Photoswitchable Fluorescent Hydrazone for Super-Resolution Cell Membrane Imaging

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.