Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy
Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy
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DOI：
10.1073/pnas.1608198113
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发表时间：
2016-10-18
影响因子：
11.1
通讯作者：
Backman, Vadim
中科院分区：
文献类型：
--
作者：
Almassalha, Luay M.;Bauer, Greta M.;Backman, Vadim
染色质的组织是包括转录、复制和DNA修复的分子过程的调节器。染色质内调节这些过程的结构跨越从核小体（10-nm）到染色体（>200-nm）水平，由于缺乏活细胞中的定量成像技术，对这些尺度之间的染色质结构的动态知之甚少。以前的工作使用分波光谱（PWS）显微镜，定量成像技术与灵敏度之间的大分子组织20和200 nm，已经表明，在这些长度尺度的染色质的转化是一个基本的事件在癌变。由于染色质的动力学可能在细胞功能中发挥关键的调节作用，因此开发可以探测染色质纳米结构的实时时间行为的活细胞成像技术至关重要。因此，我们开发了一种活细胞PWS技术，允许高通量，无标记的纳米级组织和分子功能之间的因果关系的研究在真实的时间。在这项工作中，我们使用活细胞PWS来研究由于DNA损伤引起的染色质结构的变化，并扩展了代谢功能与高阶染色质结构之间的联系。特别是，我们研究了紫外线照射过程中染色质的时间变化，表明活细胞DNA结合染料在几秒钟内诱导染色质损伤，并证明了高阶染色质结构和线粒体膜电位之间的直接联系。由于生物学功能与结构紧密结合，因此活细胞PWS是研究活细胞中纳米级结构-功能关系的有力工具。
The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.