Sequential superresolution imaging of multiple targets using a single fluorophore.
Sequential superresolution imaging of multiple targets using a single fluorophore.
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DOI：
10.1371/journal.pone.0123941
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Lidke KA
中科院分区：
文献类型：
--
作者：
Valley CC;Liu S;Lidke DS;Lidke KA
荧光超分辨(SR)显微镜或荧光纳米显微镜提供了细胞结构的纳米级细节，并允许在分子水平上对生物过程进行成像。特定的SR成像方法，如基于定位的成像，依赖于荧光团开(荧光)和关(暗)状态之间的随机转换。使用多色成像来成像多个细胞结构是复杂和受限的，因为各种有机染料的不同性质，包括它们的荧光状态占空比、每个开关事件的光子数、不可逆光漂白之前的荧光周期数以及对缓冲条件的总体敏感性。此外，多色成像需要考虑多个光路或色差，这些光路或色差可能导致在纳米尺度上很重要的微分像差。在这里，我们报告了一种顺序标记和成像的方法，允许使用单个荧光团对多个目标进行SR成像，图像之间的串扰可以忽略不计。利用Brightfield图像相关性在x-y成像平面上以~10 nm的覆盖精度配准和叠加多个图像采集，我们利用了AlexaFluor647 for dSTORM的最佳性能来成像四种不同的细胞蛋白质。我们还观察了表皮生长因子(EGF)受体和笼状蛋白在加入EGF时共存的变化，这与笼状蛋白介导的内吞作用是一致的。这些结果首次证明了使用直接随机重建显微镜(DSTORM)进行序列SR(S-SR)成像，并且这种序列成像方法可以应用于任何超分辨率技术。
Fluorescence superresolution (SR) microscopy, or fluorescence nanoscopy, provides nanometer scale detail of cellular structures and allows for imaging of biological processes at the molecular level. Specific SR imaging methods, such as localization-based imaging, rely on stochastic transitions between on (fluorescent) and off (dark) states of fluorophores. Imaging multiple cellular structures using multi-color imaging is complicated and limited by the differing properties of various organic dyes including their fluorescent state duty cycle, photons per switching event, number of fluorescent cycles before irreversible photobleaching, and overall sensitivity to buffer conditions. In addition, multiple color imaging requires consideration of multiple optical paths or chromatic aberration that can lead to differential aberrations that are important at the nanometer scale. Here, we report a method for sequential labeling and imaging that allows for SR imaging of multiple targets using a single fluorophore with negligible cross-talk between images. Using brightfield image correlation to register and overlay multiple image acquisitions with ~10 nm overlay precision in the x-y imaging plane, we have exploited the optimal properties of AlexaFluor647 for dSTORM to image four distinct cellular proteins. We also visualize the changes in co-localization of the epidermal growth factor (EGF) receptor and clathrin upon EGF addition that are consistent with clathrin-mediated endocytosis. These results are the first to demonstrate sequential SR (s-SR) imaging using direct stochastic reconstruction microscopy (dSTORM), and this method for sequential imaging can be applied to any superresolution technique.
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影响因子：
48
作者：
Dempsey, Graham T.;Vaughan, Joshua C.;Chen, Kok Hao;Bates, Mark;Zhuang, Xiaowei
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影响因子：
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作者：
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通讯作者：
Tinnefeld, Philip
影响因子：
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作者：
Cordes, Thorben;Vogelsang, Jan;Tinnefeld, Philip
通讯作者：
Tinnefeld, Philip
影响因子：
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作者：
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通讯作者：
Kittel, Robert J.