a. Images of an electroporated organoid just before sectioning at day 64 (upper left), after placement at the ALI (upper right) and stills from long-term live imaging (Supplementary Video 1) of this same tissue (bottom panels) after 2 days at the ALI. Time stamp is hrs:min. b. Temporal projection showing highly disorganized axon outgrowth at this very early stage (2 days ALI, 66 days total). a-b are representative images out of 5 similarly staged and live imaged ALI-COs. c. Temporal projection of a time-lapse (Supplementary Video 5) of a region of the ALI-CO shown in Fig. 3e and boxed in d. Overlay includes tracings of individual growth cones (colored lines) and boxed regions used for kymograph analysis (right panels) showing velocities of growth cones (yellow arrowheads). Black arrowhead points to the turning point of an axon. d. Image of the same region shown in Fig. 3e at an earlier time point of 14 days at the ALI (84 days total). e. Fluorescence image of fGFP+ tracts used for directional image analysis shown in Fig. 3f. f. Image of fGFP labelled axons with costaining for DAPI in a 90 day ALI-CO (of which 26 days at ALI), revealing tracts extending away from the main body of the organoid (outlined). g. Another example of a fGFP electroporated ALI-CO (90 days total of which 20 days at ALI) displaying both internal (yellow arrows) and escaping (white arrows) tract morphologies. h. Tracing of individual growth cones within intersecting and interdigitated fiber tracts showing crossing behaviour reminiscent of decussation (arrow) (Supplementary Video 6). Age: 17 days at the ALI, 100 days total. i. Temporal projection (left panel) and growth cone tracing (right panel) showing instances of growth cone turning (arrowheads) (Supplementary Video 7). Age: 9 days at the ALI, 79 days total. c-i are representative images out of 7 similarly staged and imaged ALI-COs. Scale bars, 500 μm (a, d, e, f, g) and 100 μm (b, c, h, i).