Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting.
Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting.
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DOI：
10.1371/journal.pone.0031457
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Baird BA
中科院分区：
文献类型：
--
作者：
Veatch SL;Machta BB;Shelby SA;Chiang EN;Holowka DA;Baird BA
我们提出了一种分析方法，使用相关函数来量化聚类在超分辨率荧光定位图像和电子显微镜图像的静态表面在两个维度。我们使用这种方法来量化标记分子的过度计数如何有助于明显的自聚簇，并计算图像的有效横向分辨率。这种治疗适用于蛋白质和脂质在细胞膜中的分布，其中有显着的兴趣，使用电子显微镜和超分辨率荧光定位技术来探测膜的异质性。当使用成对自相关函数量化图像时，由过度计数引起的明显聚类的幅度与标记分子的表面密度成反比，并且不依赖于平均分子计数的次数。与此相反，我们表明，过度计数不会引起明显的共同聚类在双标签实验时，对互相关函数进行测量。我们应用我们的分析方法定量IgE受体（Fc ε RI）在化学固定的RBL-2H3肥大细胞质膜上的分布，这些细胞来自使用随机光学重建显微镜（STORM/dSTORM）和扫描电子显微镜（SEM）获得的图像。我们发现，当IgE直接与有机荧光团缀合时，Fc ε RI结合的IgE的表观聚集由单个复合物上的过度计数标记主导。我们通过使用两种成像方法测量细胞表面上两个可重复标记的IgE-Fc ε RI池之间的成对互相关函数来验证这一观察结果。在对过度计数进行校正后，我们在荧光定位测量中观察到IgE-Fc ε RI的弱但显著的自聚簇，并且用SEM检测到没有残留的自聚簇。我们还应用这种方法定量IgE-Fc ε RI再分布后，通过与两个不同的三价配体的定义架构的交联故意聚类，我们评估的贡献，从两个过度计数的标签和蛋白质的再分布。
We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins.
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DOI：
10.1083/jcb.200503110
发表时间：
2005-11-07
期刊：
The Journal of cell biology
影响因子：
--
作者：
Larson DR;Gosse JA;Holowka DA;Baird BA;Webb WW
通讯作者：
Webb WW
影响因子：
4
作者：
Veatch, Sarah L.;Cicuta, Pietro;Baird, Barbara
通讯作者：
Baird, Barbara
影响因子：
3.7
作者：
GOMPPER, G;SCHICK, M
通讯作者：
SCHICK, M
影响因子：
48
作者：
Sengupta, Prabuddha;Jovanovic-Talisman, Tijana;Skoko, Dunja;Renz, Malte;Veatch, Sarah L.;Lippincott-Schwartz, Jennifer
通讯作者：
Lippincott-Schwartz, Jennifer
影响因子：
5.7
作者：
Annibale, P.;Scarselli, M.;Radenovic, A.
通讯作者：
Radenovic, A.