Histograms displaying how different factors affect lineage reconstruction accuracy of the automated segmentation and tracking method presented in this study. All histograms were extracted from n = 5,331 linkage annotations in the SiMView time-lapse recording of Drosophila embryogenesis. The ranges of the plots span nearly the full physical limits of parameters measured in this data set, which are as follows: the nuclei nearest neighbor distance ranges from 4 µm to 21 µm, the nuclei distance to the center of the embryo (with a diameter of ~200 µm) ranges from 0 to 106 µm, centroid displacements range from 0 µm to 44 µm between consecutive time points, and the 90th percentile of the local image contrast is 33.(a) The larger the distance between adjacent nuclei, the higher the quality of the cell lineage reconstruction.(b) The shorter the path traveled by the fluorescent signal to the detection objective, the higher the image quality and the better the cell lineage reconstruction.(c) The quality of the cell lineage reconstruction is independent of the nucleus displacement between two time points, as long as this displacement is not too large. This is the main assumption in our framework and for very large displacements the method breaks down (note that the scale of the vertical axis is substantially different compared to the other plots). Optical flow techniques can extend this range substantially and enable successful application of the automated segmentation and tracking framework also in the presence of larger displacements, if necessary. However, such an extension was not required for any of the data examples presented in this study.(d) The higher the local image contrast (ratio of nucleus brightness versus background level), the better the cell lineage reconstruction.