Intensity modulation of trichromatic split fluorescent proteins for live cell mapping

Current fluorescent protein-based multiplexed cell labeling techniques suffer from limited discrimination power due to stochastic color selection and large gene sizes from tandem repeats of multiple fluorescent proteins. We developed Caterpie, a rationally designed system using engineered split fluorescent proteins that enables deterministic identification of 20 distinct cell populations with 97% accuracy and reduced gene sizes. Through computational structure-guided design, we engineered enhanced split mNeonGreen3A and split sfCherry3C variants that achieve performance comparable to split CFP2, the best-performing split fluorescent protein. Our systematic library of trichromatic 11th β-strand tags with up to 12 tandem repeats enables predictable, high-fidelity labeling for precise cell targeting. This technology addresses critical limitations in simultaneous identification of multiple defined cell populations.