A Tailored-made BODIPY Scaffold for STED Super-resolution Microscopy

The development of bright and photostable fluorophores tailored for stimulated emission depletion (STED) microscopy remains a major challenge in bioimaging. Here, we report a rationally designed BODIPY scaffold optimized for STED applications through a three-step telescoped synthesis. The introduction of α-thiophene substituents at the BODIPY core was found to be crucial for achieving the spectral and photophysical properties required for efficient depletion using a 775 nm laser. Compared to the parent core-unsubstituted compound, the α-thiophene-modified dye exhibits a pronounced red-shifted emission and suppressed non-radiative decay, leading to high quantum yield, remarkable photostability, and minimal sensitivity to polarity or viscosity. Following incubation with HeLa Kyoto cells, efficient staining of multiple lipophilic intracellular membranes and organelles was observed. 2D and 3D STED imaging of various subcellular regions in live cells such as the nuclear membrane, endoplasmic reticulum, lipid droplets and mitochondria demonstrated that resolution below the diffraction limit of a confocal microscope was readily achievable. Notably, in the case of mitochondria both the outer and inner cristae membranes could be resolved and 3D reconstruction illustrated the tunnel like characteristics of nuclear membrane invagination. Overall, this work establishes α-thiophene substitution as a powerful molecular engineering strategy to convert environment-sensitive BODIPY fluorophores into a robust, STED-compatible platform. The modular synthetic approach further provides a versatile red-fluorescent scaffold for developing next-generation fluorophores tailored to specific biological targets and super-resolution techniques.