Improved fluorescent phytochromes for in situ imaging.

Area of Science:

• Biophysics
• Molecular Biology
• Biotechnology

Background:

• Phytochromes are pigments used in near-infrared (NIR) fluorescence applications.
• Engineered NIR fluorescent proteins (NIR-FPs) from bacteriophytochromes and cyanobacteriochromes are promising for microscopy and in vivo imaging.
• Current NIR-FPs often exhibit low fluorescence quantum yields and short fluorescence lifetimes, limiting their utility.

Purpose of the Study:

• To engineer highly fluorescent NIR-FPs with improved quantum yields and fluorescence lifetimes.
• To enhance the brightness of NIR-FPs for advanced biological imaging applications.
• To explore the potential of cyanobacterial phytochrome Cph1 variants for biosensing and optogenetics.

Main Methods:

• Rational design approach combining known fluorescence-enhancing mutations in cyanobacterial phytochrome Cph1.
• Biochemical and spectroscopic characterization of engineered variants.
• Time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM) in E. coli cells.

Main Results:

• Developed a series of highly fluorescent NIR-FP variants with fluorescence quantum yields exceeding 15%.
• Demonstrated significantly enhanced fluorescence quantum yields and prolonged fluorescence lifetimes in the engineered NIR-FPs.
• Confirmed the bright fluorescence and suitability of these NIR-FPs for in vivo imaging through FLIM in E. coli.

Conclusions:

• The engineered NIR-FPs exhibit superior fluorescence properties compared to existing probes.
• These novel NIR-FPs represent a significant advancement for fluorescence microscopy, optogenetics, and in vivo imaging.
• The rational design strategy provides a pathway for developing next-generation fluorescent protein tools for modern biology.