Cannone Fabio, Caccia Michele, Bologna Sara, Diaspro Alberto, Chirico Giuseppe
Dipartimento di Fisica, Università di Milano Bicocca, Milano, Italy.
Microsc Res Tech. 2004 Nov;65(4-5):186-93. doi: 10.1002/jemt.20125.
Green Fluorescent Protein (GFP) mutants are extensively used in optical microscopy studies of in vivo biological processes in cells. Nonetheless, blinking and bleaching of the GFP chromophore at the single molecule level greatly limits its usefulness. We have worked out what we think are the best experimental conditions for the use of the GFP mutant, GFP-mut2, as a single molecule marker in two-photon excitation measurements. We have measured molecular brightness, excited state lifetime, blinking and photo-bleaching times versus the two-photon excitation intensity on proteins embedded in silica gel matrices versus the excitation wavelength in the range 700-1,000 nm. Our results indicate that GFPmut2 can be employed as a long-lived reporter of biological processes.
绿色荧光蛋白（GFP）突变体被广泛应用于细胞内体内生物过程的光学显微镜研究。然而，GFP生色团在单分子水平上的闪烁和漂白极大地限制了其用途。我们已经找出了我们认为是使用GFP突变体GFP-mut2作为双光子激发测量中的单分子标记的最佳实验条件。我们测量了嵌入硅胶基质中的蛋白质的分子亮度、激发态寿命、闪烁和光漂白时间与双光子激发强度以及700-1000nm范围内激发波长的关系。我们的结果表明，GFPmut2可以用作生物过程的长寿命报告分子。
