Agasti Sarit S, Wang Yu, Schueder Florian, Sukumar Aishwarya, Jungmann Ralf, Yin Peng
Wyss Institute for Biologically Inspired Engineering , Harvard University , Boston , Massachusetts , USA . Email:
Department of Systems Biology , Harvard Medical School , Boston , Massachusetts , USA.
Chem Sci. 2017 Apr 1;8(4):3080-3091. doi: 10.1039/c6sc05420j. Epub 2017 Jan 30.
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 fixed cells.
超分辨率荧光成像技术的最新进展使研究人员能够克服光的经典衍射极限，并且已经开始在生物学领域产生影响。然而，传统超分辨率方法面临的一个关键挑战是其有限的多重检测能力，这阻碍了在纳米尺度上对多蛋白相互作用的系统理解。交换式点积累成像纳米显微镜（Exchange-PAINT）是一种最近开发的基于DNA的多重检测方法，理论上通过使用正交染料标记的“成像”链对目标分子进行顺序成像，实现光谱无限的多重检测。虽然这种方法对生物成像领域具有很大的潜力，但其广泛应用受到用于蛋白质标记的DNA偶联配体可用性的限制。在此，我们报告了一种通用方法，可使用多种亲和试剂（如一抗和二抗、纳米抗体和小分子结合剂）创建用于高度多重交换式点积累成像纳米显微镜成像的DNA条形码标记探针。此外，我们将用于交换式点积累成像纳米显微镜的正交成像链的可用性扩展到50多种，并在一种基于新型DNA折纸的串扰检测中测定它们之间的正交性。使用我们优化的偶联和标记策略，我们在固定细胞中展示了九色超分辨率成像。
