Focused ion beam (FIB) combined with high resolution scanning electron microscopy: A promising tool for 3D analysis of chromosome architecture
Focused ion beam (FIB) combined with high resolution scanning electron microscopy: A promising tool for 3D analysis of chromosome architecture
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DOI：
10.1016/j.jsb.2008.10.002
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发表时间：
2009-02-01
影响因子：
3
通讯作者：
Wanner, Gerhard
中科院分区：
文献类型：
--
作者：
Schroeder-Reiter, Elizabeth;Perez-Willard, Fabian;Wanner, Gerhard
聚焦离子束（FIB）铣削结合场发射扫描电子显微镜（FESEM）应用于中期大麦染色体的调查，提供了新的见解染色质包装在染色体内部和组蛋白变体的3D分布在着丝粒区域。用FIB将整个安装染色体切片，厚度在7-20 nm范围内，得到多达2000个切片，这允许高分辨率三维重建。这是第一次，它可以表明，染色体内部的特点是一个网络的相互连接的空腔，与染色体表面的开口。结合免疫金标记，可以用FIB在三维中研究组蛋白变体（磷酸化组蛋白H3，CENH 3）的着丝粒相关分布。经典的SEM分析的限制，整个安装染色体与背散射电子需要更高的加速电压，例如微弱和模糊的内部信号，可以克服与FIB研磨：从染色体内，甚至非常小的标签在10 nm的范围内可以精确地可视化。这允许在三维环境中直接定量标记分子。DNA在染色体内部的分布可以在用DNA特异性铂族无机化合物铂蓝染色后直接分析。通过原位剥离技术制备FIB薄层，然后在30 kV下用扫描透射电子检测器（STEM）在暗场中进行研究，可以进行更高分辨率的DNA分布可视化。(C)2008年爱思唯尔公司All rights reserved.
Focused ion beam (FIB) milling in combination with field emission scanning electron microscopy (FESEM) was applied to investigations of metaphase barley chromosomes, providing new insight into the chromatin packaging in the chromosome interior and 3D distribution of histone variants in the centromeric region. Whole mount chromosomes were sectioned with FIB with thicknesses in the range of 7-20 nm, resulting in up to 2000 sections, which allow high resolution three-dimensional reconstruction. For the first time, it could be shown that the chromosome interior is characterized by a network of interconnected cavities, with openings to the chromosome surface. In combination with immunogold labeling, the centromere-correlated distribution of histone variants (phosphorylated histone H3, CENH3) could be investigated with FIB in three dimensions. Limitations of classical SEM analysis of whole mount chromosomes with back-scattered electrons requiring higher accelerating voltages, e.g. faint and blurred interior signals, could be overcome with FIB milling: from within the chromosome even very small labels in the range of 10 nm could be precisely visualized. This allowed direct quantification of marker molecules in a three-dimensional context. Distribution of DNA in the chromosome interior could be directly analyzed after staining with a DNA-specific platinorganic compound Platinum Blue. Higher resolution visualization of DNA distribution could be performed by preparation of FIB lamellae with the in situ lift-out technique followed by investigation in dark field with a scanning transmission electron detector (STEM) at 30 kV. (C) 2008 Elsevier Inc. All rights reserved.