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bioRxiv · 10.1101/2022.07.02.498450

Synergizing exchangeable fluorophore labels for multi-target STED microscopy

Glogger, M.·Wang, D.·Kompa, J.·Balakrishnan, A.·Hiblot, J.·Barth, H.-D.·Johnsson, K.·Heileman, M.
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Abstract

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.

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Glogger, M., Wang, D., Kompa, J., Balakrishnan, A., Hiblot, J., Barth, H.-D., Johnsson, K., Heileman, M.. 2022-07-03. Synergizing exchangeable fluorophore labels for multi-target STED microscopy. https://doi.org/10.1101/2022.07.02.498450

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