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.
| Item Type: | Journal article |
|---|---|
| Faculties: | Physics |
| Research Centers: | Center for NanoScience (CENS) |
| Subjects: | 500 Science > 530 Physics 500 Science > 500 Science |
| ISSN: | 2041-6520 |
| Language: | English |
| Item ID: | 53535 |
| Date Deposited: | 14. Jun 2018, 09:53 |
| Last Modified: | 04. Nov 2020, 13:32 |
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