Automated single-molecule imaging in living cells.
Automated single-molecule imaging in living cells.
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DOI：
10.1038/s41467-018-05524-7
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发表时间：
2018-08-03
影响因子：
16.6
通讯作者：
Ueda M
中科院分区：
文献类型：
--
作者：
Yasui M;Hiroshima M;Kozuka J;Sako Y;Ueda M
介绍了一种基于人工智能辅助显微镜的自动化单分子成像系统。所有重要程序，即，搜索适合于观察的细胞、检测聚焦位置以及执行图像获取和单分子跟踪是完全自动化的，并且可以在活细胞中执行许多高度精确、有效和可再现的单分子成像实验。在此，将该装置用于在1天内对96孔板中的1600个细胞中的表皮生长因子受体（EGFR）进行单分子成像和分析。在单个细胞中清楚地检测到响应于各种配体和药物浓度的EGFR在质膜上的横向流动性的变化，并且确定了几个动态和药理学参数，包括扩散系数、寡聚体大小和半最大有效浓度（EC 50）。用于系统细胞信号分析的自动化单分子成像是可行的，并且可以应用于单分子筛选，从而广泛地促进生物学和药理学研究。大规模活细胞筛选通常缺乏单分子分辨率。在这里，作者提出了一种人工智能辅助的TIRF显微镜，具有自动细胞搜索和聚焦功能，并将其用于EGFR动态响应各种刺激的高通量单分子成像。
An automated single-molecule imaging system developed for live-cell analyses based on artificial intelligence-assisted microscopy is presented. All significant procedures, i.e., searching for cells suitable for observation, detecting in-focus positions, and performing image acquisition and single-molecule tracking, are fully automated, and numerous highly accurate, efficient, and reproducible single-molecule imaging experiments in living cells can be performed. Here, the apparatus is applied for single-molecule imaging and analysis of epidermal growth factor receptors (EGFRs) in 1600 cells in a 96-well plate within 1 day. Changes in the lateral mobility of EGFRs on the plasma membrane in response to various ligands and drug concentrations are clearly detected in individual cells, and several dynamic and pharmacological parameters are determined, including the diffusion coefficient, oligomer size, and half-maximal effective concentration (EC50). Automated single-molecule imaging for systematic cell signaling analyses is feasible and can be applied to single-molecule screening, thus extensively contributing to biological and pharmacological research. Large scale live cell screens often lack single-molecule resolution. Here the authors present an artificial intelligence-assisted TIRF microscope with automated cell searching and focusing, and use it for high-throughput single-molecule imaging of EGFR dynamics in response to various stimuli.
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影响因子：
4.3
作者：
Kawashima, Nagako;Nakayama, Kenichi;Biju, Vasudevanpillai
通讯作者：
Biju, Vasudevanpillai
影响因子：
6.8
作者：
AKAIKE, H
通讯作者：
AKAIKE, H
影响因子：
4.8
作者：
Carter, RE;Sorkin, A
通讯作者：
Sorkin, A
影响因子：
64.5
作者：
Lemmon MA;Schlessinger J
通讯作者：
Schlessinger J
影响因子：
2
作者：
NOCEDAL, J
通讯作者：
NOCEDAL, J