Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.
Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure.
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DOI：
10.1371/journal.pbio.0020329
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发表时间：
2004-11
期刊：
影响因子：
9.8
通讯作者：
Horstmann H
中科院分区：
文献类型：
--
作者：
Denk W;Horstmann H
许多长度尺度上的三维（3D）结构信息在生物学研究中是至关重要的。存在优秀的方法来获得分子的结构在原子，细胞器在电子显微镜，和组织在光显微镜分辨率。然而，当3D组织结构需要在数百微米的范围内重建时，存在差距，其分辨率足以跟踪最薄的细胞过程并识别突触囊泡等小细胞器。然而，这样的3D数据对于理解细胞网络是必不可少的，特别是在神经系统中，需要在大量的空间体积中完全重建。在这里，我们证明，满足这些要求的数据集可以通过自动块面成像结合连续切片室内的扫描电子显微镜。背散射对比度用于可视化使用透射电子显微镜常规技术制备的组织的重金属染色。低真空（20-60 Pa H2O）条件防止未涂覆的块体表面充电。分辨率足以追踪甚至最薄的轴突并识别突触。已经在通常低于10 nm的横向位置抖动处获得了50-70 nm厚的数百个部分的堆叠。这开启了自动获得完全重建神经元回路连接所需的电子显微镜级3D数据集的可能性。一种新的方法结合了自动成像和扫描电子显微镜室内的连续切片
Three-dimensional (3D) structural information on many length scales is of central importance in biological research. Excellent methods exist to obtain structures of molecules at atomic, organelles at electron microscopic, and tissue at light-microscopic resolution. A gap exists, however, when 3D tissue structure needs to be reconstructed over hundreds of micrometers with a resolution sufficient to follow the thinnest cellular processes and to identify small organelles such as synaptic vesicles. Such 3D data are, however, essential to understand cellular networks that, particularly in the nervous system, need to be completely reconstructed throughout a substantial spatial volume. Here we demonstrate that datasets meeting these requirements can be obtained by automated block-face imaging combined with serial sectioning inside the chamber of a scanning electron microscope. Backscattering contrast is used to visualize the heavy-metal staining of tissue prepared using techniques that are routine for transmission electron microscopy. Low-vacuum (20–60 Pa H2O) conditions prevent charging of the uncoated block face. The resolution is sufficient to trace even the thinnest axons and to identify synapses. Stacks of several hundred sections, 50–70 nm thick, have been obtained at a lateral position jitter of typically under 10 nm. This opens the possibility of automatically obtaining the electron-microscope-level 3D datasets needed to completely reconstruct the connectivity of neuronal circuits. A new method combines automated imaging with serial sectioning inside the chamber of a scanning electron microscope
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影响因子：
64.8
作者：
Euler, T;Detwiler, PB;Denk, W
通讯作者：
Denk, W
影响因子：
41.2
作者：
Donald, AM
通讯作者：
Donald, AM
DOI：
10.1046/j.1365-2818.2001.00832.x
发表时间：
2001-03-01
期刊：
JOURNAL OF MICROSCOPY-OXFORD
影响因子：
--
作者：
Alkemper, J;Voorhees, PW
通讯作者：
Voorhees, PW
影响因子：
1.8
作者：
Funsten, HO;Suszcynsky, DM;Korde, R
通讯作者：
Korde, R
影响因子：
16.2
作者：
Feng, GP;Mellor, RH;Sanes, JR
通讯作者：
Sanes, JR