Cryoelectron microscopy of vitrified sections: a new challenge for the analysis of functional nuclear architecture
Cryoelectron microscopy of vitrified sections: a new challenge for the analysis of functional nuclear architecture
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DOI：
10.1007/s00418-005-0093-x
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发表时间：
2006-01-01
影响因子：
2.3
通讯作者：
Fakan, S
中科院分区：
文献类型：
--
作者：
Bouchet-Marquis, C;Dubochet, J;Fakan, S
玻璃化切片的低温电子显微镜已成为研究细胞区室精细结构特征的有力工具。在本研究中，我们应用这种方法来探索不同哺乳动物培养细胞间期细胞核的超微结构形态。采用高压冷冻法冷冻大鼠肝癌细胞、仓鼠卵巢细胞和兔肾细胞，低温下透射电镜观察冷冻切片。我们的研究结果表明，虽然水合剖面的核结构域对比非常均匀，但由于它们的特征纹理，其中一些可以被识别出来。因此，浓缩的染色质呈细颗粒状，染色质周围区域含有相当丰富的纤维颗粒元素，表明存在分散的染色质纤维和染色质周围的原纤维和颗粒。在这些制备条件下，染色质空间看起来均匀，染色质颗粒尚未被发现。在核仁中，最显著的特征是颗粒成分，而核仁体的其他部分，对比较少，难以分辨。核包膜具有规则的核周空间和核孔复合物，很容易识别。我们的观察是在其他更传统的电子显微镜方法得到的结果的背景下讨论的。
Cryoelectron microscopy of vitrified sections has become a powerful tool for investigating the fine structural features of cellular compartments. In the present study, this approach has been applied in order to explore the ultrastructural morphology of the interphase nucleus in different mammalian cultured cells. Rat hepatoma, Chinese hamster ovary and Potorus kidney cells were cryofixed by high-pressure freezing and the cryosections were examined at low temperature by transmission electron microscopy. Our results show that while the contrast of nuclear structural domains is remarkably homogeneous in hydrated sections, some of them can be recognised due to their characteristic texture. Thus, condensed chromatin appears finely granular and the perichromatin region contains rather abundant fibro-granular elements suggesting the presence of dispersed chromatin fibres and of perichromatin fibrils and granules. The interchromatin space looks homogeneous and interchromatin granules have not been identified under these preparative conditions. In the nucleolus, the most striking feature is the granular component, while the other parts of the nucleolar body, which appear less contrasted, are difficult to resolve. The nuclear envelope is easily recognisable with its regular perinuclear space and nuclear pore complexes. Our observations are discussed in the context of results obtained by other, more conventional electron microscopic methods.