Gray Robert D M, Mercer Jason, Henriques Ricardo
MRC Laboratory for Molecular Cell Biology, University College London; Centre for Mathematics and Physics in Life Sciences and Experimental Biology (CoMPLEX), University College London.
MRC Laboratory for Molecular Cell Biology, University College London;
J Vis Exp. 2017 Apr 9(122):55471. doi: 10.3791/55471.
Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm - VirusMapper - packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.
超分辨率荧光显微镜目前正在彻底改变细胞生物学研究。它突破约300纳米分辨率极限的能力使得纳米级生物复合物和过程能够常规成像。分辨率的提高还意味着电子显微镜中常用的方法，如单颗粒分析，可以很容易地应用于超分辨率荧光显微镜。通过将这种分析方法与超分辨率光学成像相结合，利用荧光显微镜的分子特异性标记能力来生成亚稳态结构内分子元件的结构图成为可能。为此，我们开发了一种新颖的算法——VirusMapper——并将其打包为一个易于使用、高性能且高通量的ImageJ插件。本文对该软件进行了深入指南，展示了其揭示生物分子复合物新结构特征的能力。在此，我们介绍如何组装兼容数据，并提供一份关于如何使用该算法将单颗粒分析应用于超分辨率图像的分步方案。
