RESUMEN
Combining fluorescence and phosphorescence kinetics, we characterize forward and reverse intersystem crossing (FISC and RISC, respectively) between the singlet and triplet manifolds S â 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.