MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.
PLoS Biol. 2019 Aug 9;17(8):e3000388. doi: 10.1371/journal.pbio.3000388. eCollection 2019 Aug.
Methods for measuring the properties of individual cells within their native 3D environment will enable a deeper understanding of embryonic development, tissue regeneration, and tumorigenesis. However, current methods for segmenting nuclei in 3D tissues are not designed for situations in which nuclei are densely packed, nonspherical, or heterogeneous in shape, size, or texture, all of which are true of many embryonic and adult tissue types as well as in many cases for cells differentiating in culture. Here, we overcome this bottleneck by devising a novel method based on labelling the nuclear envelope (NE) and automatically distinguishing individual nuclei using a tree-structured ridge-tracing method followed by shape ranking according to a trained classifier. The method is fast and makes it possible to process images that are larger than the computer's memory. We consistently obtain accurate segmentation rates of >90%, even for challenging images such as mid-gestation embryos or 3D cultures. We provide a 3D editor and inspector for the manual curation of the segmentation results as well as a program to assess the accuracy of the segmentation. We have also generated a live reporter of the NE that can be used to track live cells in 3 dimensions over time. We use this to monitor the history of cell interactions and occurrences of neighbour exchange within cultures of pluripotent cells during differentiation. We provide these tools in an open-access user-friendly format.
用于测量其天然 3D 环境中单个细胞特性的方法将使人们能够更深入地了解胚胎发育、组织再生和肿瘤发生。然而，目前用于分割 3D 组织中细胞核的方法并不是针对细胞核密集、非球形或形状、大小或纹理不均匀的情况设计的，这些情况在许多胚胎和成人组织类型中都是真实存在的，而且在许多情况下，对于在培养中分化的细胞也是如此。在这里，我们通过设计一种基于标记核膜（NE）的新方法来克服这一瓶颈，然后使用树状结构的脊跟踪方法自动区分单个细胞核，并根据训练有素的分类器进行形状排序。该方法速度快，可处理大于计算机内存的图像。即使对于具有挑战性的图像，如中期胚胎或 3D 培养物，我们也始终获得>90%的准确分割率。我们提供了一个 3D 编辑器和检查器，用于手动编辑分割结果，并提供了一个程序来评估分割的准确性。我们还生成了一个 NE 的实时报告器，可用于随时间在 3 个维度上跟踪活细胞。我们使用它来监测多能细胞分化过程中培养物中细胞相互作用和邻居交换的历史。我们以开放访问的用户友好格式提供这些工具。
