Cremer Christoph, Szczurek Aleksander, Schock Florian, Gourram Amine, Birk Udo
Superresolution Microscopy, Institute of Molecular Biology (IMB), Mainz, Germany; Department of Physics, University of Mainz (JGU), Mainz, Germany; Institute for Pharmacy and Molecular Biotechnology (IPMB), and Kirchhoff Institute for Physics (KIP), University of Heidelberg, Heidelberg, Germany. Electronic address: http://www.optics.imb-mainz.de.
Superresolution Microscopy, Institute of Molecular Biology (IMB), Mainz, Germany.
Methods. 2017 Jul 1;123:11-32. doi: 10.1016/j.ymeth.2017.03.019. Epub 2017 Apr 6.
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. 200nm). 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.
人类基因组已被解码，但我们距离理解所有基因活动的调控仍有很大差距。基因组在细胞核中的空间组织在这些调控机制中发挥着一个很大程度上未被解释的作用，这对基因调控具有深远的功能影响。直到最近，通过光学显微镜在纳米尺度上研究这个问题似乎还是不可能的。然而，光学成像技术的新发展已从根本上超越了传统远场荧光显微镜的有限分辨率（约200纳米）。在简要回顾现有的超分辨率显微镜（SRM）方法后，我们将重点关注一种特定的SRM方法，即在单细胞/单分子水平上研究核基因组结构的光谱精确距离/位置测定显微镜（SPDM）。SPDM是定位显微镜的一种变体，它利用传统荧光蛋白或单一标准有机荧光团，并结合标准（或仅略有修改）的样本制备条件；在其实际实现模式中，相同的激光频率可用于光开关和荧光读出。目前，SPDM方法使我们能够对单个细胞中的核基因组组织进行成像，结构分辨率低至几十纳米，并对单个小染色质结构域进行定量分析；对组蛋白、染色质重塑蛋白以及与转录、剪接和修复相关因子的纳米级分布进行分析。作为一项生物医学研究应用，使用双色SPDM能够在小鼠心肌细胞中定量监测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”方法开辟了一条直接在单个细胞中直至单分子水平研究核景观的途径，从而以前所未有的分辨率测试功能基因组结构模型。
