Synergizing Exchangeable Fluorophore Labels for Multitarget STED Microscopy

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.