Perner Birgit, Schnerwitzki Danny, Graf Michael, Englert Christoph
Molecular Genetics, Leibniz Institute on Aging - Fritz Lipmann Institute (FLI).
Molecular Genetics, Leibniz Institute on Aging - Fritz Lipmann Institute (FLI); Carl Zeiss Microscopy GmbH.
J Vis Exp. 2016 Apr 7(110):e53921. doi: 10.3791/53921.
In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development. The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed. In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance. The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.
为了理解器官发生过程，需要记录组织发育过程中发生的空间和时间变化。本文所述方法通过使用配备结构光照和z轴堆叠采集功能的荧光解剖显微镜，对斑马鱼胚胎正常和受损的肾脏发育进行延时分析。为了可视化肾发生过程，使用了具有荧光标记肾脏结构的转基因斑马鱼（Tg(wt1b:GFP)）。通过注射针对威尔姆斯肿瘤基因wt1a的反义吗啉代寡核苷酸来引发肾脏缺陷，已知该基因对肾脏发育至关重要。该实验设置的优点是将变焦显微镜与简单的策略相结合，无需额外设备即可在x、y或z方向重新调整移动。为了避免由温度变化和机械振动引起的焦点漂移，采用了自动聚焦策略，而不是使用通常需要的环境箱。为了重新调整由于xy轴漂移引起的位置变化，使用了带有印记重定位网格的成像室。与使用共聚焦激光扫描或光片显微镜等更复杂的光学切片延时记录设置相比，变焦显微镜易于操作。此外，它还具有解剖显微镜特有的优点，如高景深和长工作距离。本文介绍的研究器官发生的方法也可用于不具备光学切片能力的荧光立体显微镜。尽管该技术在高通量方面存在局限性，但它为通常用于发育组织和器官动态延时记录的更复杂设备提供了一种替代方案。
