Mohler William A, Isaacson Ariel B
Cold Spring Harb Protoc. 2010 Mar;2010(3):pdb.prot5388. doi: 10.1101/pdb.prot5388.
This protocol describes the preparation of Caenorhabditis elegans embryos for four-dimensional (4D) imaging. Embryos are excised from gravid hermaphrodites and allowed to adhere directly to a poly-L-lysine-coated coverslip. The coverslip is then used to create a slide chamber in which the embryos are suspended in buffer. These suspended mounts are better for 4D recordings than agar mounts because, although agar mounts produce more uniformly oriented embryos, evaporation from the agar pads tends to shift the embryos gradually over time. 4D spatial registration with suspended mounts is quite stable, and it is possible to digitally reorient embryos within the recorded volume of the three-dimensional (3D) stack during post-processing of the data.
本方案描述了用于四维（4D）成像的秀丽隐杆线虫胚胎的制备方法。从怀孕的雌雄同体线虫中取出胚胎，使其直接附着在聚-L-赖氨酸包被的盖玻片上。然后使用盖玻片制作一个载玻片腔室，将胚胎悬浮在缓冲液中。与琼脂固定相比，这些悬浮固定更适合进行4D记录，因为尽管琼脂固定能使胚胎的取向更均匀，但随着时间的推移，琼脂垫上的蒸发往往会使胚胎逐渐移位。悬浮固定的4D空间配准相当稳定，并且在数据后处理过程中，可以在三维（3D）堆栈的记录体积内对胚胎进行数字重新定向。
