Quantitative Single-Molecule Localization Microscopy (qSMLM) of Membrane Proteins Based on Kinetic Analysis of Fluorophore Blinking Cycles.
Quantitative Single-Molecule Localization Microscopy (qSMLM) of Membrane Proteins Based on Kinetic Analysis of Fluorophore Blinking Cycles.
复制标题
基于荧光团闪烁循环动力学分析的膜蛋白定量单分子定位显微镜 (qSMLM)。
DOI：
10.1007/978-1-4939-7265-4_10
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发表时间：
2017
影响因子：
--
通讯作者：
M. Heilemann
中科院分区：
文献类型：
--
作者：
F. Fricke;J. Beaudouin;Sebastian Malkusch;R. Eils;M. Heilemann
光开关或光激活荧光团是单分子定位显微镜的关键。除了提供具有亚衍射空间分辨率的荧光图像之外，还可以通过观察单个荧光团随时间的变化获得额外的信息。这包括由单个荧光蛋白或荧光团表现出的特征性生物物理现象“闪烁”，并且遵循明确的动力学定律。分析“闪烁”的动力学允许确定多分子复合物中荧光团的数量。因此，可以提取分子水平的定量信息，代表单分子超分辨率显微镜的非常有用的扩展。这个概念是特别有用的研究同源和异源寡聚信号蛋白复合物在质膜的完整细胞与分子分辨率。在这里，我们提供了一个实验框架破译的化学计量的膜蛋白的SMLM和光开关统计的基础上。
Photoswitchable or photoactivatable fluorophores are the key in single-molecule localization microscopy. Next to providing fluorescence images with subdiffraction spatial resolution, additional information is available from observing single fluorophores over time. This includes the characteristic photophysical phenomenon of "blinking" that is exhibited by single fluorescent proteins or fluorophores and follows well-defined kinetic laws. Analyzing the kinetics of "blinking" allows determining the number of fluorophores in a multi-molecular complex. As such, quantitative information at the molecular level can be extracted, representing a tremendously useful extension of single-molecule super-resolution microscopy. This concept is in particular useful to study homo- and heterooligomeric signaling protein complexes in the plasma membrane of an intact cell with molecular resolution. Here, we provide an experimental framework for deciphering the stoichiometry of membrane proteins on the basis of SMLM and photoswitching statistics.