a reference labeling in cleared fish. We developed a mounting method based on 3D-printed stamps used to create a grid of wells in an agarose cast. We demonstrate that the quality and homogeneity of the fluorescence signal allows an unequivocal segmentation of the whole specimen and the white matter. From there, we built an automated image registration and analysis pipeline for the volumetric analysis of morphological defects. The platform, termed ZeBraInspector, is focused on the analysis of the shape of the whole EE or the white-matter of the central nervous system. A user-friendly software to visualize at large scale registered and segmented 3D images can be downloaded from https://tefor.net/portfolio/zebrainspector. We finally provide two examples of application: the characterization of a phenotype associated with a neuro-developmental mutation, and the defects caused by treatments with a toxic anti-cancer compound. ZeBraInspector can be adapted to a wide variety of high-content analyses.

Generation of Self-Induced Myocardial Ischemia in Large-Sized Cardiac Spheroids without Alteration of Environmental Conditions Recreates Fibrotic Remodeling and Tissue Stiffening Revealed by Constriction Assays

“…Initially, cellular damage at the ischemic core was assessed by live/dead staining at day 2 and day 10 of culture, but cellular density prevented fluorescent signal visualization a few micrometers below the surface. Thus, after staining, spheroids were fixed and subjected to a clearing process, 11 which enabled the visualization of inner layers of the spheroid with confocal microscopy (Figure 3A). Z-stack projection of confocal images (Figure 3B) showed the external distribution of living cells and internal congregation of dead cells since day 2 of spheroid culture.…”