Hoshi Osamu, Shigeno Masatsugu, Ushiki Tatsuo
Division of Microscopic Anatomy and Bio-imaging, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
Arch Histol Cytol. 2006 Mar;69(1):73-8. doi: 10.1679/aohc.69.73.
The present study introduces a method for obtaining three-dimensional images of native (i.e., unfixed) chromosomes by atomic force microscopy (AFM) in a liquid. Human metaphase chromosomes were isolated from a human lymphoblast-like cell line, K562, by the hexylene glycol procedure according to Wray and Stubble- field (1970), adsorbed on a silane-coated glass slide, and observed in a dynamic force mode (i.e., intermittent contact mode) of AFM in a hexylene buffer solution. In adequate operating conditions, the shape of chromosomes with paired chromatids was clearly and three-dimensionally observed by AFM. At high magnification, globular or fibrous structures about 50 nm thick could be found on the surface of each chromaid, implying that chromatin fibers were strongly wound or twisted in the chromatid. Thus, AFM imaging enabled the direct visualization of native chromosomes in a liquid at high resolution--which is comparable with that of scanning electron microscopy--and can serve to analyze the mechanism of chromosome condensation and separation in relation to the structure of chromosomes.
本研究介绍了一种通过原子力显微镜（AFM）在液体中获取天然（即未固定）染色体三维图像的方法。根据Wray和Stubblefield（1970）的己二醇法，从人淋巴母细胞样细胞系K562中分离出人中期染色体，吸附在硅烷包被的载玻片上，并在己二醇缓冲溶液中以AFM的动态力模式（即间歇接触模式）进行观察。在适当的操作条件下，通过AFM可以清晰地三维观察到具有配对染色单体的染色体形状。在高倍放大下，可以在每个染色单体表面发现约50纳米厚的球状或纤维状结构，这意味着染色质纤维在染色单体中强烈缠绕或扭曲。因此，AFM成像能够在液体中以高分辨率直接观察天然染色体，其分辨率与扫描电子显微镜相当，可用于分析与染色体结构相关的染色体凝聚和分离机制。
