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 () 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 (), 3,3-difluoroazetidine (), and julolidine (). This structural change resulted in enhanced brightness (i.e., extinction coefficient multiplied by fluorescence quantum yield), specifically in because of its rigidity and ED capability. Next, three electron push-pull hydrazones (-) were designed based on the scaffold of , 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 was developed based on parent compound , 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.