PDF(Pubmed)
Abstract：
For cryo-electron tomography (cryo-ET) of beam-sensitive biological specimens, a planar sample geometry is typically used. As the sample is tilted, the effective thickness of the sample along the direction of the electron beam increases and the signal-to-noise ratio concomitantly decreases, limiting the transfer of information at high tilt angles. In addition, the tilt range where data can be collected is limited by a combination of various sample-environment constraints, including the limited space in the objective lens pole piece and the possible use of fixed conductive braids to cool the specimen. Consequently, most tilt series are limited to a maximum of ±70°, leading to the presence of a missing wedge in Fourier space. The acquisition of cryo-ET data without a missing wedge, for example using a cylindrical sample geometry, is hence attractive for volumetric analysis of low-symmetry structures such as organelles or vesicles, lysis events, pore formation or filaments for which the missing information cannot be compensated by averaging techniques. Irrespective of the geometry, electron-beam damage to the specimen is an issue and the first images acquired will transfer more high-resolution information than those acquired last. There is also an inherent trade-off between higher sampling in Fourier space and avoiding beam damage to the sample. Finally, the necessity of using a sufficient electron fluence to align the tilt images means that this fluence needs to be fractionated across a small number of images; therefore, the order of data acquisition is also a factor to consider. Here, an n-helix tilt scheme is described and simulated which uses overlapping and interleaved tilt series to maximize the use of a pillar geometry, allowing the entire pillar volume to be reconstructed as a single unit. Three related tilt schemes are also evaluated that extend the continuous and classic dose-symmetric tilt schemes for cryo-ET to pillar samples to enable the collection of isotropic information across all spatial frequencies. A fourfold dose-symmetric scheme is proposed which provides a practical compromise between uniform information transfer and complexity of data acquisition.
摘要：
对于束敏感生物标本的低温电子层析成像（cryo-ET），通常使用平面样品几何形状。当样品倾斜时，样品沿电子束方向的有效厚度增加，信噪比随之降低，限制信息在高倾斜角度的传输。此外,可以收集数据的倾斜范围受到各种样本环境约束的组合的限制，包括物镜极片中的有限空间和可能使用固定导电编织物来冷却样品。因此,大多数倾斜系列限制在±70°的最大值，导致傅里叶空间中缺失的楔形物的存在。在没有缺失楔形的情况下获取低温ET数据，例如，使用圆柱形样品几何形状，因此，对于低对称性结构如细胞器或囊泡的体积分析具有吸引力，裂解事件，无法通过平均技术补偿丢失信息的孔形成或细丝。无论几何形状如何，电子束损伤的标本是一个问题，获取的第一个图像将传递更多的高分辨率信息比最后获得。在傅立叶空间中的较高采样与避免对样品的光束损坏之间也存在固有的折衷。最后,必须使用足够的电子注量来对准倾斜图像，这意味着该注量需要在少量图像上进行分割；因此，数据采集的顺序也是一个需要考虑的因素。这里,描述和模拟了n螺旋倾斜方案，该方案使用重叠和交错的倾斜系列来最大限度地利用支柱几何形状，允许整个支柱体积被重建为一个单元。还评估了三种相关的倾斜方案，这些方案将用于cryo-ET的连续和经典剂量对称倾斜方案扩展到支柱样品，以能够收集所有空间频率上的各向同性信息。提出了一种四倍剂量对称方案，该方案在均匀的信息传递和数据采集的复杂性之间提供了实际的折衷。
