DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging.
DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging.
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DOI：
10.1039/c6sc05420j
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发表时间：
2017-04-01
期刊：
影响因子：
8.4
通讯作者：
Yin P
中科院分区：
文献类型：
--
作者：
Agasti SS;Wang Y;Schueder F;Sukumar A;Jungmann R;Yin P
我们报告了使用DNA条形码绑定剂的多路复用细胞超分辨率成像的发展。超分辨率荧光成像的最新进展使研究人员能够克服光的经典衍射极限，并已开始对生物学产生影响。然而，传统的超分辨方法面临的一个关键挑战是它们有限的多路复用能力，这阻碍了对纳米尺度上多蛋白质相互作用的系统理解。最近开发的一种基于DNA的多路复用方法Exchange-Paint，理论上通过使用正交化的染料标记的‘成像器’链顺序成像目标分子，从而促进了光谱无限多路复用。虽然这种方法为生物成像领域带来了巨大的希望，但它的广泛应用受到了蛋白质标记DNA偶联配体的可用性的阻碍。在这里，我们报告了一种通用的方法来创建DNA条形码标记探针，用于高度复合的Exchange-Paint成像，使用各种亲和剂，如一抗和二抗、纳米体和小分子结合剂。此外，我们将用于交换油漆的正交成像器链的可用性扩展到50以上，并在一种新的基于DNA折纸的串扰测试中分析了它们的正交性。使用我们优化的共轭和标记策略，我们展示了在固定细胞中原位九色超分辨率成像。
We report the development of multiplexed cellular super-resolution imaging using DNA-barcoded binders. Recent advances in super-resolution fluorescence imaging allow researchers to overcome the classical diffraction limit of light, and are already starting to make an impact in biology. However, a key challenge for traditional super-resolution methods is their limited multiplexing capability, which prevents a systematic understanding of multi-protein interactions on the nanoscale. Exchange-PAINT, a recently developed DNA-based multiplexing approach, in theory facilitates spectrally-unlimited multiplexing by sequentially imaging target molecules using orthogonal dye-labeled ‘imager’ strands. While this approach holds great promise for the bioimaging community, its widespread application has been hampered by the availability of DNA-conjugated ligands for protein labeling. Herein, we report a universal approach for the creation of DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging, using a variety of affinity reagents such as primary and secondary antibodies, nanobodies, and small molecule binders. Furthermore, we extend the availability of orthogonal imager strands for Exchange-PAINT to over 50 and assay their orthogonality in a novel DNA origami-based crosstalk assay. Using our optimized conjugation and labeling strategies, we demonstrate nine-color super-resolution imaging in situ in fixed cells.