Detection of single fluorescent proteins inside eukaryotic cells using two-photon fluorescence.
Detection of single fluorescent proteins inside eukaryotic cells using two-photon fluorescence.
复制标题
使用两光子荧光检测真核细胞内部荧光蛋白。
DOI：
10.1364/boe.3.000340
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发表时间：
2012-02-01
影响因子：
3.4
通讯作者：
Cheng W
中科院分区：
文献类型：
--
作者：
Hou X;Cheng W
活细胞中的单个荧光蛋白的成像是一项具有挑战性的任务，因为强烈的细胞自发荧光。可以通过减少荧光激发体积来最小化自体荧光。全内反射荧光（TIRF）显微镜已被常规用于减少激发体积并检测细胞膜中或接近细胞膜的单个蛋白质分子。然而，渐逝场的有限穿透深度排除了驻留在真核细胞内部深处的单个荧光蛋白的成像。在这里，我们报告检测单荧光蛋白真核细胞内的双光子荧光（TPF）显微镜。TPF具有小于0.1飞升（fL）的激发体积。TPF下的细胞自发荧光较低，因此使我们能够检测单个增强型绿色荧光蛋白（EGFP）和单个单体青色荧光蛋白（mTFP1.0），它们位于细胞内几微米深处。离散逐步光漂白TPF观察细胞内的两种蛋白质。单分子荧光轨迹的定量分析表明，mTFP1.0比EGFP亮约两倍，而其荧光漂白前的时间约短10倍。这些发现证明了TPF在单分子水平上对真核细胞成像的敏感性，并将有助于测量细胞内的蛋白质化学计量。
Imaging single fluorescent proteins in a live cell is a challenging task because of the strong cellular autofluorescence. Autofluorescence can be minimized by reducing fluorescence excitation volume. Total internal reflection fluorescence (TIRF) microscopy has been routinely used to reduce excitation volume and detect single protein molecules in or close to cell membrane. However, the limited penetration depth of evanescent field excludes imaging of single fluorescent proteins that reside deep inside a eukaryotic cell. Here we report detection of single fluorescent proteins inside eukaryotic cells by two-photon fluorescence (TPF) microscopy. TPF has an excitation volume less than 0.1 femtoliter (fL). Cell autofluorescence under TPF is low and thus enables us to detect single enhanced green fluorescent proteins (EGFP) and single monomeric teal fluorescent proteins (mTFP1.0) that reside several microns deep inside the cell. Discrete stepwise photobleaching of TPF was observed for both proteins inside the cell. Quantitative analysis of single-molecule fluorescence trajectories show that mTFP1.0 is about twofold brighter than EGFP, while its fluorescence on-time before bleaching is about 10 fold shorter. These findings demonstrate the sensitivity of TPF for imaging of eukaryotic cells at single-molecule level and will be useful for measurement of protein stoichiometry inside the cell.