Electron cryotomography of vitrified cells with a Volta phase plate
Electron cryotomography of vitrified cells with a Volta phase plate
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DOI：
10.1016/j.jsb.2015.03.004
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发表时间：
2015-05-01
影响因子：
3
通讯作者：
Danev, Radostin
中科院分区：
文献类型：
--
作者：
Fukuda, Yoshiyuki;Laugks, Ulrike;Danev, Radostin
电子冷冻层析成像提供了一种研究多形性物体（如细胞器或细胞）三维结构的方法，分辨率为1-3纳米。研究辐射敏感生物样品的一个限制是层析图的低信噪比，这可能会掩盖细节。为了克服这一限制，最近开发的Volta相板（VPP）被应用于广泛的细胞结构的电子冷冻层析研究，从趋磁细菌到原代培养的神经元。结果表明，VPP显著提高了层析图的对比度，从而提高了层析图的信噪比，并且避免了泽尼克相板典型的干扰条纹伪影。VPP提供的对比度改善也在相对较厚（类似于400 nm）样品的投影图像中得到证实。为了探讨VPP和能量滤波器各自的贡献，对两者不同组合获得的图像进行了比较。零损耗能量滤波降低了样品较厚区域的背景噪声，提高了趋磁细菌中聚β -羟基丁酸颗粒等特征的对比度，而VPP对所有样品区域的对比度都有所提高。三维重建后，结合VPP和能量滤波器获得的层析图清晰地显示了神经元过程的结构特征。我们还表明，VPP可以与聚焦离子束铣削相结合，以检查嵌入细胞深处的结构。因此，我们期望VPP将成为电子冷冻层析工作流程的标准元素。(C) 2015爱思唯尔公司版权所有。
Electron cryotomography provides a means of studying the three dimensional structure of pleomorphic objects, such as organelles or cells, with a resolution of 1-3 nm. A limitation in the study of radiation sensitive biological samples is the low signal-to-noise ratio of the tomograms which may obscure fine details. To overcome this limitation, the recently developed Volta phase plate (VPP) was applied in electron cryotomographic studies of a wide range of cellular structures, from magnetotactic bacteria to primary cultured neurons. The results show that the VPP improves contrast significantly and consequently the signal-to-noise ratio of the tomograms, moreover it avoids disturbing fringing artifacts typical for Zernike phase plates. The contrast improvement provided by the VPP was also confirmed in projection images of relatively thick (similar to 400 nm) samples. In order to investigate the respective contributions of the VPP and the energy filter, images acquired with different combinations of the two were compared. Zero-loss energy filtering reduced the background noise in thicker areas of the sample and improved the contrast of features such as poly-beta-hydroxybutyrate granules in magnetotactic bacteria, whereas the VPP provided an overall contrast improvement for all sample areas. After 3D reconstruction, tomograms acquired with the combination of a VPP and an energy filter showed structural features in neuronal processes with outstanding clarity. We also show that the VPP can be combined with focused ion beam milling to examine structures embedded deeply inside cells. Thus, we expect that VPP will become a standard element of the electron cryotomography workflow. (C) 2015 Elsevier Inc. All rights reserved.