Synergizing Exchangeable Fluorophore Labels for Multitarget STED Microscopy

• Published in: ACS nano Vol. 16; no. 11; pp. 17991 - 17997
• Main Authors: Glogger, Marius, Wang, Dongni, Kompa, Julian, Balakrishnan, Ashwin, Hiblot, Julien, Barth, Hans-Dieter, Johnsson, Kai, Heilemann, Mike
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22.11.2022
• Subjects: Fluorescent Dyes - chemistry; Microscopy, Fluorescence - methods; Proteins; Super-resolution microscopy; multicolor imaging; DNA-PAINT; STED; live-cell imaging; Halo-Tag; exchangeable fluorophores
• ISSN: 1936-0851; 1936-086X
• DOI: 10.1021/acsnano.2c07212

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.