Max Planck Institute of Molecular Cell Biology and Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany.
Development. 2012 Sep;139(17):3242-7. doi: 10.1242/dev.082586.
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).
光片显微镜技术，如选择平面照明显微镜（SPIM），非常适合对持续数小时到数天的发育过程进行延时成像。这项有前途的技术的成功主要受到缺乏适合脆弱样本安装的技术的限制。在琼脂糖中嵌入斑马鱼胚胎，这在传统的共聚焦显微镜中很常见，但会导致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移动性。我们发现，填充低浓度琼脂糖或甲基纤维素的氟化乙烯丙烯（FEP）管在 3 天内为活体胚胎提供了足够的生理环境限制和适合荧光成像的光学清晰度之间提供了最佳平衡。我们还比较了不同浓度的 Tricaine 对斑马鱼发育的影响，并根据应用提供了最佳使用的指南。我们的研究结果将使光片显微镜技术更适用于发育生物学的更多领域，特别是斑马鱼胚胎和其他小型生物的多视图长期成像。此外，对整体和体内成像的样本制备的改进将促进其他新兴的光学成像技术，如光学投影断层扫描（OPT）。
