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

Michael Weber,Marcel Leutenegger,Vladimir N Belov,Lukas J Patalag,Stefan W Hell,Mariano Bossi,Taukeer A Khan

doi:10.1002/chem.202004645

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.

Highlights

The conversion of a non-fluorescent compound into a fluorescent form and back is at the heart of fluorescence microscopy with diffraction-unlimited spatial resolution
Chemistry - A European Journal published by Wiley-VCH GmbH
Without the use of complex solubilizing modifications of the structure, we introduce a general conjugation and purification technique, which overcomes the limitations associated with low water-solubility and dye aggregation that would lead to low degrees of labelling (DOL) and unspecific antibody staining

Summary

Introduction

The conversion of a non-fluorescent compound into a fluorescent form and back is at the heart of fluorescence microscopy (nanoscopy) with diffraction-unlimited spatial resolution. The switching capacity for these structures is often achieved by transfer to a poorly conjugated system; in particular, cyclic and uncharged derivatives (esters, amides) formed upon an intramolecular nucleophilic attack While such transformations significantly decrease water-solubility, making bioconjugation quite challenging, the hydrophilic properties can be restored by the use of polar groups and linkers.[12] Such „masked“ fluorophores have been extensively used for single-molecule localization microscopy.[13] the combination of a photoactivation and STED imaging offers unique possibilities in superresolution, such as counting the number of fluorophores participating in STED imaging.[14] The spatial control of the active fluorophores can be used to protect the fluorophores from bleaching by the STED laser and to increase the spatial resolution. Ble and efficient for bioconjugation of any hydrophobic dye or molecule, far beyond caged xanthene fluorophores

Results and Discussion

Conclusions

Conflict of interest