Agasti, Sarit S.Wang, YuSchueder, FlorianSukumar, AishwaryaJungmann, RalfYin, Peng2017-05-012017Agasti, Sarit S., Yu Wang, Florian Schueder, Aishwarya Sukumar, Ralf Jungmann, and Peng Yin. 2017. “DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc05420j Click here for additional data file.” Chemical Science 8 (4): 3080-3091. doi:10.1039/c6sc05420j. http://dx.doi.org/10.1039/c6sc05420j.http://nrs.harvard.edu/urn-3:HUL.InstRepos:32630473Recent 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.en-USJournal Article2017-05-0110.1039/c6sc05420j