Pereira Pedro M, Almada Pedro, Henriques Ricardo
MRC Laboratory for Molecular Cell Biology and Department of Cell and Developmental Biology, University College London, London, UK.
Methods Cell Biol. 2015;125:95-117. doi: 10.1016/bs.mcb.2014.10.004. Epub 2015 Jan 7.
Super-resolution (SR) methodologies permit the visualization of cellular structures at near-molecular scale (1-30 nm), enabling novel mechanistic analysis of key events in cell biology not resolvable by conventional fluorescence imaging (∼300-nm resolution). When this level of detail is combined with computing power and fast and reliable analysis software, high-content screenings using SR becomes a practical option to address multiple biological questions. The importance of combining these powerful analytical techniques cannot be ignored, as they can address phenotypic changes on the molecular scale and in a statistically robust manner. In this work, we suggest an easy-to-implement protocol that can be applied to set up a high-content 3D SR experiment with user-friendly and freely available software. The protocol can be divided into two main parts: chamber and sample preparation, where a protocol to set up a direct STORM (dSTORM) sample is presented; and a second part where a protocol for image acquisition and analysis is described. We intend to take the reader step-by-step through the experimental process highlighting possible experimental bottlenecks and possible improvements based on recent developments in the field.
超分辨率（SR）方法能够在近分子尺度（1-30纳米）下可视化细胞结构，从而对细胞生物学中的关键事件进行新颖的机制分析，而这些事件是传统荧光成像（分辨率约为300纳米）无法解析的。当这种细节水平与计算能力以及快速可靠的分析软件相结合时，使用SR进行高内涵筛选就成为解决多个生物学问题的切实可行的选择。不能忽视将这些强大分析技术相结合的重要性，因为它们能够在分子尺度上以统计学上可靠的方式解决表型变化问题。在这项工作中，我们提出了一个易于实施的方案，该方案可应用于使用用户友好且免费的软件建立高内涵3D SR实验。该方案可分为两个主要部分：腔室和样品制备，其中介绍了设置直接STORM（dSTORM）样品的方案；第二部分描述了图像采集和分析的方案。我们打算引导读者逐步了解实验过程，突出可能的实验瓶颈以及基于该领域最新进展的可能改进。
