Issue 4, 2017

DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging


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

Supplementary files

Article information

Article type
Edge Article
11 Dec 2016
28 Jan 2017
First published
30 Jan 2017
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2017,8, 3080-3091

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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