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

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons197583

Schueder,  Florian
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons172959

Jungmann,  Ralf
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;

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Volltexte (frei zugänglich)

c6sc05420j.pdf
(Verlagsversion), 3MB

Ergänzendes Material (frei zugänglich)

c6sc05420j1.pdf
(Ergänzendes Material), 44MB

Zitation

Agasti, S. S., Wang, Y., Schueder, F., Sukumar, A., Jungmann, R., & Yin, P. (2017). DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging. Chemical Science, 8(4), 3080-3091. doi:10.1039/c6sc05420j.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-002D-E73C-F
Zusammenfassung
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.