期刊
ACS NANO
卷 16, 期 11, 页码 17991-17997出版社
AMER CHEMICAL SOC
DOI: 10.1021/acsnano.2c07212
关键词
Super-resolution microscopy; DNA-PAINT; STED; exchangeable fluorophores; multicolor imaging; live-cell imaging; Halo-Tag
类别
资金
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [SFB1177, HE 6166/17-1, INST 161/926-1 FUGG, INST 161/1020-1, TRR 186]
- Max Planck Society
- Ecole Polytechnique Federale de Lausanne (EPFL)
Combining label exchange and the synergy of different types of exchangeable labels can increase the multiplexing capabilities in fluorescence microscopy and the information content of microscopy images.
Investigating the interplay of cellular proteins with optical microscopy requires multitarget labeling. Spectral multiplexing using high-affinity or covalent labels is limited in the number of fluorophores that can be discriminated in a single imaging experiment. Advanced microscopy methods such as STED microscopy additionally demand balanced excitation, depletion, and emission wavelengths for all fluorophores, further reducing multiplexing capabilities. Noncovalent, weak-affinity labels bypass this spectral barrier through label exchange and sequential imaging of different targets. Here, we combine exchangeable HaloTag ligands, weak-affinity DNA hybridization, and hydrophophic and protein-peptide interactions to increase labeling flexibility and demonstrate six-target STED microscopy in single cells. We further show that exchangeable labels reduce photobleaching as well as facilitate long acquisition times and multicolor live-cell and high-fidelity 3D STED microscopy. The synergy of different types of exchangeable labels increases the multiplexing capabilities in fluorescence microscopy, and by that, the information content of microscopy images.
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