Fast and Multiplexed Super Resolution Imaging of Fixed and Immunostained Cells with DNA-PAINT-ERS.
Fast and Multiplexed Super Resolution Imaging of Fixed and Immunostained Cells with DNA-PAINT-ERS.
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DOI：
10.1002/cpz1.618
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发表时间：
2022-11
期刊：
影响因子：
--
通讯作者：
Nan, Xiaolin
中科院分区：
文献类型：
--
作者：
Koester, Anna M.;Szczepaniak, Malwina;Nan, Xiaolin
超分辨率显微镜的最新进展使光学显微镜能够在10-20纳米尺度上成像，提供了完整和水合样品中自然生物结构和过程的前所未有的细节。在现有的策略中，DNA点积累成像纳米级地形(DNA-PAINT)提供了方便的多路复用，这是询问复杂生物系统的重要特征。然而，DNA-PAINT的一个实际局限性是成像速度慢。在最初的形式下，每个目标的DNA-Paint成像需要几十分钟到几个小时才能完成。为了应对这一挑战，已经引入了几个改进的实现。其中包括DNA-PAINT-ERS(其中E=碳酸亚乙酯；R=重复序列；S=间隔区)，这是一套既能加快DNA-PAINT成像速度又能改善图像质量的策略。使用DNA-PAINT-ERS，对微管等典型细胞目标进行成像只需5-10分钟。重要的是，DNA-PAINT-ERS还促进了多路传输，并且可以很容易地集成到当前的生物样本荧光染色工作流程中。在这里，我们提供了一个详细的，一步一步的指南，快速和多重的DNA-PAINT-ERS成像固定和免疫染色细胞生长在玻璃基质上作为粘附单分子层。该方案应容易地扩展到不同形式的生物样品(例如组织切片)或染色机制(例如使用纳米体)。基本方案1：DNA-PAINT-ERS探针的制备基本方案2：在固定细胞中用DNA-PAINT-ERS成像膜靶的样品制备替代方案1：提取的U2OS细胞的免疫染色基本方案3：超分辨率图像采集和分析
Recent advances in super resolution microscopy have enabled imaging at the 10–20 nm scale on a light microscope, providing unprecedented details of native biological structures and processes in intact and hydrated samples. Of the existing strategies, DNA points accumulation in imaging nanoscale topography (DNA-PAINT) affords convenient multiplexing, an important feature in interrogating complex biological systems. A practical limitation of DNA-PAINT, however, was the slow imaging speed. In its original form, DNA-PAINT imaging of each target takes tens of minutes to hours to complete. To address this challenge, several improved implementations have been introduced. These include DNA-PAINT-ERS (where E = ethylene carbonate; R = repeat sequence; S = spacer), a set of strategies that lead to both accelerated DNA-PAINT imaging speed and improved image quality. With DNA-PAINT-ERS, imaging of typical cellular targets such as microtubules takes only 5–10 minutes. Importantly, DNA-PAINT-ERS also facilitates multiplexing and can be easily integrated into current workflows for fluorescence staining of biological samples. Here we provide a detailed, step-by-step guide for fast and multiplexed DNA-PAINT-ERS imaging of fixed and immunostained cells grown on glass substrates as adherent monolayers. The protocol should be readily extended to biological samples of a different format (for example tissue sections) or staining mechanisms (for example using nanobodies). Basic Protocol 1: Preparation of probes for DNA-PAINT-ERS Basic Protocol 2: Sample preparation for imaging membrane targets with DNA-PAINT-ERS in fixed cells Alternate Protocol 1: Immunostaining of extracted U2OS cells Basic Protocol 3: Super resolution image acquisition and analysis
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影响因子：
48
作者：
Dempsey, Graham T.;Vaughan, Joshua C.;Chen, Kok Hao;Bates, Mark;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
影响因子：
1.2
作者：
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通讯作者：
Nan, Xioalin
影响因子：
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作者：
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通讯作者：
Jungmann, Ralf
影响因子：
16.6
作者：
Civitci, Fehmi;Shangguan, Julia;Nan, Xiaolin
通讯作者：
Nan, Xiaolin
影响因子：
3.6
作者：
Lee J;Park S;Hohng S
通讯作者：
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