RT Journal Article
SR Electronic
T1 Synergizing exchangeable fluorophore labels for multi-target STED microscopy
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.07.02.498450
DO 10.1101/2022.07.02.498450
A1 M. Glogger
A1 D. Wang
A1 J. Kompa
A1 A. Balakrishnan
A1 J. Hiblot
A1 H.D. Barth
A1 K. Johnsson
A1 M. Heilemann
YR 2022
UL http://biorxiv.org/content/early/2022/07/03/2022.07.02.498450.abstract
AB Investigating the interplay of cellular proteins with optical microscopy requires multi-target 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. Non-covalent, 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 6-target STED microscopy in single cells. We further show that exchangeable labels reduce photobleaching, facilitate long acquisition times and multi-color live-cell and high-fidelity 3D STED microscopy. The synergy of different types of exchangeable labels increase the multiplexing capabilities in fluorescence microscopy, and by that, the information content of microscopy images.Competing Interest StatementJ.K., J.H. and K.J. are listed as inventors for the patent nrHTL: Non covalent Halotag ligands filed by the Max Planck Society and related to the present work.