预标记追踪:采用双标记方法对活体样本进行可靠的体积单细胞追踪和谱系追踪
Primed Track: Reliable Volumetric Single-cell Tracking and Lineage Tracing of Living Specimen with Dual-labeling Approaches.
作者信息
Welling Maaike, Kalyviotis Konstantinos, Pantazis Periklis
机构信息
Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, Basel, Switzerland.
Department of Bioengineering, Imperial College London, London, United Kingdom.
出版信息
Bio Protoc. 2020 Jun 5;10(11):e3645. doi: 10.21769/BioProtoc.3645.
Mammalian embryonic development starts with a single fertilized zygote that develops into a blastocyst embryo consisting of three cell types that evolve into either embryonic or extra-embryonic tissues. Lineage tracing of these cells can provide important information about the molecular and cellular dynamics contributing to fate allocation during early development. While global labeling techniques allow for visualization of all cells at the same time, lineage tracing of cells over several divisions can become complicated due to embryo movement and rotation as well as increasing cell densities. Here, we use green-to-red photoconvertible proteins for both global and sparse labeling of cells of interest in the developing murine embryo. We use primed conversion to achieve precise photoconversion of single nuclei in 4-cell stage embryos followed by volumetric live imaging to capture development up to the blastocyst stage. We developed an image analysis pipeline, called , that uses the dual labeling strategy for both straightforward segmentation and registration of all cells in the embryo as well as correction of rotational and spatial drift. Together, this strategy allows for reliable and fast tracking and lineage tracing of individual cells, even over increased imaging time intervals that result in a major reduction in data volume, all essential conditions for volumetric long-term imaging techniques.
哺乳动物的胚胎发育始于单个受精的受精卵,该受精卵发育成一个囊胚胚胎,由三种细胞类型组成,这些细胞类型会发育成胚胎组织或胚外组织。对这些细胞进行谱系追踪可以提供有关早期发育过程中细胞命运分配的分子和细胞动力学的重要信息。虽然全局标记技术可以同时可视化所有细胞,但由于胚胎的移动和旋转以及细胞密度的增加,对细胞进行多个分裂周期的谱系追踪可能会变得复杂。在这里,我们使用绿色到红色的光转换蛋白对发育中的小鼠胚胎中感兴趣的细胞进行全局和稀疏标记。我们使用引发转换在4细胞期胚胎中实现单核的精确光转换,然后进行体积实时成像以捕获直至囊胚期的发育过程。我们开发了一种名为的图像分析流程,该流程使用双重标记策略对胚胎中的所有细胞进行直接分割和配准,以及校正旋转和空间漂移。总之,这种策略允许对单个细胞进行可靠且快速的跟踪和谱系追踪,即使在成像时间间隔增加导致数据量大幅减少的情况下也是如此,而这些都是体积长期成像技术的所有基本条件。