Strobl Frederic, Klees Selina, Stelzer Ernst H K
Physical Biology, Buchmann Institute for Molecular Life Sciences (BMLS); Cluster of Excellence Frankfurt, Macromolecular Complexes; Goethe-Universität Frankfurt am Main - Campus Riedberg.
Physical Biology, Buchmann Institute for Molecular Life Sciences (BMLS); Cluster of Excellence Frankfurt, Macromolecular Complexes; Goethe-Universität Frankfurt am Main - Campus Riedberg;
J Vis Exp. 2017 Apr 28(122):55629. doi: 10.3791/55629.
The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species. This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.
赤拟谷盗已成为发育遗传学和进化发育生物学领域重要的昆虫模式生物。与传统的宽视野和共聚焦荧光显微镜相比，使用基于光片的荧光显微镜观察赤拟谷盗胚胎具有多种优势。由于基于光片的显微镜具有独特的特性，可以在数天的时间内，沿多个方向以高信噪比记录活体标本的三维图像，同时显著减少光漂白和光毒性。经过四年多的方法开发以及数据的不断增加，现在似乎是为赤拟谷盗群体乃至整个昆虫群体建立光片技术使用标准操作规程的合适时机。本方案描述了适用于不同目的的三种固定技术，展示了两种适用于长期活体成像的新型定制转基因赤拟谷盗品系，推荐了五种用于标记固定胚胎细胞内结构的荧光染料，并提供了关于数据后处理的信息，以便及时评估所记录的数据。代表性结果集中在长期活体成像、光学切片以及沿多个方向观察同一个胚胎。相应的数据集作为可下载资源提供。最后，该方案讨论了活体成像实验的质量控制、当前的局限性以及所概述程序对其他昆虫物种的适用性。本方案主要面向寻求优于标准实验室设备的成像解决方案的发育生物学家。它推动了持续努力，以弥合在方法上开发和完善显微镜技术的技术导向型实验室/群体与需要应对技术挑战的“即插即用”解决方案的生命科学实验室/群体之间的差距。此外，它支持一种将生物学问题置于关注中心的公理方法。
