[Two-photon fluorescence from recombinant green fluorescent protein].

The photoconversion process of recombinant green fluorescent protein (rGFP) was investigated by two-photon excitation. The results indicated that the rGFP had very strong two-photon excitation fluorescence. The changes of two-photon-induced fluorescence polarization suggest that there is a proton transfer process between two different proton states of rGFP chromophores. The conformation of rGFP chromophores could be changed upon illumination, which partly block the energy transfer processes from amino acid residues to chromophores in rGFP, and result in the decrease of two-photon-induced fluorescence intensity. The fluorescence from amino acid residues in rGFP was also observed by three-photon excitation, which resulted from the blocked amino acid residues in rGFP. These results suggested that it is necessary to optimize rGFP excitation and detection for quantitative fluorescence microscopy.