Photoswitchable Fluorescent Hydrazone for Super-Resolution Cell Membrane Imaging
Qingkai Qi, Yunshu Liu, Vedang Puranik, S. Patra, Zdeněk Švindrych, Xiayi Gong, Ziwei She, Yang Zhang, Ivan Aprahamian
Abstract
Advancing the field of super-resolution microscopy will require the design and optimization of new molecular probes whose emission can be toggled “ON” and “OFF” using light. Recently, we reported on a hydrazone photochrome ( 1 ) whose emission can be photoswitched on demand, although its low brightness and UV light-dependent back isomerization limited its use in such applications. Here, we report on the optimization of this parent fluorophore by replacing its dimethylamine electron-donating group with conformationally more rigid groups, namely, azetidine ( 2 ), 3,3-difluoroazetidine ( 3 ), and julolidine ( 4 ). This structural change resulted in enhanced brightness (i.e., extinction coefficient multiplied by fluorescence quantum yield), specifically in 4 because of its rigidity and ED capability. Next, three electron push–pull hydrazones ( 5 – 7 ) were designed based on the scaffold of 4, using cyano, nitro, or dicyanovinyl, respectively, as the electron-withdrawing groups, resulting in the progressive red-shifting of the photoswitching wavelengths into the visible region and further enhancement in brightness. Finally, fluorogenic probe 8 was developed based on parent compound 7, which could be activated solely with visible light and used in the super-resolution imaging of fixed-cell and live-cell plasma membranes with average localization precisions of 17 and 25 nm, respectively.