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. 2022 Aug 18;13:4864. doi: 10.1038/s41467-022-32465-z

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Fixation and backtracking strategy for light-sheet imaging of organoid growth (shown recording 007). b After fixation a registration step may be needed to overlap the fixed nuclei with the nuclei as shown in the last timepoint of the timelapse. Left: midplane of raw data from last time-point H2B-mCherry recording (cyan) against same plane after fixation (red). Center: registration maps the fixed volume into the last time-point volume. Although overlap is not perfect, it is sufficient to maps each nucleus back. Right: After registration additional information from immunolabelling can be overlayed onto last time-point of the live recording, and so cells of interest can be backtracked. A total of four different experiments were performed with similar results. c Lineage tree depicting the backtracking of two sister cells. d After fixation, staining for DLL1 and Lysozyme shows two cells expressing these markers. Backtracking of them is shown in (a). e Nuclei volume distribution for the backtracked cells against all other cells per generation. f Evaluation of nuclei and cell volumes for the backtracked cells against all other cells during generations 5 and 6, as depicted in (c). For all other cells, the midline corresponds to the mean, whereas the gray region is the standard distribution. g Nearest neighbor evaluation of the backtracked Lys+ cell (red). All nearest neighbors are depicting in magenta, with all remaining cells in gray. Dashed line on the lineage tree is presented as corresponding segmented meshes on the right. A dividing cell can be recognized by the interkinetic movement of its nucleus further apically. Source data are provided as a Source Data file.