Cryo-FIB-SEM serial milling and block face imaging: Large volume structural analysis of biological tissues preserved close to their native state
Cryo-FIB-SEM serial milling and block face imaging: Large volume structural analysis of biological tissues preserved close to their native state
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DOI：
10.1016/j.jsb.2016.09.016
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发表时间：
2016-12-01
影响因子：
3
通讯作者：
Schertel, Andreas
中科院分区：
文献类型：
--
作者：
Vidavsky, Netta;Akiva, Anat;Schertel, Andreas
许多重要的生物学问题可以通过在高分辨率（5-20 nm）下对大量完整的冷冻固定水合组织（>= 10,000 μ m(3)）进行3D研究来解决。这可以通过在低温条件下使用连续FIB铣削和块表面成像来实现。在这里，我们展示了冷冻fib - sem方法的独特潜力，使用两个广泛研究的模型系统；海胆胚胎和斑马鱼幼体的尾鳍。我们特别关注矿物沉积地点的环境。细胞器，包括线粒体、高尔基体、内质网、细胞核和核孔，通过不同生化成分表面电位的差异产生的图像对比度可见。图像堆栈的自动分割和/或体积渲染以及骨骼和细胞环境的3D重建，提供了组织/细胞组件在空间中的相对分布的详细视图，从而提供了它们之间的相互作用。同时获取二次和反向散射电子图像增加了额外的信息。例如，斑马鱼尾巴的连续视图显示，在区块中延伸超过20 μ m深度的线粒体网络中存在电子密集矿物颗粒。如图所示，使用低温FIB扫描电镜进行大体积成像，可以为了解各种生物组织的结构和功能做出重大贡献。(C) 2016 Elsevier Inc.版权所有。
Many important biological questions can be addressed by studying in 3D large volumes of intact, cryo fixed hydrated tissues ( >= 10,000 mu m(3)) at high resolution (5-20 nm). This can be achieved using serial FIB milling and block face surface imaging under cryo conditions. Here we demonstrate the unique potential of the cryo-FIB-SEM approach using two extensively studied model systems; sea urchin embryos and the tail fin of zebrafish larvae. We focus in particular on the environment of mineral deposition sites.The cellular organelles, including mitochondria, Golgi, ER, nuclei and nuclear pores are made visible by the image contrast created by differences in surface potential of different biochemical components. Auto segmentation and/or volume rendering of the image stacks and 3D reconstruction of the skeleton and the cellular environment, provides a detailed view of the relative distribution in space of the tissue/cellular components, and thus of their interactions. Simultaneous acquisition of secondary and back-scattered electron images adds additional information. For example, a serial view of the zebrafish tail reveals the presence of electron dense mineral particles inside mitochondrial networks extending more than 20 mu m in depth in the block. Large volume imaging using cryo FIB SEM, as demonstrated here, can contribute significantly to the understanding of the structures and functions of diverse biological tissues. (C) 2016 Elsevier Inc. All rights reserved.