Near-infrared PAINT localization microscopy via chromophore replenishment of phytochrome-derived fluorescent tag

Bacterial phytochromes are attractive molecular templates for engineering fluorescent proteins (FPs) because their near-infrared (NIR) emission significantly extends the spectral coverage of GFP-like FPs. Existing phytochrome-based FPs covalently bind heme-derived tetrapyrrole chromophores and exhibit constitutive fluorescence. Here we introduce Rep-miRFP, an NIR imaging probe derived from bacterial phytochrome, which interacts non-covalently and reversibly with biliverdin chromophore. In Rep-miRFP, the photobleached non-covalent adduct can be replenished with fresh biliverdin, restoring fluorescence. By exploiting this chromophore renewal capability, we demonstrate NIR PAINT nanoscopy in mammalian cells using Rep-miRFP. Reversible non-covalent interaction between a bacterial phytochrome-derived fluorescent protein and its biliverdin chromophore enables near-infrared localization nanoscopy in mammalian cells.