Stochastic optical reconstruction microscopy-based relative localization analysis (STORM-RLA) for quantitative nanoscale assessment of spatial protein organization
Stochastic optical reconstruction microscopy-based relative localization analysis (STORM-RLA) for quantitative nanoscale assessment of spatial protein organization
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DOI：
10.1091/mbc.e16-02-0125
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发表时间：
2016-11-07
影响因子：
3.3
通讯作者：
Gourdie, Robert G.
中科院分区：
文献类型：
--
作者：
Veeraraghavan, Rengasayee;Gourdie, Robert G.
蛋白质之间的空间关联对于理解它们在生物系统中的功能至关重要。荧光显微镜图像的共定位分析被广泛用于评估这一点。然而，对具有衍射极限分辨率的二维图像进行的共定位分析仅表明蛋白质在 xy 平面上彼此的距离在 200300 nm 以内，并且沿 z 轴彼此的距离在 500-700 nm 以内。在这里，我们展示了一种适用于单分子位置数据的新型三维定量分析：基于随机光学重建显微镜的相对定位分析（STORM-RLA）。该方法具有显着优势：1) STORM 成像在 xy 平面上提供 20 nm 的分辨率，沿 z 轴提供 < 50 nm 的分辨率； 2) STORM-RLA 提供了共标记蛋白簇之间重叠频率和程度的定量评估； 3) STORM-RLA 还计算三维空间中重叠和非重叠簇之间的精确距离。因此，STORM-RLA 代表了蛋白质空间组织高通量定量评估的重大进步。
The spatial association between proteins is crucial to understanding how they function in biological systems. Colocalization analysis of fluorescence microscopy images is widely used to assess this. However, colocalization analysis performed on two-dimensional images with diffraction-limited resolution merely indicates that the proteins are within 200300 nm of each other in the xy-plane and within 500-700 nm of each other along the z-axis. Here we demonstrate a novel three-dimensional quantitative analysis applicable to single-molecule positional data: stochastic optical reconstruction microscopy-based relative localization analysis (STORM-RLA). This method offers significant advantages: 1) STORM imaging affords 20-nm resolution in the xy-plane and < 50 nm along the z-axis; 2) STORM-RLA provides a quantitative assessment of the frequency and degree of overlap between clusters of colabeled proteins; and 3) STORM-RLA also calculates the precise distances between both overlapping and nonoverlapping clusters in three dimensions. Thus STORM-RLA represents a significant advance in the high-throughput quantitative assessment of the spatial organization of proteins.