McGurk Patrick D, Lovely C Ben, Eberhart Johann K
Institute for Cell and Molecular Biology, The University of Texas at Austin.
J Vis Exp. 2014 Jan 30(83):e51190. doi: 10.3791/51190.
Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.
延时成像技术能够直接观察形态发生过程，即形状的形成过程。斑马鱼胚胎因其光学透明性以及易于进行基因操作，已成为一种常用的模式生物，用于对活体胚胎的形态发生进行延时分析。对活斑马鱼胚胎进行共聚焦成像，需要用荧光标记物（如转基因或注射染料）持续标记感兴趣的组织。这个过程要求胚胎被麻醉并固定在合适位置，以便其正常健康发育。成像参数的设置必须考虑到三维生长情况，并在分辨单个细胞的需求与快速获取发育快照之间取得平衡。我们的研究结果表明，能够对荧光标记的斑马鱼胚胎进行长期的体内成像，并检测颅神经嵴中导致颅面异常的各种组织行为。麻醉和固定造成的发育延迟极小，且胚胎在此过程中未受损伤。经延时成像的胚胎可放回液体培养基中，随后在发育的后续阶段进行成像或固定。随着转基因斑马鱼品系的日益丰富以及特征明确的命运图谱和移植技术的发展，对任何所需组织进行成像成为可能。因此，体内延时成像与斑马鱼遗传方法（包括对突变体和显微注射胚胎的分析）能有力地结合起来。
