Department of Biological Sciences, Tata Institute of Fundamental Research.
Department of Biological Sciences, Tata Institute of Fundamental Research;
J Vis Exp. 2024 Jul 19(209). doi: 10.3791/66735.
Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.
光片显微镜已成为在最小光毒性条件下对斑马鱼胚胎进行长时间活成像的首选方法。特别是，多视角系统允许样品旋转，可从不同角度对整个胚胎进行成像。然而，在多视角系统的大多数成像过程中，由于样品通常被制备在聚合物管中，因此样品安装是一个麻烦的过程。为了帮助这一过程，本方案描述了在 70%胚层内卷和早期体节阶段之间成像早期斑马鱼发育的基本安装策略。具体而言，该研究提供了关于在绒毛膜内 70%胚层内卷和芽期胚胎默认位置的各种位置的统计数据。此外，它还讨论了在发育早期对整个斑马鱼胚胎进行成像所需的最佳角度数量和角度间隔，以便通过融合不同的视图提取细胞尺度信息。最后，由于胚胎覆盖了相机的整个视场，这是获得细胞尺度分辨率所必需的，因此该方案详细介绍了使用胚胎上方或下方的珠粒信息来注册不同视图的过程。
