Gladstein Scott, Stawarz Andrew, Almassalha Luay M, Cherkezyan Lusik, Chandler John E, Zhou Xiang, Subramanian Hariharan, Backman Vadim
Department of Biomedical Engineering, Northwestern University, Evanston, IL, USA.
Methods Mol Biol. 2018;1745:337-360. doi: 10.1007/978-1-4939-7680-5_19.
Despite extensive research in the area, current understanding of the structural organization of higher-order chromatin topology (between 20 and 200 nm) is limited due to a lack of proper imaging techniques at these length scales. The organization of chromatin at these scales defines the physical context (nanoenvironment) in which many important biological processes occur. Improving our understanding of the nanoenvironment is crucial because it has been shown to play a critical functional role in the regulation of chemical reactions. Recent progress in partial wave spectroscopic (PWS) microscopy enables real-time measurement of higher-order chromatin organization within label-free live cells. Specifically, PWS quantifies the nanoscale variations in mass density (heterogeneity) within the cell. These advancements have made it possible to study the functional role of chromatin topology, such as its regulation of the global transcriptional state of the cell and its role in the development of cancer. In this chapter, the importance of studying chromatin topology is explained, the theory and instrumentation of PWS are described, the measurements and analysis processes for PWS are laid out in detail, and common issues, troubleshooting steps, and validation techniques are provided.
尽管在该领域进行了广泛研究，但由于在这些长度尺度上缺乏合适的成像技术，目前对高阶染色质拓扑结构（20至200纳米之间）的结构组织的了解仍然有限。这些尺度上的染色质组织定义了许多重要生物过程发生的物理环境（纳米环境）。增进我们对纳米环境的理解至关重要，因为已证明它在化学反应调控中起着关键的功能作用。部分波谱（PWS）显微镜技术的最新进展使得在无标记活细胞内实时测量高阶染色质组织成为可能。具体而言，PWS可量化细胞内质量密度的纳米级变化（异质性）。这些进展使得研究染色质拓扑结构的功能作用成为可能，例如其对细胞全局转录状态的调控及其在癌症发展中的作用。在本章中，将解释研究染色质拓扑结构的重要性，描述PWS的理论和仪器，详细阐述PWS的测量和分析过程，并提供常见问题、故障排除步骤和验证技术。
