Figure 1.

Robotically articulated colonoid microinjection maintains atmospheric control facilitating long-term sampling of large batches of colonoids. (A) Organoids grown from adult stem cells in 3D culture form complex monolayers organized around a hollow, mucus-filled lumen cavity analogous to the colon lumen. (B) 3D-printed customized fittings were used to mount robotic microinjection hardware within an atmospheric imaging chamber of an automated imaging system. (C) A 90° bend in the injection needle allows for vertical articulation of the microinjection needle, minimizing hydrogel disruption and needle breaking during injections. (D) Wet etching mechanically pulled borosilicate capillaries produces clean, fine aperture needles capable of injecting large batches of organoids without disturbing monolayer integrity. (E) Computer vision made and measured the area of masks from images of the smallest droplets of fluorescent cargo delivered reproducibly by 5 replicate needles, facilitating volume estimation. (F) Optimized needles produce varying volumes at the same injection duration (black) but similar minimal volumes when the minimal duration reproducibly delivering cargo from each needle is used (blue). (G) Organoid-microbe injections can be performed visually by locating an organoid of interest (1), aligning the needle to the organoid lumen (2), articulating the needle against the organoid monolayer distorting its appearance (3), puncturing the monolayer and injecting cargo (4), and retracting the needle from the organoid lumen (5) to visualize specific transplantation of DsRED-expressing E coli within the lumen (needle tip is marked with an asterisk). EpCAM, Epithelial cell adhesion molecule; Muc2, Mucin 2; Ortho, Orthogonal view; RFP, Red Fluorescent Protein.