Department of Physiology, McGill University, Montreal, QC, Canada.
Methods Mol Biol. 2022;2515:171-191. doi: 10.1007/978-1-0716-2409-8_11.
Emerging evidence suggests that neurodegeneration is directly linked to dysfunction of cytoskeleton; however, visualizing the organization of cytoskeletal structures in brain tissues remains challenging due to the limitation of resolution of light microscopy. Superresolution imaging overcomes this limitation and resolves subcellular structures below the diffraction barrier of light (20-200 nm), while retaining the advantages of fluorescent microscopy such as simultaneous visualization of multiple proteins and increased signal sensitivity and contrast. However, superresolution imaging approaches have been largely limited to very thin samples such as cultured cells growing as a single monolayer. Analysis of thicker tissue sections represents a technical challenge due to high background fluorescence and quality of the tissue preservation methods. Among superresolution microscopy approaches, structured illumination microscopy is one of the most compatible methods for analyzing thicker native tissue samples. We have developed a methodology that allows maximal preservation and quantitative analyses of cytoskeletal networks in tissue sections from a rodent brain. This methodology includes a specialized fixation protocol, tissue preparation, and image acquisition procedures optimized for the characterization of subcellular cytoskeletal structures using superresolution with structured illumination microscopy.
新出现的证据表明，神经退行性变与细胞骨架功能障碍直接相关；然而，由于光显微镜的分辨率限制，观察脑组织中细胞骨架结构的组织仍然具有挑战性。超分辨率成像克服了这一限制，可解析低于光衍射极限（20-200nm）的亚细胞结构，同时保留荧光显微镜的优点，如同时可视化多个蛋白质以及提高信号灵敏度和对比度。然而，超分辨率成像方法在很大程度上仅限于非常薄的样本，例如作为单层生长的培养细胞。由于高背景荧光和组织保存方法的质量，对较厚的组织切片进行分析代表了一个技术挑战。在超分辨率显微镜方法中，结构照明显微镜是分析较厚的天然组织样本的最兼容方法之一。我们已经开发了一种方法，允许最大限度地保存和定量分析来自啮齿动物大脑的组织切片中的细胞骨架网络。该方法包括专门的固定方案、组织准备和图像采集程序，这些程序经过优化，可使用结构光照明显微镜进行超分辨率对亚细胞细胞骨架结构进行特征化。
