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Fig. 1 Overview of the zebrafish egg injection robot and experimental outline. (A) A representative image of the integrated egg injector. The needle holder is used to load needles and the motorized stage enables the movement of plates containing embryos during injections. A 96-well plate is for droplet calibration and the agarose gel grid plate is for the placement of zebrafish eggs. A touch screen allows for the operation and visualization of all the injection processes. (B–H) The procedure of zebrafish embryo injection using the robot. (B) Step1: preparation of an agarose grid plate using a mold. Different molds (e.g., 9 × 100, 1024, 360 grid wells) can be used based on research purposes. (C) Step 2: placement of zebrafish eggs on the agarose grid. Zebrafish embryos can be placed randomly at any wells of the grid, as empty wells can be recognized and skipped by the robot. (D) Step 3: needle calibration for both xy direction and z direction, as shown on the screenshots from the touch screen. (E) Step 4: droplet calibration is performed by injection of suspension into mineral oil and droplet size can be determined automatically by the robot. (F) Step 5: injection positions should be chosen, either yolk center or close to the first cell; subsequently, the robot can do injections automatically for wells that users have selected. Different injection distances (e.g., 0 μm, 30 μm and 50 μm) can be chosen for close to the first cell injection. (G) Step 6: noninjected eggs can be killed by loading a blunt needle. (H) Step 7: after the injection is finished, the injected zebrafish eggs can be transferred to petri dishes for culture and dead eggs should be removed.