Super-resolution microscopy with DNA-PAINT
Super-resolution microscopy with DNA-PAINT
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DOI：
10.1038/nprot.2017.024
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发表时间：
2017-06-01
期刊：
影响因子：
14.8
通讯作者：
Jungmann, Ralf
中科院分区：
文献类型：
--
作者：
Schnitzbauer, Joerg;Strauss, Maximilian T.;Jungmann, Ralf
超分辨率技术已经开始改变生物学和生物医学研究，它允许研究人员观察远低于经典衍射极限的结构。由于使用了DNA探针，纳米级地形成像的DNA点积累（DNA PAINT）为基于定位的超分辨率显微镜提供了一种易于实现的方法。在DNA-PAINT中，短染料标记（“成像仪”）寡核苷酸与互补目标（“对接”）链的短暂结合产生了必要的“闪烁”，从而实现了随机超分辨率显微镜。利用DNA分子的可编程性和特异性作为成像和标记探针，研究人员可以将闪烁从染料光物理中分离出来，减轻了当前超分辨率技术的局限性，使它们与几乎任何单分子兼容的染料兼容。DNA-PAINT的最新发展已经实现了光谱无限复用、精确分子计数和超高分子尺度（亚5纳米）空间分辨率，达到1纳米定位精度。通过将对接链与抗体连接，DNA-PAINT可以应用于多种体外和细胞应用。在这里，我们为新手和专家用户提供了DNA PAINT框架的关键方面的协议。本协议描述了DNA折纸测试样品的创建，原位样品制备，多路数据采集，数据模拟，超分辨率图像重建和后处理，如漂移校正，分子计数（qPAINT）和粒子平均。此外，我们还提供了一个名为Picasso的集成软件包，用于所涉及的计算步骤。该协议被设计成模块化的，因此可以根据特定应用程序的需求选择和实现单个组件。该过程可在1-2天内完成。
Super-resolution techniques have begun to transform biological and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topography (DNA PAINT) offers an easy-to-implement approach to localization-based super-resolution microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking to enable stochastic super-resolution microscopy. Using the programmability and specificity of DNA molecules as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resolution techniques, making them compatible with virtually any single-molecule-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise molecule counting and ultra-high, molecular scale (sub-5-nm) spatial resolution, reaching 1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample preparation, multiplexed data acquisition, data simulation, super-resolution image reconstruction and post-processing such as drift correction, molecule counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1-2 d.