Long-Term, Six-Dimensional Live-Cell Imaging for the Mouse Preimplantation Embryo That Does Not Affect Full-Term Development
Long-Term, Six-Dimensional Live-Cell Imaging for the Mouse Preimplantation Embryo That Does Not Affect Full-Term Development
复制标题
DOI：
10.1262/jrd.20166
复制
发表时间：
2009-06-01
影响因子：
1.8
通讯作者：
Wakayama, Teruhiko
中科院分区：
文献类型：
--
作者：
Yamagata, Kazuo;Suetsugu, Rinako;Wakayama, Teruhiko
哺乳动物植入前胚胎发育是通过多种时间和空间的紧密协调调控实现的。变化因此，对这些事件进行三维动态的分析是很有价值的。我们以前已经开发了一种活细胞成像方法的基础上表达的荧光蛋白，使用mRNA注射和延时荧光显微镜。然而，使用传统的荧光显微镜，由于胚胎的厚度和光学问题，无法消除“焦点模糊”，因此无法获得三维图像。此外，由于细胞反复暴露于强激发光会产生光毒性，因此长期观察对胚胎发育不利。在这里，我们改进了我们的成像系统，以实现小鼠植入前胚胎的六维活细胞成像（x，y和z轴，延时，重复和多样本）。重要的是，通过改进成像设备和优化成像条件，如激发强度和图像采集的时间间隔，该程序本身对足月发育无害，尽管它是一个延长的成像过程。例如，从以7.5分钟间隔施加两种不同波长的激发（488和561 nm）约70小时的胚胎中获得活的幼崽，并且在每个时间点在z轴上获得51个图像;因此，总共拍摄了56，814个荧光图像。所有的幼崽都是健康的，生殖正常，没有转基因。因此，这种活细胞成像技术对于小鼠的发育是安全的。这为发育和生殖研究提供了一种新的方法，因为它可以对发育进行回顾性和前瞻性分析。它也可能适用于人类生殖技术和生产动物研究等领域的胚胎质量评估。
Mammalian preimplantation embryonic development is achieved by tightly coordinated regulation of a great variety of temporal and spatial. changes. Therefore, it would be valuable to analyze these events three-dimensionally and dynamically. We have previously developed a live-cell imaging method based on the expression of fluorescent proteins, using mRNA injection and time-lapse florescence microscopy. However, with conventional fluorescent microscopy, three-dimensional images could not be obtained due to the thickness of the embryos and the optical problem in which 'out-of focus blur' cannot be eliminated. Moreover, as the repeated exposure of intense excitation light to the cell yields phototoxicity, long-term observation was detrimental to embryonic development. Here, we improved our imaging system to enable six-dimensional live-cell imaging of mouse preimplantation embryos (x, y and z axes, time-lapse, multicolor and multisample). Importantly, by improving the imaging devices and optimizing the conditions for imaging, such as intensity of excitation and time intervals for image acquisition, the procedure itself was not detrimental to full-term development, although it is a prolonged imaging process. For example, live pups were obtained from embryos to which two different wavelengths of excitation (488 and 561 nm) were applied at 7.5-min intervals for about 70 h, and 51 images were acquired in the z axis at each time point; thus, a total of 56,814 fluorescent images were taken. All the pups were healthy, reproductively normal and not transgenic. Thus, this live-cell imaging technology is safe for full-term mouse development. This offers a novel approach for developmental and reproductive research in that it enables both retrospective and prospective analyses of development. It might also be applicable to assessment of embryo quality in fields such as human reproductive technology and production animal research.