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

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.