The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues.

中文翻译：

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HiCTMap：通过高通量成像检测分析染色体域结构和位置


核空间中染色体的空间组织是一个广泛研究的领域，它依赖于对染色体结构特征和 3D 位置的高精度和鲁棒性测量。然而，目前还没有可用的工具以高通量格式对染色体区域进行成像和分析。在这里，我们开发了高通量染色体区域定位 (HiCTMap)，这是一种对哺乳动物细胞中 2D 和 3D 染色体区域定位进行稳健且快速分析的方法。 HiCTMap是一种基于高通量成像的染色体检测方法，可以对染色体结构和核位置进行常规分析。 HiCTMap 采用 384 孔板形式的优化 FISH 染色方案，结合定制的自动图像分析工作流程，可以在每个实验条件下忠实地检测大量细胞中的染色体区域及其 2D 和 3D 位置。我们应用这项新技术来可视化男性和女性原代人类皮肤成纤维细胞中的 18、X 和 Y 号染色体，并准确检测各自基因型中正确的染色体数量。鉴于能够可视化和定量分析大量细胞核，我们使用 HiCTMap 高精度测量染色体区域面积和体积，并使用质心或等距壳分析确定染色体区域的径向位置。 HiCTMap 协议还与 RNA FISH 兼容，女性细胞中 X 染色体和 Xist RNA 的同时标记证明了这一点。我们认为 HiCTMap 将成为对多种细胞类型和组织中染色体区域进行常规精确绘图的有用工具。