Computational method for calculating fluorescence intensities within three-dimensional structures in cells.
Computational method for calculating fluorescence intensities within three-dimensional structures in cells.
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DOI：
10.4161/cl.23150
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发表时间：
2012-10-01
期刊：
影响因子：
--
通讯作者：
Kahn RA
中科院分区：
文献类型：
--
作者：
Caster AH;Kahn RA
一般来说，荧光显微镜的使用是细胞生物学的核心，对许多领域（例如，膜交通）至关重要，这些领域依赖于荧光显微镜来识别所研究分子的细胞位置以及它们与其他分子共定位的程度。严格的定位或共定位数据需要定量的图像分析，在不同的领域和实验室之间差异很大。虽然大多数公布的数据使用二维图像，但越来越多的人认识到收集三维数据集的优势。这些包括评估整个细胞和避免焦平面偏差的能力。这在成像和量化具有不规则边界的细胞器的变化时尤其重要，这些细胞器在种群中具有不同的外观，例如高尔基体。我们描述了一种方法，用于量化任何三维结构中一种蛋白质的信号强度变化，由不同标记物的存在所定义。我们以这种方法为例，量化了高尔基体跨膜蛋白载体的衔接子募集，尽管它可以直接应用于细胞中的任何部位。总之，尽管细胞器结构存在差异，但这些优势有助于对不同条件下的差异进行严格的统计测试，我们相信这种荧光数据的定量方法可以有效地应用于广泛的实验问题。
The use of fluorescence microscopy is central to cell biology in general, and essential to many fields (e.g., membrane traffic) that rely upon it to identify cellular locations of molecules under study and the extent to which they co-localize with others. Rigorous localization or co-localization data require quantitative image analyses that can vary widely between fields and laboratories. While most published data use two-dimensional images, there is an increasing appreciation for the advantages of collecting three-dimensional data sets. These include the ability to evaluate the entire cell and avoidance of focal plane bias. This is particularly important when imaging and quantifying changes in organelles with irregular borders and which vary in appearance between cells in a population, e.g., the Golgi. We describe a method developed for quantifying changes in signal intensity of one protein within any three-dimensional structure, defined by the presence of a different marker. We use as examples of this method the quantification of adaptor recruitment to transmembrane protein cargos at the Golgi though it can be directly applied to any site in the cell. Together, these advantages facilitate rigorous statistical testing of differences between conditions, despite variations in organelle structure, and we believe that this method of quantification of fluorescence data can be productively applied to a wide array of experimental questions.