Photoactivatable Fluorophore for Stimulated Emission Depletion (STED) Microscopy and Bioconjugation Technique for Hydrophobic Labels

The use of photoactivatable dyes in STED microscopy has so far been limited by two-photon activation through the STED beam and by the fact that photoactivatable dyes are poorly solvable in water. Herein, we report ONB-2SiR, a fluorophore that can be both photoactivated in the UV and specifically de-excited by STED at 775 nm. Likewise, we introduce a conjugation and purification protocol to effectively label primary and secondary antibodies with moderately water-soluble dyes. Greatly reducing dye aggregation, our technique provides a defined and tunable degree of labeling, and improves the imaging performance of dye conjugates in general.