Vamping: Stereology-based automated quantification of fluorescent puncta size and density
Vamping: Stereology-based automated quantification of fluorescent puncta size and density
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DOI：
10.1016/j.jneumeth.2012.05.031
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发表时间：
2012-07-30
影响因子：
3
通讯作者：
Morrison, John H.
中科院分区：
文献类型：
--
作者：
Dumitriu, Dani;Berger, Seth I.;Morrison, John H.
众所周知，树突棘的大小和突触后密度（PSD）与突触的分子和功能特征相关。因此，开发能够高通量定量和测量 PSD 的显微镜方法是神经生物学领域的当代需求。虽然测量亚微米结构的黄金标准仍然是电子显微镜 (EM)，但这种方法非常费力，因此并不总是可行。免疫组织化学 (IHC) 是一种更快的技术，可用于识别 PSD 等生物结构，但其产生的荧光图像传统上更难以解释和量化。在这里，我们报告了两种新的图像分析工具，它们可以精确测量荧光斑点的尺寸和密度。抗 PSD-95 染色用于靶向突触。 vamping 新技术基于体视学原理，分别使用 2 维和 3 维（VAMP2D 和 VAMP3D）的点状体积辅助测量。全自动图像分析工具在我们之前获得 PSD 尺寸和/或密度的 EM 测量值的同一对象上进行了测试。基于每种方法获得的数据之间高度一致的结果，vamping 提供了一种 EM 的便捷替代方案，但仍可以在测量亚细胞结构时提供高精度。 (c) 2012 Elsevier B.V. 保留所有权利。
The size of dendritic spines and postsynaptic densities (PSDs) is well known to be correlated with molecular and functional characteristics of the synapse. Thus, the development of microscopy methods that allow high throughput quantification and measurement of PSDs is a contemporary need in the field of neurobiology. While the gold standard for measurement of sub-micrometer structures remains electron microscopy (EM), this method is exceedingly laborious and therefore not always feasible. Immunohistochemistry (IHC) is a much faster technique for identifying biological structures such as PSDs, but the fluorescent images resulting from it have traditionally been harder to interpret and quantify. Here, we report on two new image analysis tools that result in accurate size and density measurements of fluorescent puncta. Anti-PSD-95 staining was used to target synapses. The new technique of vamping, using Volume Assisted Measurement of Puncta in 2 and 3 Dimensions (VAMP2D and VAMP3D) respectively, is based on stereological principles. The fully automated image analysis tool was tested on the same subjects for whom we had previously obtained EM measurements of PSD size and/or density. Based on highly consistent results between data obtained by each of these methods, vamping offers an expedient alternative to EM that can nonetheless deliver a high level of accuracy in measuring sub-cellular structures. (c) 2012 Elsevier B.V. All rights reserved.