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.

中文翻译：

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具有DNA-PAINT的超高分辨率显微镜。

通过允许研究人员观察远低于经典光衍射极限的结构，超分辨率技术已开始改变生物学和生物医学研究。由于使用了DNA探针，用于纳米级地形（DNA-PAINT）成像的DNA点积累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，短染料标记的（“成像子”）寡核苷酸与其互补靶标（“对接”）链的瞬时结合会产生必要的“闪烁”，以实现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，研究人员可以使眨眼与染料光物理脱钩，从而减轻了当前超分辨率技术的局限性，使它们几乎可以与任何单分子兼容的染料相容。DNA-PAINT的最新发展已经实现了光谱上无限的多路复用，精确的分子计数和超高的分子级（5纳米以下）空间分辨率，达到了约1纳米的定位精度。通过将对接链与抗体连接，DNA-PAINT可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框架关键方面的协议。该协议描述了DNA折纸测试样品的创建，原位样品制备，多路复用数据采集，数据模拟，超分辨率图像重建以及后处理，例如漂移校正，分子计数（qPAINT）和颗粒平均。此外，我们提供了一个名为Picasso的集成软件包，对于所涉及的计算步骤。该协议被设计为模块化的，因此可以根据特定应用程序的需求选择和实现各个组件。该过程可以在1-2天之内完成。