RhodaMal Dyes: Novel Red Emissive Fluorophores as Probes for High Contrast Live-Cell Imaging

Red and near-infrared emitting dyes are of high interest in fluorescence microscopy. Herein, we report two new red emissive dyes, namely RhodaMal 620 and RhodaMal 638 by incorporation of malononitrile, a highly electron-withdrawing and π-extending moiety into the traditional rhodol framework. The dyes have their absorption and emission maxima at 620/638 and 664/708 nm in aqueous solution. By increasing the viscosity of the medium to mimic the microenvironment of crowded and viscous subcellular compartments, we showed a drastic 20-fold increase in the fluorescence signal. Conjugating the dyes to triphenylphosphonium cation (TPP⁺) enabled efficient mitochondrial targeting. RhodaMal 620 was further used in live cell STED microscopy with a 775 nm depletion laser, enabling super resolution with raw full-width at half maximum values down to 77 nm.