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

Combining fluorescence and phosphorescence kinetics, we characterize forward and reverse intersystem crossing (FISC and RISC, respectively) between the singlet and triplet manifolds S ↔ T in photoswitchable (rsEGFP2) and non-photoswitchable (EGFP) green fluorescent proteins upon continuous 488 nm laser excitation at cryogenic temperatures (CTs). Both proteins behave very similarly, with T₁ absorption spectra showing a visible peak at 490 nm (10 mM^-1 cm^-1) and a vibrational progression in the near-infrared (720 to 905 nm). The dark lifetime of T₁ is 21-24 ms at 100 K and very weakly temperature-dependent up to 180 K. Above 180 K, T₁ lifetimes reduce rapidly to few milliseconds as found at room temperature (RT). FISC and RISC quantum yields are 0.3 and 0.1%, respectively, for both proteins. The light-induced RISC channel becomes faster than the dark reversal at power densities as low as 20 W cm^-2. We discuss implications for fluorescence (super resolution-) microscopy at CT and RT.