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

ABSTRACT 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.