An automated segmentation methodology for quantifying immunoreactive puncta number and fluorescence intensity in tissue sections.
An automated segmentation methodology for quantifying immunoreactive puncta number and fluorescence intensity in tissue sections.
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DOI：
10.1016/j.brainres.2008.08.060
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发表时间：
2008-11-13
期刊：
影响因子：
2.9
通讯作者：
Lewis, David A.
中科院分区：
文献类型：
--
作者：
Fish, Kenneth N.;Sweet, Robert A.;Deo, Anthony J.;Lewis, David A.
关键词：
许多人脑疾病与皮质突触的结构和功能紊乱有关。在这些疾病状态下，对突触机制的基本问题进行研究需要能够对组织切片中的小突触结构进行成像和量化，并评估这些主要功能部位的蛋白质水平。我们开发了一种新的自动分割成像方法，专门回答这些基本问题。该方法利用旋转盘共聚焦显微镜的进步，并结合从荧光强度/形态分割协议的多次迭代的信息来构建免疫反应（IR）斑点的三维对象掩模。这种新方法的独特之处在于，高荧光和低荧光IR斑点被同等地掩蔽，从而允许定量组织切片中荧光标记的斑点的数量。此外，最终对象掩模的形状高度代表其对应的原始数据。因此，对象掩模可用于提取有关IR斑点的信息（例如，目标蛋白的平均荧光强度）。重要的是，所提出的分割方法可以很容易地适用于大多数现有的显微镜分析包。
A number of human brain diseases have been associated with disturbances in the structure and function of cortical synapses. Answering fundamental questions about the synaptic machinery in these disease states requires the ability to image and quantify small synaptic structures in tissue sections and to evaluate protein levels at these major sites of function. We developed a new automated segmentation imaging method specifically to answer such fundamental questions. The method takes advantage of advances in spinning disk confocal microscopy, and combines information from multiple iterations of a fluorescence intensity/morphological segmentation protocol to construct three-dimensional object masks of immunoreactive (IR) puncta. This new methodology is unique in that high- and low-fluorescing IR puncta are equally masked, allowing for quantification of the number of fluorescently-labeled puncta in tissue sections. In addition, the shape of the final object masks highly represents their corresponding original data. Thus, the object masks can be used to extract information about the IR puncta (e.g., average fluorescence intensity of proteins of interest). Importantly, the segmentation method presented can be easily adapted for use with most existing microscopy analysis packages.
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