A comparison of the fluorescence dynamics of single molecules of a green fluorescent protein: One- versus two-photon excitation
A comparison of the fluorescence dynamics of single molecules of a green fluorescent protein: One- versus two-photon excitation
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DOI：
10.1002/cphc.200500247
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发表时间：
2006-01-16
期刊：
影响因子：
2.9
通讯作者：
Werner, JH
中科院分区：
文献类型：
--
作者：
Cotlet, M;Goodwin, PM;Werner, JH
我们报告了来自维多利亚水母的野生型绿色荧光蛋白 (GFP) 超级文件夹 GFP (SFGFP) 突变体的单个分子的荧光动态。 SFGFP 是一种新颖且强大的变体，专为蛋白质表达水平的体内高通量筛选而设计。它显示出更高的热稳定性，并且在与折叠性差的蛋白质融合时能够保留其荧光。我们使用最近开发的单分子技术，结合荧光涨落光谱和时间相关单光子计数来表征 SFGFP 在一个（OPE）和两个（TPE）光子激发条件下的光物理性质。我们使用罗丹明 170 作为模型发色团来验证方法并解释 SFGFP 的单分子结果。在 OPE 下，单个 SFGFP 分子分别由于三重态形成和基态质子化-去质子化而在μs和数十μs的时间尺度上经历荧光闪烁，如激发强度和pH依赖的实验所证明的。 OPE 单分子荧光寿命表明 SFGFP 群体中的异质性，表明 SFGFP 发色团的去质子化 I 和 B 形式的存在。单个 SFGFP 分子的 TPE 导致发色团的光转换。由于三重态的形成，单个 SFGFP 分子的 TPE 在μs 时间尺度上显示出荧光闪烁。与 SFGFP 发色团的质子化-去质子化相关的闪烁仅在低 pH 条件下才能检测到。我们的结果表明，SFGFP 是一种很有前途的融合报告基因，可用于 OPE 和 TPE 显微镜的细胞内应用。
We report on the dynamics of fluorescence from individual molecules of a mutant of the wild-type green fluorescent protein (GFP) from Aequorea victoria, super folder GFP (SFGFP). SFGFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. It shows increased thermal stability and is able to retain its fluorescence when fused to poorly folding proteins. We use a recently developed single-molecule technique which combines fluorescence-fluctuation spectroscopy and time-correlated single photon counting in order to characterize the photophysical properties of SFGFP under one(OPE) and two- (TPE) photon excitation conditions. We use Rhodamine 170 as a model chromophore to validate the methodology and to explain the single-molecule results of SFGFP Under OPE, single SFGFP molecules undergo fluorescence flickering on the time scale of mu s and tens of mu s due to triplet formation and ground-state protonation-deprotonation, respectively, as demonstrated by excitation intensity- and pH-dependent experiments. OPE single-molecule fluorescence lifetimes indicate heterogeneity in the population of SFGFP, indicating the presence of the deprotonated I and B forms of the SFGFP chromophore. TPE of single SFGFP molecules results in the photoconversion of the chromophore. TPE of single SFGFP molecules show fluorescence flickering on the time scale of mu s due to triplet formation. A flicker connected with protonation-deprotonation of the SFGFP chromophore is detected only at low pH. Our results show that SFGFP is a promising fusion reporter for intracellular applications using OPE and TPE microscopy.