A guide to accurate fluorescence microscopy colocalization measurements
A guide to accurate fluorescence microscopy colocalization measurements
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DOI：
10.1529/biophysj.106.089441
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发表时间：
2006-12-01
影响因子：
3.4
通讯作者：
Wiseman, Paul W.
中科院分区：
文献类型：
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作者：
Comeau, Jonathan W. D.;Costantino, Santiago;Wiseman, Paul W.
生物分子相互作用是绝大多数细胞过程的基础，并且鉴定主要相互作用组分通常是理解控制各种细胞功能的机制的第一步。因此，可以对固定或活细胞的荧光显微镜图像进行的统计图像分析已被常规地应用于生物物理学和细胞生物学研究。这些方法通过分析共定位像素的双色荧光图像来测量相互作用粒子的分数。共定位算法已被证明是有效的，虽然这些测量的动态范围和准确性从来没有得到很好的建立。空间图像互相关光谱（ICCS），交叉相关的空间强度波动记录在图像中的两个检测通道同时，最近也被证明是一个有效的措施，以及共定位。通过模拟，成像的荧光抗体吸附在玻璃和细胞测量，我们表明，ICCS表现得比标准的共定位算法在中等到高密度的粒子，这是经常遇到的细胞系统。此外，发现两个标记的感兴趣的物种之间的密度比在共定位分析的准确性中起主要作用。通过应用标准，荧光显微镜共定位算法和空间ICCS之间的直接和系统的比较，我们显示了每种方法都适用的制度，更重要的是，它们不能产生准确的结果。
Biomolecular interactions are fundamental to the vast majority of cellular processes, and identification of the major interacting components is usually the first step toward an understanding of the mechanisms that govern various cell functions. Thus, statistical image analyses that can be performed on fluorescence microscopy images of fixed or live cells have been routinely applied for biophysical and cell biological studies. These approaches measure the fraction of interacting particles by analyzing dual color fluorescence images for colocalized pixels. Colocalization algorithms have proven to be effective, although the dynamic range and accuracy of these measurements has never been well established. Spatial image cross-correlation spectroscopy (ICCS), which cross-correlates spatial intensity fluctuations recorded in images from two detection channels simultaneously, has also recently been shown to be an effective measure of colocalization as well. Through simulations, imaging of fluorescent antibodies adsorbed on glass and cell measurements, we show that ICCS performs much better than standard colocalization algorithms at moderate to high densities of particles, which are often encountered in cellular systems. Furthermore, it was found that the density ratio between the two labeled species of interest plays a major role in the accuracy of the colocalization analysis. By applying a direct and systematic comparison between the standard, fluorescence microscopy colocalization algorithm and spatial ICCS, we show regimes where each approach is applicable, and more importantly, where they fail to yield accurate results.