Polarization modulation with optical lock-in detection reveals universal fluorescence anisotropy of subcellular structures in live cells.
Polarization modulation with optical lock-in detection reveals universal fluorescence anisotropy of subcellular structures in live cells.
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DOI：
10.1038/s41377-021-00689-1
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发表时间：
2022-01-01
期刊：
影响因子：
--
通讯作者：
Gao J
中科院分区：
文献类型：
--
作者：
Guan M;Wang M;Zhanghao K;Zhang X;Li M;Liu W;Niu J;Yang X;Chen L;Jing Z;Zhang MQ;Jin D;Xi P;Gao J
荧光团的取向可以揭示有关其相关亚细胞细胞器的结构和动力学的关键信息。尽管在超分辨率方面取得了重大进展，但荧光偏振显微镜仍然局限于具有较强偏振调制的独特样品，并且由于背景噪声过大而不适用于样品中较弱的偏振信号。本文采用光锁相检测的方法对微弱偏振调制进行超分辨率放大。这种新技术被称为光学锁定检测超分辨率偶极子定向映射（OLID-SDOM），可以实现每秒100帧的二维定向快速提取，并分辨距离达50 nm，适用于监测体内有关定向变化的结构动力学。OLID-SDOM用于探索多种gfp标记的亚细胞器的普遍各向异性，包括线粒体、溶酶体、高尔基体、核内体等。我们发现OLID-SDOM的取向均匀性因子（Orientation Uniformity Factor， OUF）对不同的亚细胞细胞器具有特异性，表明其各向异性与细胞器的功能有关，OUF有可能成为区分正常细胞和异常细胞（甚至癌细胞）的指标。此外，溶酶体和肌动蛋白的双色超分辨率OLID-SDOM成像显示了其在研究动态分子相互作用方面的潜力。利用实时OLID-SDOM观察活体神经元树突棘扩张和收缩的细微各向异性变化。OLID-SDOM揭示了各种样品中以前无法观察到的荧光各向异性，并指出了它们潜在的动态分子结构变化，扩展了活细胞研究的工具包。
The orientation of fluorophores can reveal crucial information about the structure and dynamics of their associated subcellular organelles. Despite significant progress in super-resolution, fluorescence polarization microscopy remains limited to unique samples with relatively strong polarization modulation and not applicable to the weak polarization signals in samples due to the excessive background noise. Here we apply optical lock-in detection to amplify the weak polarization modulation with super-resolution. This novel technique, termed optical lock-in detection super-resolution dipole orientation mapping (OLID-SDOM), could achieve a maximum of 100 frames per second and rapid extraction of 2D orientation, and distinguish distance up to 50 nm, making it suitable for monitoring structural dynamics concerning orientation changes in vivo. OLID-SDOM was employed to explore the universal anisotropy of a large variety of GFP-tagged subcellular organelles, including mitochondria, lysosome, Golgi, endosome, etc. We found that OUF (Orientation Uniformity Factor) of OLID-SDOM can be specific for different subcellular organelles, indicating that the anisotropy was related to the function of the organelles, and OUF can potentially be an indicator to distinguish normal and abnormal cells (even cancer cells). Furthermore, dual-color super-resolution OLID-SDOM imaging of lysosomes and actins demonstrates its potential in studying dynamic molecular interactions. The subtle anisotropy changes of expanding and shrinking dendritic spines in live neurons were observed with real-time OLID-SDOM. Revealing previously unobservable fluorescence anisotropy in various samples and indicating their underlying dynamic molecular structural changes, OLID-SDOM expands the toolkit for live cell research.
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DOI：
10.1083/jcb.201012143
发表时间：
2011-06-13
期刊：
The Journal of cell biology
影响因子：
--
作者：
DeMay BS;Bai X;Howard L;Occhipinti P;Meseroll RA;Spiliotis ET;Oldenbourg R;Gladfelter AS
通讯作者：
Gladfelter AS
影响因子：
16.2
作者：
Bosch M;Castro J;Saneyoshi T;Matsuno H;Sur M;Hayashi Y
通讯作者：
Hayashi Y
影响因子：
56.9
作者：
Balzarotti, Francisco;Eilers, Yvan;Hell, Stefan W.
通讯作者：
Hell, Stefan W.
带有单个相机的双视图显微镜：分子取向和钙的实时成像。
影响因子：
7.8
作者：
Kinosita, K Jr;Itoh, H;Ishiwata, S;Hirano, K;Nishizaka, T;Hayakawa, T
通讯作者：
Hayakawa, T
影响因子：
16.8
作者：
Kampmann, Martin;Atkinson, Claire E.;Mattheyses, Alexa L.;Simon, Sanford M.
通讯作者：
Simon, Sanford M.