Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope
Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope
复制标题
DOI：
10.1242/dev.082586
复制
发表时间：
2012-09-01
期刊：
影响因子：
4.6
通讯作者：
Huisken, Jan
中科院分区：
文献类型：
--
作者：
Kaufmann, Anna;Mickoleit, Michaela;Huisken, Jan
光片显微镜技术，如选择性平面照明显微镜（SPIM），非常适合持续几个小时到几天的发育过程的延时成像。这种有前途的技术的成功主要受到缺乏合适的技术来安装易碎样品的限制。将斑马鱼胚胎包埋在琼脂糖中，这在传统的共聚焦显微镜中很常见，导致了严重的生长缺陷和不可靠的结果。在这项研究中，我们系统地量化了在更合适的条件下安装的斑马鱼胚胎的活力和流动性。我们发现，填充有低浓度琼脂糖或甲基纤维素的氟化乙烯丙烯（FEP）制成的管提供了在生理环境中超过3天的活胚胎的充分限制与适合于荧光成像的光学清晰度之间的最佳平衡。我们还比较了不同浓度的三卡因对斑马鱼发育的影响，并根据应用情况提供了最佳使用指南。我们的研究结果将使光片显微镜技术适用于更多的发育生物学领域，特别是斑马鱼胚胎和其他小生物的多视图长期成像。此外，用于整体和体内成像的样品制备的改进将促进其他新兴的光学成像技术，例如光学投影断层扫描（OPT）。
Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).