Asymmetric Sulfhydryl-Substituted Squaraine Fluorophores for Sustained SIM Imaging in Live Cells

Recognized for its excellent temporal and spatial resolution, structured illumination microscopy (SIM) requires photostable fluorophores to enable dynamic imaging. Squaraine fluorophores are promising candidates for fluorescence imaging because of their intriguing photophysical properties. However, traditional squaraine fluorophores, based on an oxocyclobutenolate ring with a symmetric structure, suffer from poor photostability, limiting their application in SIM super-resolution imaging, especially for sustained tracking of biological events. To address this issue, we developed a strategy for improving the photostability of squaraine by synthesizing asymmetric molecular antennae and substituting the hydroxyl with sulfhydryl. The enhancement of intramolecular charge transfer (ICT) in the asymmetric structure enhances resistance to photobleaching, and the thiolation prolongs the fluorescence lifetime, resulting in a highly stable squaraine with remarkable optical performance. As such, we used our asymmetric thio-squaraine for tracking the dynamic changes of mitochondria during interactions between mitochondria and lysosomes. Significantly, our design strategy enables the synthesis of various thio-squaraine derivatives with superior photostability, long emission wavelengths, and high brightness, broadening the potential applicability of squaraine fluorophores in the field of SIM imaging.