绿荧光蛋白的双光子激发的荧光特性研究

Two-photon Fluorescence from Recombinant Green Fluorescent Protein

利用双光子激发方式研究了重组绿荧光蛋白 (recombinantgreenfluorescentprotein,简称rGFP)的光转换特性 ,研究结果表明rGFP具有较强的双光子激发荧光。双光子激发的荧光偏振光谱表明rGFP在辐照前质子态和去质子态之间存在着有效的能量转移过程。rGFP辐照后导致生色团构象的变化 ,部分阻断了rGFP内源的氨基酸与生色团之间的能量转移过程 ,导致rGFP的双光子激发的荧光强度下降。观察到rGFP的三光子激发的荧光特性 ,这种三光子激发的荧光主要来源于rGFP内源的氨基酸 (色氨酸 ,酪氨酸等 )的吸收。研究结果对在实际使用定量的荧光显微镜时具有一定的指导意义。

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-photoninduced 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.