Energy filtered electron tomography of ice-embedded actin and vesicles
Energy filtered electron tomography of ice-embedded actin and vesicles
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DOI：
10.1016/s0006-3495(97)78689-2
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发表时间：
1997-01-01
影响因子：
3.4
通讯作者：
Baumeister, W
中科院分区：
文献类型：
--
作者：
Grimm, R;Barmann, M;Baumeister, W
用半自动单轴倾斜电子断层扫描技术显示了肌动蛋白细丝在“体模细胞”即脂泡中的三维结构。该仪器由120千伏电子显微镜组成，配有柱后能量滤光器，用于零损失成像模式。除改变倾斜角度外，自动断层扫描所需的所有步骤，如重新对准图像区域、重新聚焦、对中能量选择狭缝，都由外部计算机控制进行。这种设置允许对多达800 nm厚的冰包埋样品进行成像，与使用常规电子显微镜进行断层扫描产生的图像对比度相比，具有更好的图像对比度，尽管在膜-细丝相互作用的研究中尤其明显的缺失楔形效应，但单轴倾斜断层扫描被发现是这种研究的合适的(事实上是唯一可用的)方法，与在肌动蛋白凝胶中发现的随机肌动蛋白网络相比，弯曲半径小于2微米的囊泡中和囊泡上的肌动蛋白细丝往往排列在单层平行的细丝中，并且通常导致小泡的拉长形状。位于外部的肌动蛋白细丝通常与囊泡膜伴生。
Semiautomatic single-axis tilt electron tomography has been used to visualize the three-dimensional organization of actin filaments in ''phantom cells,'' i.e. lipid vesicles, The instrumentation consisted of a 120-kV electron microscope equipped with a postcolumn energy filter, which was used in the zero-loss imaging mode, Apart from changing the tilt angle, all steps required for automated tomography, such as recentering the image area, refocusing, and centering the energy-selecting slit, were performed by external computer control, This setup permitted imaging of ice-embedded samples up to a thickness of 800 nm with improved image contrast compared with that produced by tomography with a conventional electron microscope, In spite of the missing-wedge effect that is especially obvious in the study of membrane-filament interaction, single-axis tilt tomography was found to be an appropriate (in fact the only available) method for this kind of investigation, In contrast to random actin networks found in actin gels, actin filaments in and on vesicles with a bending radius of less than similar to 2 mu m tend to be arranged in single layers of parallel filaments and often induce an elongated shape of the vesicles. Actin filaments located on the outside usually associate with the vesicle membrane.