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

Lukas Rane, Jip Wulffele,Dominique Bourgeois,Oleksandr Glushonkov, Angela M R Mantovanelli,Ninon Zala,Martin Byrdin

The journal of physical chemistry. B(2023)

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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 T absorption spectra showing a visible peak at 490 nm (10 mM cm) and a vibrational progression in the near-infrared (720 to 905 nm). The dark lifetime of T is 21-24 ms at 100 K and very weakly temperature-dependent up to 180 K. Above 180 K, T 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. We discuss implications for fluorescence (super resolution-) microscopy at CT and RT.
green fluorescent proteins,cryogenic temperatures,reverse intersystem crossing,light-induced
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