Extended Data Fig. 2: An automated platform for fixing, staining, and imaging Drosophila embryos.

a–d, Collecting Drosophila embryos. (a) Custom fly chambers were made, holding up to 24 different strains. (b) An explosion-view of the fly chambers. Embryo meshes (red) can attach and detach from the fly chambers and are suspended above an apple juice-agar plate. (c) Embryos are collected onto the embryo meshes and washed with saline solution and bleached. (d) Embryos are loaded into a fixation plate. e–h, Components of the robot. (e) The fluid-dispensing manifold. Seven pneumo-hydraulic syringe pumps are coupled to the fluid-dispensing manifold; one pump for priming chemicals into the fluid-dispensing manifold, and six pumps for dispensing chemicals into the fixation plate. (f) The fluid-separating manifold uses 24 small syringes to aspirate fluid from the isotonic shocking attachments. (g and g’) Different components of the robot. (h) Cross-section of the fixation plate and aspiration tips and syringes. 24 small aspiration tips draw fluid from the top of each well within the isotonic shocking attachment and six main dual-purpose tips dispense and aspirate fluid into and out of the bottom of the wells. i–k, The adaptive feedback imaging pipeline. (i) Samples are mounted on multi-well slides. (j) An overview tile-scan of each well is taken and x,y coordinates for embryos (green) are identified either manually or computationally. (k) For each coordinate, a fast, low-resolution confocal stack is automatically acquired. An algorithm determines the embryo’s z position and rotation, yielding a bounding box within which a high-resolution, 3D stack of the entire embryo is acquired. See also Methods. l, Control E3N WT embryos were fixed and stained on the robot. A single embryo in the same orientation and age from each well was selected and the individual nuclear fluorescence intensities were measured in AU, arbitrary units of fluorescence intensity. In plots, centre line is mean, upper and lower limits are standard deviation.