The genome is hierarchically organized into several 3D architectures, including chromatin loops, domains, compartments and regions associated with nuclear lamina and nucleoli. Changes in these architectures have been associated with normal development, aging and a wide range of diseases. Despite its critical importance, understanding how the genome is spatially organized in single cells, how organization varies in different cell types in mammalian tissue and how organization affects gene expression remains a major challenge. Previous approaches have been limited by a lack of capacity to directly trace chromatin folding in 3D and to simultaneously measure genomic organization in relation to other nuclear components and gene expression in the same single cells. We have developed an image-based 3D genomics technique termed ‘chromatin tracing’, which enables direct 3D tracing of chromatin folding along individual chromosomes in single cells. More recently, we also developed multiplexed imaging of nucleome architectures (MINA), which enables simultaneous measurements of multiscale chromatin folding, associations of genomic regions with nuclear lamina and nucleoli and copy numbers of numerous RNA species in the same single cells in mammalian tissue. Here, we provide detailed protocols for chromatin tracing in cell lines and MINA in mammalian tissue, which take 3–4 d for experimental work and 2–3 d for data analysis. We expect these developments to be broadly applicable and to affect many lines of research on 3D genomics by depicting multiscale genomic architectures associated with gene expression, in different types of cells and tissue undergoing different biological processes.

基因组按层次组织成几个 3D 结构，包括染色质环、结构域、区室以及与核层和核仁相关的区域。这些结构的变化与正常发育、衰老和多种疾病有关。尽管它至关重要，但了解基因组在单细胞中的空间组织方式、哺乳动物组织中不同细胞类型的组织方式如何变化以及组织方式如何影响基因表达仍然是一个重大挑战。以前的方法因缺乏直接追踪 3D 染色质折叠以及同时测量与其他核成分和同一单细胞中基因表达相关的基因组组织的能力而受到限制。我们开发了一种基于图像的 3D 基因组学技术，称为“染色质追踪”，这使得染色质沿着单个细胞中的单个染色体折叠的直接 3D 追踪成为可能。最近，我们还开发了核组结构 (MINA) 的多重成像，它可以同时测量多尺度染色质折叠、基因组区域与核层和核仁的关联以及哺乳动物组织中相同单细胞中众多 RNA 种类的拷贝数。在这里，我们提供了细胞系染色质追踪和哺乳动物组织 MINA 的详细方案，实验工作需要 3-4 天，数据分析需要 2-3 天。我们希望这些发展能够广泛适用，并通过描绘与基因表达相关的多尺度基因组结构，在经历不同生物过程的不同类型的细胞和组织中，影响 3D 基因组学的许多研究方向。