Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
J Microsc. 2010 Jun 1;238(3):254-64. doi: 10.1111/j.1365-2818.2009.03350.x.
Telomeres are the complex end structures that confer functional integrity and positional stability to human chromosomes. Telomere research has long been dominated by length measurements and biochemical analyses. Recently, interest has shifted towards the role of their three-dimensional organization and dynamics within the nuclear volume. In the mammalian interphase nucleus, there is increasing evidence for a telomeric configuration that is non-random and is cell cycle and cell type dependent. This has functional implications for genome stability. Objective and reproducible representation of the spatiotemporal organization of telomeres, under different experimental conditions, requires quantification by reliable automated image processing techniques. In this paper, we describe methods for quantitative telomere analysis in cell nuclei of living human cells expressing telomere-binding fusion proteins. We present a toolbox for determining telomere positions within the nucleus with subresolution accuracy and tracking telomeres in 4D controlled light exposure microscopy (CLEM) recordings. The use of CLEM allowed for durable imaging and thereby improved segmentation performance considerably. With minor modifications, the underlying algorithms can be expanded to the analysis of other intranuclear features, such as nuclear bodies or DNA double stranded break foci.
端粒是赋予人类染色体功能完整性和位置稳定性的复杂末端结构。端粒研究长期以来一直以长度测量和生化分析为主导。最近，人们对其在核体积内的三维组织和动力学的作用产生了兴趣。在哺乳动物间期核中，越来越多的证据表明端粒的构象是非随机的，并且依赖于细胞周期和细胞类型。这对基因组稳定性具有功能意义。在不同的实验条件下，需要通过可靠的自动图像处理技术进行定量表示，以客观和可重复的方式呈现端粒的时空组织。在本文中，我们描述了在表达端粒结合融合蛋白的活人体细胞核中进行定量端粒分析的方法。我们提出了一种工具包，用于以亚分辨率精度确定核内端粒的位置，并在 4D 受控光暴露显微镜 (CLEM) 记录中跟踪端粒。CLEM 的使用允许持久成像，从而大大提高了分割性能。通过微小的修改，基础算法可以扩展到分析其他核内特征，如核体或 DNA 双链断裂焦点。
