Accurate lineage reconstruction of mammalian pre-implantation development is
essential for inferring the earliest cell fate decisions. Lineage tracing using global fluorescence
labeling techniques is complicated by increasing cell density and rapid embryo rotation, which
hampers automatic alignment and accurate cell tracking of obtained four-dimensional imaging data
sets. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (prpcFPs) to simultaneously visualize the global green and the photoconverted red population in order
to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of
H2B-pr-mEosFP-labeled nuclei combined with light-sheet imaging greatly facilitates segmentation,
classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using
green and red labels as fiducial markers, we computationally correct for rotational and translational
drift, reduce overall data size, and accomplish high-fidelity lineage tracing even for increased
imaging time intervals – addressing major concerns in the field of volumetric embryo imaging.