Jusuf Patricia, Harris William A, Poggi Lucia
Cold Spring Harb Protoc. 2013 Mar 1;2013(3):pdb.prot073544. doi: 10.1101/pdb.prot073544.
In this protocol, we describe how to make and analyze four dimensional (4D) movies of retinal lineage in the zebrafish embryo in vivo. 4D consists of three spatial dimensions (3D) reconstructed from stacks of confocal planes plus one time dimension. Our imaging is performed on transgenic cells that express fluorescent proteins under the control of cell-specific promoters or on cells that transiently express such reporters in specific retinal cell progenitors. An important aspect of lineage tracing is the ability to follow individual cells as they undergo multiple cell divisions, final migration, and differentiation. This may mean many hours of 4D imaging, requiring that cells be kept healthy and maintained under conditions suitable for normal development. The longest movies we have made are ∼50 h. By analyzing these movies, we can see when a specific cell was born and who its sister was, allowing us to reconstruct its retinal lineages in vivo.
在本实验方案中，我们描述了如何在斑马鱼胚胎体内制作和分析视网膜谱系的四维（4D）电影。4D由从共聚焦平面堆栈重建的三个空间维度（3D）加上一个时间维度组成。我们的成像在转基因细胞上进行，这些细胞在细胞特异性启动子的控制下表达荧光蛋白，或者在特定视网膜细胞祖细胞中瞬时表达此类报告基因的细胞上进行。谱系追踪的一个重要方面是能够跟踪单个细胞经历多次细胞分裂、最终迁移和分化的过程。这可能意味着需要数小时的4D成像，要求细胞保持健康并在适合正常发育的条件下维持。我们制作的最长电影约为50小时。通过分析这些电影，我们可以看到特定细胞何时产生以及它的姐妹细胞是谁，从而能够在体内重建其视网膜谱系。
