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Updated: Mar 29, 2026

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

Published on: April 22, 2021

Intensity modulation of trichromatic split fluorescent proteins for live cell mapping.

Mamoru Ishii¹, Tomoaki Kinjo², Yohei Kondo³

¹Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan; Institute of Industrial Science, The University of Tokyo, Tokyo, Japan.

Cell Reports Methods

|March 27, 2026

Summary

This summary is machine-generated.

Researchers developed Caterpie, a novel cell labeling system using engineered split fluorescent proteins. This technology allows for accurate identification of 20 cell populations, overcoming limitations of current multiplexed cell labeling methods.

Keywords:

CP: imaging Rosetta cell labeling cell population discrimination fluorescence intensity modulation fluorescence microscopy multicolor imaging multiplexed spectral labeling protein design protein tagging split fluorescent proteins

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Area of Science:

Cell biology
Biotechnology
Molecular imaging

Background:

Multiplexed cell labeling is crucial for understanding complex biological systems.
Current fluorescent protein techniques face challenges with limited discrimination and large gene sizes.
Stochastic color selection and tandem repeats hinder precise cell population identification.

Purpose of the Study:

To develop a novel cell labeling system with enhanced discrimination power and reduced gene size.
To engineer split fluorescent proteins for deterministic and high-fidelity cell population identification.
To overcome limitations of existing multiplexed cell labeling methods.

Main Methods:

Development of Caterpie, a rationally designed system utilizing engineered split fluorescent proteins.
Computational structure-guided design of enhanced split mNeonGreen3A and split sfCherry3C variants.
Creation of a systematic library of trichromatic 11th β-strand tags with up to 12 tandem repeats.

Main Results:

Deterministic identification of 20 distinct cell populations with 97% accuracy.
Engineered split fluorescent proteins achieved performance comparable to split CFP2.
Reduced gene sizes compared to traditional tandem repeat fluorescent proteins.
Predictable, high-fidelity labeling for precise cell targeting.

Conclusions:

Caterpie system significantly improves multiplexed cell labeling capabilities.
The technology enables precise targeting and simultaneous identification of multiple defined cell populations.
Engineered split fluorescent proteins offer a powerful tool for advanced cell biology research.