(a) Overview of the pipeline used for reliable automated segmentation, tracking, and lineage tracing of the imaged embryos; (1) Segmentation: low thresholds are used for the spot detection in both the green and red channel to enable detection of dimmer cells at later developmental time points. Incorrectly segmented spots are excluded by defined filters: (i) exclusion of spots outside of a defined radius of the embryo, (ii) replacement of incorrectly segmented double spots by one spot per one nucleus, and (ii) exclusion of red spots that do not colocalize with green nuclear spots. (2) Tracking: Spatial drift as well as rapid embryo rotation complicates tracking nuclei over prolonged time windows. The segmented nuclei are used for defining reference frames based on the center of mass of the green nuclei and the orientation of the red nuclei. The alignment of the references frames of each time point compensates the spatial and rotational drifts. (3) Lineage tracing: Automated lineage tree reconstruction can make false connections when cells are dividing. By separating the calculation of the lineage trees in the photoconverted red channel from the green channel, the less complex datasets for each channel result in more consistent lineage tracing. pr-mEosFP fluorescence (green) and primed converted pr-mEosFP fluorescence (magenta) overlaid with segmentation results (green and Magenta spheres); Scale bar, 20 µm (b) Lineage trees from the same embryo (corresponding to Videos 1 and 4) reconstructed from segmented nuclei before correction for rotational and translational drift (left), after correction for rotational and translational drift for the red channel (second left), after correction for rotational and translational drift for the green channel minus the spots that colocalize with the red spots (second right), and after final manual lineage reconstruction (right). The embryo was imaged every 15 min.