ZEMs: Zebrafish embedding molds for high-throughput imaging of zebrafish embryos and larvae.

Abstract

Zebrafish imaging is a powerful tool for observing physiological responses in real time, from the whole organism to the organ, tissue, and cellular levels. It enables researchers to derive biological meaning by observing morphological and histological changes, cell migration, and more. To analyze such dynamic phenomena, the acquisition of high-quality and consistent images is essential. However, it remains challenging to acquire standardized images at specific regions of interest in zebrafish. In this study, we developed a customized imaging platform, the zebrafish embedding mold (ZEM), designed to facilitate imaging of zebrafish embryos and larvae. Three types of molds were fabricated to accommodate different developmental stages and imaging orientations. The ZEM provided stable positioning of embryos (0-2 days post-fertilization, dpf) and larvae (3-7 dpf), enabling improved imaging of developmental stages, morphological changes, and fluorescence signals. Using this platform, we successfully analyzed the biodistribution and accumulation patterns of fluorescent polystyrene nanoplastics, as well as morphological alteration induced by exposure to the environmental pollutant benzo[a]pyrene. The ZEM ensured consistent specimen orientation in lateral, dorsal and ventral view, enabling quantitative image-based analysis and reliable toxicological assessment. This platform has the potential to be utilized for image-based screening and mechanistic studies, supporting multi-time point observations, reproducible image acquisition, and statistical analysis using the zebrafish model.