A Compact Fluorogenic Peptide Tag for Bright Wash‐Free Live‐Cell Imaging

ABSTRACT Live‐cell fluorescence imaging offers profound insights into cellular biology, yet achieving strong fluorescence signals with minimal labeling footprint and perturbation to proteins of interest remains a persistent challenge. Here we introduce FluoP‐tag, a compact fluorogenic peptide‐tag platform for tandem‐array engineering in live cells. FluoP‐tag combines a 31‐residue peptide (∼3.4 kDa) with a cell‐permeable fluorogenic dye, FluoP580 (568 Da), rationally engineered to undergo peptide‐triggered switching from a non‐emissive spirolactam state to an emissive zwitterionic state. This compact design enables wash‐free intracellular labeling and copy‐number‐dependent signal amplification in tandem‐array formats. An 8×FluoP array produced 3.37‐fold higher brightness than HaloTag labeled with a Rhodamine X‐derived probe while remaining smaller overall (∼29.5 kDa vs. ∼33 kDa). FluoP‐tag arrays further enabled protein imaging across diverse intracellular compartments, spanning biomolecular condensates in stress granules, the nucleus, and the nucleolus, and cytoskeletal architectures resolved by live‐cell structured illumination microscopy (SIM). FluoP‐tag provides a compact fluorogenic tag‐array platform for protein labeling and imaging in living cells.