Schmid Volker J, Cremer Marion, Cremer Thomas
BioImaging Group, Department of Statistics, Ludwig Maximilians-Universität München, Ludwigstrasse 33, 80539 Munich, Germany.
Biocenter, Department Biology II, Ludwig Maximilians-Universität München, Großhadernerstrasse 2, 82152 Martinsried, Germany.
Methods. 2017 Jul 1;123:33-46. doi: 10.1016/j.ymeth.2017.03.013. Epub 2017 Mar 18.
Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction. In addition, these tools allow 3D mapping of specific DNA/RNA sequences and nuclear proteins relative to the 3D chromatin compaction maps and comparisons of multiple cell nuclei. The tools are available in the free and open source R packages nucim and bioimagetools. We discuss the use of masks for the segmentation of nuclei and the use of DNA stains, such as DAPI, as a proxy for local differences in chromatin compaction. We further discuss the limitations of 3D maps of the nuclear landscape as well as problems of the biological interpretation of such data.
超分辨荧光显微镜技术的最新进展彻底改变了细胞微观研究，包括细胞核在空间和时间上极其复杂的结构组织。在本文中，我们描述并讨论了用于（半）自动、定量三维分析细胞核空间组织的工具。这些工具能够对单个细胞核中高度分辨的不同染色质压缩水平进行定量评估，这些水平反映了三维核景观中功能不同的区域或子隔室，并能测量不同染色质压缩位点之间的绝对距离。此外，这些工具还能相对于三维染色质压缩图谱对特定DNA/RNA序列和核蛋白进行三维映射，并对多个细胞核进行比较。这些工具可在免费开源的R包nucim和bioimagetools中获取。我们讨论了用于细胞核分割的掩码的使用以及使用DNA染色剂（如DAPI）作为染色质压缩局部差异的替代指标。我们还进一步讨论了核景观三维图谱的局限性以及此类数据生物学解释方面的问题。
