Dual-axis target mapping and automated sequential acquisition of dual-axis EM tomographic data
Dual-axis target mapping and automated sequential acquisition of dual-axis EM tomographic data
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DOI：
10.1016/j.jsb.2009.06.010
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发表时间：
2009-11-01
影响因子：
3
通讯作者：
Agard, David A.
中科院分区：
文献类型：
--
作者：
Zheng, Shawn Q.;Matsuda, Atsushi;Agard, David A.
双轴电子显微层析成像通过沿沿着两个正交轴获取相同目标的两个互补倾斜数据集来最小化缺失楔形引起的分辨率损失。这种强大的方法的潜力已经阻碍了固有的实际挑战，在寻找原来的目标，显着位移，由于非euccentric标本旋转。人工搜索原始靶点不仅费时费力，而且人工搜索过程中所增加的剂量是不可控的。我们已经开发了一个分层对齐方案，允许从任意数量的目标网站在一个网格方向收集断层扫描数据，然后找到并收集正交数据集很少或没有用户干预。受Leginon成功的多尺度映射的启发，我们的对齐在三个级别上进行，以逐渐确定原始目标。在最低级别的网格晶格是用来确定旋转角度和平移位移所造成的标本旋转通过自动和交叉相关分析的一对图谱构建之前和之后的标本旋转。使用一对较小的地图集地图在下一级进一步细化目标位置。目标位置的最终细化是通过对准目标包含的图像块来完成的。考虑到这种分层对准的批处理性质，最初在一组中选择多个目标，然后依次获取。在完成沿沿着第一轴的所有目标上的数据收集之后并且在样本旋转之后，执行分层对准以重新定位原始目标。然后在第二轴的这些目标上恢复数据收集。因此，只需要一个样本旋转收集多个双轴层析数据集。在我们的FEI Tecnai Polara TF30电子显微镜上以86，000x CCD放大率获取玻璃冰中的20S蛋白酶体双轴层析数据集的实验表明，所开发的方案非常稳健。用于寻找和定位原始目标的额外剂量几乎可以忽略不计。该方案已集成到UCSF断层扫描软件套件中，该软件套件可在www.msg.ucsf.edu/tomography免费下载供学术使用。(C)2009 Elsevier Inc. All rights reserved.
Dual-axis electron microscopic tomography minimizes the missing wedge-induced resolution loss by taking two complementary tilt data sets of the same target along two orthogonal axes. The potential of this powerful approach has been hampered by the practical challenges inherent in finding the original targets that are dramatically displaced due to non-eucentric specimen rotation. Not only is the manual search for the original targets time consuming and tedious but the added dose during manual searching is uncontrollable. We have developed a hierarchical alignment scheme that allows tomographic data to be collected from an arbitrary number of target sites in one grid orientation and then to find and collect orthogonal data sets with little or no user intervention. Inspired by the successful multi-scale mapping in Leginon, our alignment is performed in three levels to gradually pinpoint the original targets. At the lowest level the grid lattice is used to determine the rotation angle and translational shift resulting from specimen rotation via auto- and cross-correlative analysis of a pair of atlas maps constructed before and after specimen rotation. The target locations are further refined at the next level using a pair of smaller atlas maps. The final refinement of target positions is done by aligning the target contained image tiles. Given the batch processing nature of this hierarchical alignment, multiple targets are initially selected in a group and then sequentially acquired. Upon completion of the data collection on all the targets along the first axis and after specimen rotation, the hierarchical alignment is performed to relocate the original targets. The data collection is then resumed on these targets for the second axis. Therefore, only one specimen rotation is needed for collecting multiple dual-axis tomographic data sets. The experiment of acquiring 20S Proteasomes dual-axis tomographic data sets in vitreous ice at 86,000x CCD magnification on our FEI Tecnai Polara TF30 electron microscope has suggested that the developed scheme is very robust. The extra doses for finding and centering the original targets are almost negligible. This scheme has been integrated into UCSF Tomography software suite that can be downloaded at www.msg.ucsf.edu/tomography free for academic use. (C) 2009 Elsevier Inc. All rights reserved.