Super-resolution microscopy approaches to nuclear nanostructure imaging
Super-resolution microscopy approaches to nuclear nanostructure imaging
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DOI：
10.1016/j.ymeth.2017.03.019
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发表时间：
2017-07-01
期刊：
影响因子：
4.8
通讯作者：
Birk, Udo
中科院分区：
文献类型：
--
作者：
Cremer, Christoph;Szczurek, Aleksander;Birk, Udo
人类基因组已经被解码，但我们离了解所有基因活动的调控还很遥远。细胞核中基因组的空间组织在这些调控机制中发挥了很大程度上无法解释的作用，这对基因调控具有深远的功能影响。直到最近，用光学显微镜在纳米尺度上研究这个问题似乎是不可能的。然而，光学成像技术的新发展已经从根本上超越了传统远场荧光显微镜（约200纳米）的有限分辨率。在简要回顾了现有的超分辨率显微镜（SRM）方法之后，我们重点介绍了在单细胞/单分子水平上研究核基因组结构的一种特殊的SRM方法，光谱精密距离/位置测定显微镜（SPDM）。SPDM是定位显微镜的一种变体，利用常规荧光蛋白或单一标准有机荧光团与标准（或仅稍加修改）标本制备条件相结合；在其实际实现模式中，可以使用相同的激光频率进行光开关和荧光读出。目前，SPDM方法使我们能够将单个细胞中的核基因组组织成像到几十纳米（nm）的结构分辨率，并对单个小染色质结构域进行定量分析；组蛋白、染色质重塑蛋白的纳米级分布，以及转录、剪接和修复相关因子。双色SPDM作为一种生物医学研究应用，可以定量监测小鼠心肌细胞缺血条件对染色质纳米结构（DNA）的影响。这些新颖的“分子光学”方法开辟了一条途径，可以直接研究单个细胞中的核景观，直至单分子水平，从而以前所未有的分辨率测试功能基因组结构模型。(C) 2017年Elsevier Inc.出版。
The human genome has been decoded, but we are still far from understanding the regulation of all gene activities. A largely unexplained role in these regulatory mechanisms is played by the spatial organization of the genome in the cell nucleus which has far-reaching functional consequences for gene regulation. Until recently, it appeared to be impossible to study this problem on the nanoscale by light microscopy. However, novel developments in optical imaging technology have radically surpassed the limited resolution of conventional far-field fluorescence microscopy (ca. 200 nm). After a brief review of available super-resolution microscopy (SRM) methods, we focus on a specific SRM approach to study nuclear genome structure at the single cell/single molecule level, Spectral Precision Distance/Position Determination Microscopy (SPDM). SPDM, a variant of localization microscopy, makes use of conventional fluorescent proteins or single standard organic fluorophores in combination with standard (or only slightly modified) specimen preparation conditions; in its actual realization mode, the same laser frequency can be used for both photoswitching and fluorescence read out. Presently, the SPDM method allows us to image nuclear genome organization in individual cells down to few tens of nanometer (nm) of structural resolution, and to perform quantitative analyses of individual small chromatin domains; of the nanoscale distribution of histones, chromatin remodeling proteins, and transcription, splicing and repair related factors. As a biomedical research application, using dual-color SPDM, it became possible to monitor in mouse cardiomyocyte cells quantitatively the effects of ischemia conditions on the chromatin nanostructure (DNA). These novel "molecular optics" approaches open an avenue to study the nuclear landscape directly in individual cells down to the single molecule level and thus to test models of functional genome architecture at unprecedented resolution. (C) 2017 Published by Elsevier Inc.