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 (<b>1</b>) 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 (<b>2</b>), 3,3-difluoroazetidine (<b>3</b>), and julolidine (<b>4</b>). This structural change resulted in enhanced brightness (i.e., extinction coefficient multiplied by fluorescence quantum yield), specifically in <b>4</b> because of its rigidity and ED capability. Next, three electron push-pull hydrazones (<b>5</b>-<b>7</b>) were designed based on the scaffold of <b>4</b>, 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 <b>8</b> was developed based on parent compound <b>7</b>, 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.