Imaging cellular activity across all organs reveals body-wide circuits

Key Points:

• Zebrafish and Danionella cerebrum were reared under controlled temperature and light-dark cycles with specific feeding protocols; sex was undetermined in young fish and randomization was applied where relevant, but no blinding was used during data collection or analysis.
• Transgenic lines in zebrafish and Danionella cerebrum were generated using Tol2 transposase and CRISPR–Cas9 techniques, with codon-optimized calcium indicators for functional imaging; founders were screened for dense expression to ensure experimental quality.
• Functional imaging involved embedding fish in low-melting agarose, using spinning-disk confocal microscopy for volumetric scans, and applying treatments such as ketamine, tricaine, cold stimulus, and hypoxia with controlled oxygen levels; optogenetic stimulation was performed via a digital micromirror device.
• Data processing included a novel iterative patch-wise optical flow registration algorithm (WHOLISTIC) validated against synthetic benchmarks and other methods, combined with non-negative matrix factorization for cellular segmentation and coherence-based spectral clustering for identifying functional tissue ensembles.
• Whole-body expansion microscopy (WB-ExM) protocols with immunofluorescence and fluorescence in situ hybridization were developed for high-resolution anatomical and molecular imaging, complemented by PhotoMap for unbiased quantification of gel deformation during expansion; 3D models and quantitative analyses were constructed using these data.