Colocalization of fluorescent probes: accurate and precise registration with nanometer resolution.
Colocalization of fluorescent probes: accurate and precise registration with nanometer resolution.
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DOI：
10.1101/pdb.top067918
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发表时间：
2012-02-01
影响因子：
--
通讯作者：
Spudich, James A
中科院分区：
文献类型：
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作者：
Churchman, L Stirling;Spudich, James A
荧光探针的共定位通常用于细胞生物学中来辨别细胞中两种蛋白质的接近程度。考虑到光学显微镜的分辨率极限约为 250 nm，因此不需要高分辨率共定位技术。然而，随着细胞生物学和单分子生物物理学更高分辨率技术的出现，共定位也必须得到改善。对于衍射限制的应用，通常对一个颜色通道应用几何变换（即平移、缩放和旋转）以将其与另一个颜色通道对齐；然而，要实现高分辨率共定位，这还不够。单探针的单分子高分辨率共定位 (SHREC) 使用局部加权平均变换来实现至少 10 nm 的共定位分辨率。本文介绍了收集基准点校准数据集的过程以及确定共定位变换的适当分析。
Colocalization of fluorescent probes is commonly used in cell biology to discern the proximity of two proteins in the cell. Considering that the resolution limit of optical microscopy is on the order of 250 nm, there has not been a need for high-resolution colocalization techniques. However, with the advent of higher resolution techniques for cell biology and single-molecule biophysics, colocalization must also improve. For diffraction-limited applications, a geometric transformation (i.e., translation, scaling, and rotation) is typically applied to one color channel to align it with the other; however, to achieve high-resolution colocalization, this is not sufficient. Single-molecule high-resolution colocalization (SHREC) of single probes uses the local weighted mean transformation to achieve a colocalization resolution of at least 10 nm. This article describes the process of collecting a calibration data set of fiducials and the appropriate analysis to determine the transformation for colocalization.