Mapping the nervous system of the Idiosepius hallami pygmy squid: insights from whole-animal X-ray nanotomography imaging.

The study of a nervous system as big as the cephalopods requires multimodal imaging approaches capable of capturing neural architecture across scales. Here, we present a whole-animal volume of the pygmy squid hatchling Idiosepius hallami, acquired using X-ray holographic nanotomography at the beamline ID16A of the European Synchrotron. The reconstructed 3D volume comprises 40 tiled scans acquired at a voxel size of 125 nm. While individual neurons are not resolved at this resolution, we segmented major body regions and mapped the large-scale connectivity by tracing afferent and efferent nerve bundles, including projections from the olfactory organs, chromatophore lobes, and arm ganglia to the brain. The acquisition of this dataset represents a significant milestone for X-ray nanotomography, being the largest whole animal volume imaged at this spatial resolution. The volume serves as a resource for comparative neuroscience and cephalopod biology.