Nanostructure analysis using spatially modulated illumination microscopy.
Nanostructure analysis using spatially modulated illumination microscopy.
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DOI：
10.1159/000070464
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发表时间：
2003-01-01
期刊：
影响因子：
--
通讯作者：
Cremer, C.
中科院分区：
文献类型：
--
作者：
Failla, A. V.;Albrecht, B.;Cremer, C.
为了更好地理解细胞过程，发展光光学方法来分析生物纳米结构及其在三维(3D)保守细胞内部的动力学是非常必要的。这里，要考虑的重要结构参数是拓扑，即相互位置和距离，以及构成亚单元的大小。由于一种新的远场光荧光显微镜方法--空间调制照明(SMI)显微镜的发展，这已经成为可能。使用这种方法，可以测量荧光标记目标之间的轴向距离，精度接近1纳米；它们的大小可以确定到几十纳米。该方法可以扩展到确定由于它们的光谱特征而能够被区分的任意数量的小对象/子单元的3D位置和相互3D距离和大小。因此，这种新的方法允许“原位”纳米结构的阐明，到目前为止，人们认为这超出了远场光学显微镜的可能性。讨论的应用实例包括：大型蛋白质-蛋白质复合体、核酸-蛋白质复合体(如转录工厂)或高度复杂的DNA-蛋白质纳米结构的共址/纳米尺寸和拓扑分析，这些纳米结构构成了真核细胞核中活性/非活性基因区域。
For an improved understanding of cellular processes, it is highly desirable to develop light optical methods for the analysis of biological nanostructures and their dynamics in the interior of three-dimensionally (3D) conserved cells. Here, important structural parameters to be considered are the topology, i.e. the mutual positions and distances, as well as the sizes of the constituting subunits. This has become possible by the development of a novel method of far-field light fluorescence microscopy, spatially modulated illumination (SMI) microscopy. Using this approach, axial distances between fluorescence-labeled targets can be measured with an accuracy close to 1 nm; their sizes can be determined down to a few tens of nanometers. This approach can be extended to the determination of 3D positions and mutual 3D distances and sizes of any number of small objects/subunits that can be discriminated due to their spectral signatures. Consequently, the new approach allows an 'in situ' nanostructure elucidation, until now regarded to be beyond the possibilities of far-field light microscopy. Application examples discussed are: colocalization/nanosizing and topological analysis of large protein-protein complexes, of nucleic acid-protein complexes (such as transcription factories), or of the highly complex DNA-protein nanostructures of which active/inactive gene regions in the eukaryotic cell nucleus are constituted.