Light-Induced Forward and Reverse Intersystem Crossing in Green Fluorescent Proteins at Cryogenic Temperatures

Area of Science:

• Photophysics
• Biophysics
• Spectroscopy

Background:

• Green fluorescent proteins (GFPs) are crucial tools in biological imaging.
• Understanding intersystem crossing (ISC) is vital for advanced microscopy techniques.
• Cryogenic temperatures offer unique environments for studying protein dynamics.

Purpose of the Study:

• To characterize forward and reverse intersystem crossing (FISC and RISC) in GFP variants.
• To investigate the temperature dependence of ISC processes.
• To explore implications for fluorescence microscopy.

Main Methods:

• Kinetics analysis combining fluorescence and phosphorescence.
• Continuous 488 nm laser excitation at cryogenic temperatures (CTs).
• Spectroscopic characterization of triplet state absorption.

Main Results:

• Both photoswitchable (rsEGFP2) and non-photoswitchable (EGFP) proteins exhibit similar ISC behavior.
• Triplet state (T₁) absorption spectra show peaks at 490 nm and in the near-infrared.
• T₁ dark lifetime is ~21-24 ms at 100 K, weakly temperature-dependent up to 180 K.
• FISC and RISC quantum yields are 0.3% and 0.1%, respectively.
• Light-induced RISC becomes faster than dark reversal at 20 W cm^-2.

Conclusions:

• ISC kinetics in GFPs are largely conserved across temperature ranges.
• Cryogenic conditions reveal distinct triplet state properties.
• Findings inform the use of GFPs in super-resolution microscopy at CT and RT.