Synergizing Exchangeable Fluorophore Labels for Multitarget STED Microscopy.
ACS Nano
; 16(11): 17991-17997, 2022 11 22.
Article
em En
| MEDLINE
| ID: mdl-36223885
ABSTRACT
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas
/
Corantes Fluorescentes
Idioma:
En
Revista:
ACS Nano
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Alemanha