Fig. 2.

Single-organoid tracking. Organoid tracking over multiple time points was achieved with a registration mark and single-particle tracking. (a) A registration mark was inscribed on the bottom of the first well of the 24-well plate and referenced at each time point to ensure accurate $XYZ$ positioning. (b) Organoid tracking was achieved by registering the NAD(P)H image time series, segmenting the organoids in each frame, and detecting the centroids of each segmented organoid. These centroids were tracked using an open-source single-particle tracking tool (TrackMate). (c) A segmented organoid mask at 48-h posttreatment is overlaid with the centroid tracking results at each imaging time point. Scale bar: $500\mu \mathrm{m}$.