Journal
CHEMISTRY-A EUROPEAN JOURNAL
Volume 27, Issue 1, Pages 451-458Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.202004645
Keywords
fluorescence; optical superresolution; photoactivation; protein labelling; stimulated emission depletion (STED)
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Funding
- German Bundesministerium fur Bildung und Forschung [FKZ 13N14122]
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The study introduces ONB-2SiR, a photoactivatable fluorophore that can be activated in UV and specifically de-excited by STED at 775 nm. Additionally, a conjugation and purification protocol is presented to effectively label primary and secondary antibodies with moderately water-soluble dyes, reducing dye aggregation and improving imaging performance.
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.
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