Customizable FDM-based zebrafish larva mold for live imaging

Accurate and reproducible orientation of zebrafish larvae is essential for high-resolution live imaging but remains difficult with standard agarose mounting. Although previous methods have used 3D-printed tools, often fabricated via stereolithography, here, we present an orientation tool made via fused deposition modeling and enhanced with a thin resin coating to improve surface smoothness and performance. This design creates consistent, larval-shaped wells that orient larvae dorsoventrally. The molds, designed to fit standard glass-bottom imaging dishes, are inexpensive to produce on common desktop 3D printers and can be easily customized to fit different imaging setups using the open-source design files. This approach enables reliable, long-term live imaging of multiple larvae and extends the use of low-cost additive manufacturing in zebrafish imaging workflows.