Generation of orientation tools for automated zebrafish screening assays using desktop 3D printing

Background: The zebrafish has been established as the main vertebrate model system for whole organism screening applications. However, the lack of consistent positioning of zebrafish embryos within wells of microtiter plates remains an obstacle for the comparative analysis of images acquired in automated screening assays. While technical solutions to the orientation problem exist, dissemination is often hindered by the lack of simple and inexpensive ways of distributing and duplicating tools. Results: Here, we provide a cost effective method for the production of 96-well plate compatible zebrafish orientation tools using a desktop 3D printer. The printed tools enable the positioning and orientation of zebrafish embryos within cavities formed in agarose. Their applicability is demonstrated by acquiring lateral and dorsal views of zebrafish embryos arrayed within microtiter plates using an automated screening microscope. This enables the consistent visualization of morphological phenotypes and reporter gene expression patterns. Conclusions: The designs are refined versions of previously demonstrated devices with added functionality and strongly reduced production costs. All corresponding 3D models are freely available and digital design can be easily shared electronically. In combination with the increasingly widespread usage of 3D printers, this provides access to the developed tools to a wide range of zebrafish users. Finally, the design files can serve as templates for other additive and subtractive fabrication methods.