Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast
Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast
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DOI：
10.1073/pnas.1602202113
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发表时间：
2016-08-30
影响因子：
11.1
通讯作者：
Backman, Vadim
中科院分区：
文献类型：
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作者：
Dong, Biqin;Almassalha, Luay M.;Backman, Vadim
将染色质等核酸在非扰动、结构和动态复杂的细胞系统中形成的纳米级细胞内结构可视化，将有助于扩大我们对生物过程的理解，并为生物学发现开辟下一个前沿。传统的超分辨技术用于显示核酸形成的亚衍射大分子结构需要外源标记，这可能会扰乱细胞功能并改变他们打算研究的分子过程，特别是在超分辨所需的极高标记密度下。然而，尽管有极大的兴趣和明显的需求，在天然非扰动条件下的核苷酸拓扑的无标记光学超分辨成像一直是不可能的。在这里，我们研究了天然核苷酸的光开关过程，并基于单分子光子定位显微镜(PLM)的原理，展示了利用未经修饰的DNA的本征对比度对细胞结构进行亚衍射分辨率成像的方法。利用DNA-PLM，我们实现了间期核和有丝分裂染色体的纳米成像，允许对DNA占有率进行定量分析，并对染色体组织进行次衍射分析。这一研究可能为具有核苷酸拓扑结构的大分子结构的无标记超分辨纳米成像提供新的途径，并有助于开发新的基于DNA的超分辨成像造影剂。
Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize sub-diffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging.