Ultrafast Photoconversion of the Green Fluorescent Protein Studied by Accumulative Femtosecond Spectroscopy

The irreversible photoconversion of T203V green fluorescent protein (GFP) via decarboxylation is studied under femtosecond excitation using an accumulative product detection method that allows us to measure small conversion efficiencies of down to ΔOD = 10⁻⁷ absorbance change per pulse. Power studies with 800- and 400-nm pulse excitation reveal that excitation to higher states of the neutral form of the GFP chromophore induces photoconversion very efficiently. The singly excited neutral chromophore is a resonant intermediate of the two-step excitation process that leads to efficient photoconversion. We determine the dynamics of this two-step process by separating the excitation step of the neutral chromophore from the further excitation step to the reactive state in a time-resolved two-color experiment. The dynamics show that a further excitation to the very reactive higher excited state is only possible from the initially excited neutral chromophore and not from the fluorescent intermediate state. For applications of GFP in two-photon fluorescence microscopy, the found photochemical behavior implies that the high intensity conditions used in microscopy can lead to photoconversion easily and care has to be taken to avoid unwanted photoconversion.