Intramolecularly quenched fluorogens enable multispectral activation with a fluorescein-targeting single-chain variable fragment.

Fluorogen-activating proteins enable low-background fluorescence imaging by inducing emission only upon fluorogen binding. However, spectral diversification often involves coordinated optimization of fluorogens and their protein partners, while compatibility with structurally diverse fluorophore scaffolds remains limited. Here, we present a fluorogen-centered strategy for multispectral fluorescence activation using a single fluorescein-targeting single-chain variable fragment (scFv), E2. Intramolecularly quenched fluorogens were constructed through a heterodimeric architecture integrating a fluorescein recognition module with spectrally distinct fluorophores, including rhodamine, dicyanoisophorone, and Cy7. Systematic optimization of the linker structure and fluorophore identity enabled efficient self-quenching in the free state and fluorescence activation upon E2 binding, producing enhancements of 63.7-, 28.2-, and 45.0-fold across visible to near-infrared channels. Spectroscopic and fluorescence lifetime analyses suggested that fluorescence activation is associated with perturbation of intramolecular quenching interactions. The optimized fluorogen L4-Cy7 binds E2 with a Kd value of 1.8 μM. Genetic fusion of E2 to tumor-targeting scFvs enabled receptor-specific activation of L4-Cy7 and rapid imaging of prostate and breast cancer cells without washing after fluorogen addition. Overall, this work establishes a modular fluorogen-centered framework for multispectral activation using a unified protein scaffold and provides an optimized near-infrared fluorogen for targeted cellular imaging.