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Issue 4, 2017
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DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging

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Abstract

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.

Graphical abstract: DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging

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Publication details

The article was received on 11 Dec 2016, accepted on 28 Jan 2017 and first published on 30 Jan 2017


Article type: Edge Article
DOI: 10.1039/C6SC05420J
Citation: Chem. Sci., 2017,8, 3080-3091
  • Open access: Creative Commons BY license
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    DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging

    S. S. Agasti, Y. Wang, F. Schueder, A. Sukumar, R. Jungmann and P. Yin, Chem. Sci., 2017, 8, 3080
    DOI: 10.1039/C6SC05420J

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