Optically Modulated and Optically Activated Delayed Fluorescent Proteins through Dark State Engineering.

Area of Science:

• Biophysics
• Molecular Biology
• Optical Engineering

Background:

• Modulating fluorescent protein emission enhances sensitivity in biological imaging.
• Yellow fluorescent proteins (YFPs) possess dark states that can be modulated by secondary illumination.

Purpose of the Study:

• To engineer yellow fluorescent proteins (YFPs) with distinct modulation profiles and optically gated, delayed fluorescence.
• To develop novel fluorescent proteins for background- and reference-free, high-sensitivity cellular imaging.

Main Methods:

• Engineering EYFP and mVenus YFPs through single point mutations and double mutations.
• Utilizing secondary illumination (near-IR coexcitation) to induce optically activated delayed fluorescence (OADF).
• Spectroscopic analysis to characterize modulation pathways and dark states.

Main Results:

• Identified single point mutations creating longer-lived modulatable dark states and double mutations rendering EYFP unmodulatable.
• Demonstrated OADF via secondary laser-induced reverse intersystem crossing (RISC) from a triplet state.
• Developed Optically Modulated and Optically Activated Delayed Fluorescent Proteins (OMFPs and OADFPs) with unique modulation spectra and OADF properties.

Conclusions:

• Engineered YFPs offer distinct modulation profiles and optically gated delayed fluorescence for advanced bioimaging.
• OMFPs and OADFPs provide a pathway for background-free, high-sensitivity detection.
• Structural and spectroscopic data offer mechanistic insights for designing future optically modulated fluorescent proteins.