Applied Physics Program, Northwestern University, Evanston, IL, 60208, USA.
Department of Biomedical Engineering, Northwestern University, Evanston, IL, 60208, USA.
Sci Rep. 2022 Jul 16;12(1):12198. doi: 10.1038/s41598-022-16028-2.
Chromatin organization over multiple length scales plays a critical role in the regulation of transcription. Deciphering the interplay of these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein, we introduce ChromSTEM, a method that utilizes high-angle annular dark-field imaging and tomography in scanning transmission electron microscopy combined with DNA-specific staining for electron microscopy. We utilized ChromSTEM for an in-depth quantification of 3D chromatin conformation with high spatial resolution and contrast, allowing for characterization of higher-order chromatin structure almost down to the level of the DNA base pair. Employing mass scaling analysis on ChromSTEM mass density tomograms, we observed that chromatin forms spatially well-defined higher-order domains, around 80 nm in radius. Within domains, chromatin exhibits a polymeric fractal-like behavior and a radially decreasing mass-density from the center to the periphery. Unlike other nanoimaging and analysis techniques, we demonstrate that our unique combination of this high-resolution imaging technique with polymer physics-based analysis enables us to (i) investigate the chromatin conformation within packing domains and (ii) quantify statistical descriptors of chromatin structure that are relevant to transcription. We observe that packing domains have heterogeneous morphological properties even within the same cell line, underlying the potential role of statistical chromatin packing in regulating gene expression within eukaryotic nuclei.
多种长度尺度上的染色质组织在转录调控中起着关键作用。要揭示这些过程的相互作用，需要在体外对染色质结构进行高分辨率、三维、定量成像。在此，我们介绍了 ChromSTEM 方法，该方法结合了高角度环形暗场成像和层析技术在扫描透射电子显微镜中的应用，以及 DNA 特异性染色用于电子显微镜。我们利用 ChromSTEM 进行了深入的三维染色质构象定量分析，具有高空间分辨率和对比度，几乎可以达到 DNA 碱基对的水平来表征高级别染色质结构。我们对 ChromSTEM 质量密度层析图像进行了质量缩放分析，观察到染色质形成了空间上定义明确的高级别结构域，半径约为 80nm。在结构域内，染色质表现出聚合分形样的行为，以及从中心到外围的质量密度逐渐减小。与其他纳米成像和分析技术不同，我们证明了我们将这种高分辨率成像技术与基于聚合物物理的分析相结合的独特组合，使我们能够 (i) 研究包装结构域内的染色质构象，以及 (ii) 量化与转录相关的染色质结构的统计描述符。我们观察到，即使在同一细胞系内，包装结构域也具有异质的形态特征，这表明统计上的染色质包装在调节真核细胞核内的基因表达方面可能起着重要作用。
