Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers
Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers
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DOI：
10.1038/srep43621
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发表时间：
2017-03-02
影响因子：
4.6
通讯作者：
Hoogenboom, Jacob P.
中科院分区：
文献类型：
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作者：
Haring, Martijn T.;Liv, Nalan;Hoogenboom, Jacob P.
在生物科学中，来自荧光和电子显微镜的数据被关联以允许在活细胞成像之后的细胞超微结构和/或超微结构分析内的荧光生物分子识别。高精度（100 nm以下）图像叠加需要添加基准标记，这使得叠加精度取决于感兴趣区域中存在的基准点的数量。在这里，我们报告了一种自动化的方法，光电子图像重叠在高精度，即低于5纳米。我们的方法依赖于直接可视化的电子束的位置在荧光检测通道使用阴极发光指针。我们表明，图像覆盖使用阴极发光指针校正图像失真，是独立的用户解释，并不需要基准，允许图像相关的分子精度的样品上的任何地方。
In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.