McGovern Institute for Brain Research and Koch Institute, MIT, Cambridge, MA, USA.
Department of Biological Engineering, MIT, Cambridge, MA, USA.
Methods Mol Biol. 2022;2468:141-203. doi: 10.1007/978-1-0716-2181-3_9.
Studies of C. elegans will benefit from a powerful method for super-resolution imaging of proteins and mRNAs at any 3-D locations throughout the entire animal. Conventional methods of super-resolution imaging in C. elegans, such as STORM, PALM, SR-SIM and STED, are limited by imaging depths that are insufficient to map the entire depth of adult worms, and involve hardware that may not be accessible to all labs. We recently developed expansion of C. elegans (ExCel), a method for physically magnifying fixed whole animals of C. elegans with high isotropy, which provides effective resolutions finer than the diffraction limit, across the entire animal, on conventional confocal microscopes. In this chapter, we present a family of three detailed ExCel protocols. The standard ExCel protocol features simultaneous readout of diverse molecules (fluorescent proteins, RNA, DNA, and general anatomy), all at 70 nm resolution (3.5× linear expansion). The epitope-preserving ExCel protocol enables imaging of endogenous proteins with off-the-shelf antibodies, at a ~ 100 nm resolution (2.8× linear expansion). The iterative ExCel protocol allows readout of fluorescent proteins at ~25 nm resolution (20× linear expansion). The protocols described here comprise a versatile toolbox for super-resolution imaging of C. elegans.
秀丽隐杆线虫的研究将受益于一种强大的方法，可以在整个动物体内的任何 3D 位置对蛋白质和 mRNAs 进行超分辨率成像。秀丽隐杆线虫中传统的超分辨率成像方法，如 STORM、PALM、SR-SIM 和 STED，受到成像深度的限制，不足以绘制成年线虫的整个深度，并且涉及到可能不是所有实验室都能获得的硬件。我们最近开发了秀丽隐杆线虫的扩展（ExCel）方法，该方法可以物理放大秀丽隐杆线虫的整个固定动物，具有高度各向同性，在传统共聚焦显微镜上，在整个动物体内提供有效分辨率优于衍射极限的超分辨率成像。在这一章中，我们提出了一系列三种详细的 ExCel 协议。标准 ExCel 协议具有同时读出多种分子（荧光蛋白、RNA、DNA 和一般解剖结构）的功能，所有这些都具有约 70nm 的分辨率（约 3.5 倍线性扩展）。表位保留的 ExCel 协议允许使用现成的抗体对内源性蛋白质进行成像，分辨率约为 100nm（约 2.8 倍线性扩展）。迭代 ExCel 协议允许以约 25nm 的分辨率（约 20 倍线性扩展）读取荧光蛋白。这里描述的协议构成了秀丽隐杆线虫超分辨率成像的多功能工具包。
