A machine vision guided robot for fully automated embryonic microinjection

Abstract

Microinjection is a widely used technique for transgenesis, mutagenesis, cell labeling, cryopreservation, and in vitro fertilization in multiple single and multicellular organisms. Microinjection requires specialized skills acquired for each target organism and involves rate limiting and labor-intensive preparatory steps. Here we constructed a machine vision (MV) guided generalized robot that fully automates the process of microinjection in fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio) embryos. The robot uses machine learning (ML) models trained to detect individual embryos in images of agar plates, and models trained to identify specific anatomical locations within each embryo in 3D space using dual view microscopes. The robot uses this information to serially perform microinjection in each detected embryo without any human intervention. We constructed and used three such robots to automatically microinject tens of thousands of Drosophila and zebrafish embryos. We systematically optimized robotic microinjection for each species and validated the use of the robot by performing routine transgenesis with proficiency comparable to highly skilled human practitioners while achieving up to 4x increases in microinjection throughput in Drosophila. The automated microinjection robot was utilized to microinject pools of over 20,000 uniquely barcoded plasmids into 1,713 embryos in two days to rapidly generate more than 400 unique transgenic Drosophila lines. This high throughput microinjection experiment enabled a novel measurement of the number of independent germline integration events per successfully injected embryo. Finally, we showed that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and survival of cryopreserved embryos post-thaw as compared to manual microinjection, opening the tantalizing possibility of large-scale cryobanking of aquatic species at an industrial scale. We anticipate that this versatile automated microinjection system can be applied to carry out microinjection for genome-wide manipulation and cryopreservation at scale in a wide range of other organisms.

Conduction‐Dominated Cryomesh for Organism Vitrification

“…[58,59,65] Zebrafish embryos were microinjected with 10 nL of CPA at the high cell stage (80% PG and 20% MeOH) using robotic microinjection. [69] The embryos were cultured in an incubator at 28 °C for 3 h after microinjection. The zebrafish embryos were transferred to the cryomesh with care.…”