PAINTpro: Protein‐Based PAINT in Living Cells Using Engineered Reversible Bilirubin‐Binding Fluorescent Protein

ABSTRACT

Point accumulation for imaging in nanoscale topography (PAINT) is a single‐molecule localization microscopy (SMLM) technique that achieves nanometer‐level resolution, enabling direct observation of complex subcellular structures. However, its application in living cells remains limited by the lack of suitable labeling strategies and the challenge of imaging dynamic cellular structures over extended periods. In this study, we introduce PAINTpro, a protein‐based PAINT technique that facilitates live‐cell SMLM imaging and visualizes dynamic structures at the nanoscale. PAINTpro utilizes an engineered reversible bilirubin (BR)‐binding protein, Fluorescent protein for Maximizing Imaging with Nano‐resolution (FluoroMIN), derived from the green fluorescent protein UnaG. FluoroMIN incorporates mutations based on in silico molecular dynamics simulations to accelerate BR exchange, resulting in reversible fluorescence, high single‐molecule signal yield, and prolonged switching lifetime. These properties enable both live‐cell labeling and super‐resolution PAINT imaging with a resolution of ∼50 nm. Moreover, PAINTpro supports long‐term imaging of dynamic structures, providing time‐resolved super‐resolution images of microtubules with a resolution of up to 60 nm. Notably, PAINTpro is readily adaptable from existing protein‐PAINT protocols, offering a powerful tool for super‐resolution visualization of biological processes.