Two-photon directed evolution of green fluorescent proteins
Caleb R Stoltzfus1, Lauren M Barnett2, Mikhail Drobizhev1
1Physics Department, Montana State University, Bozeman MT. 59717.
Scientific Reports
|July 7, 2015
Summary
Directed evolution enhanced fluorescent proteins for improved two-photon absorption (2PA) properties. New variants show up to 50% greater brightness for advanced two-photon imaging in living tissues.
Area of Science:
- Biophysics
- Molecular Biology
- Microscopy
Background:
- Fluorescent proteins (FPs) are crucial for biological imaging.
- Directed evolution has improved FP properties, but not two-photon absorption (2PA).
- Enhanced 2PA is vital for deep-tissue and brain imaging via two-photon microscopy.
Purpose of the Study:
- To develop and apply directed evolution for improving FP two-photon absorption (2PA) properties.
- To create novel FP variants with enhanced performance for two-photon imaging.
Main Methods:
- Developed a high-throughput screening method for quantifying two-photon excited fluorescence (2PEF) efficiency and 2PA cross-section in mutant FPs.
- Applied this screening method to EGFP through three rounds of directed evolution.
- Utilized E. coli expression for generating and testing mutant FP libraries.
Main Results:
- Successfully screened tens of thousands of mutant FPs.
- Achieved up to a 50% enhancement in peak 2PA cross-section for evolved EGFP variants.
- Demonstrated increased brightness within the near-infrared (NIR) spectral region.
Conclusions:
- Directed evolution is effective for improving FP two-photon absorption (2PA) properties.
- The developed screening technique enables rapid optimization of FPs for two-photon imaging.
- New FP variants offer significant advantages for deep-tissue imaging applications.
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