1. Photoactivatable Fluorophore for Stimulated Emission Depletion (STED) Microscopy and Bioconjugation Technique for Hydrophobic Labels
- Author
-
Stefan W. Hell, Taukeer A. Khan, Lukas J. Patalag, Marcel Leutenegger, Michael Weber, Mariano L. Bossi, Vladimir N. Belov, and Synthetic Organic Chemistry
- Subjects
Fluorophore ,stimulated emission depletion (STED) ,010402 general chemistry ,01 natural sciences ,Catalysis ,protein labelling ,chemistry.chemical_compound ,Stimulated emission ,Fluorescent Dyes ,Bioconjugation ,Ionophores ,Full Paper ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,STED microscopy ,General Chemistry ,Full Papers ,Photochemical Processes ,Fluorescence ,optical superresolution ,0104 chemical sciences ,Microscopy, Fluorescence ,photoactivation ,Biophysics ,fluorescence ,Hydrophobic and Hydrophilic Interactions - Abstract
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., ONB‐2SIR, a photoactivatable dye resistant against two‐photon uncaging by 775 nm STED light is synthesized and used to perform STED microscopy before and after photoactivation without undesired activation by the STED light. The effects of the low water‐solubility of ONB‐2SIR on antibody conjugation was overcome by glycan labelling with defined degree of labelling (DOL) and a phase separation purification method.
- Published
- 2021