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.

中文翻译：

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光束敏感生物样品冷冻电子断层扫描的支柱数据采集策略。


对于光束敏感生物样本的冷冻电子断层扫描 (cryo-ET)，通常使用平面样本几何形状。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，限制了高倾斜角度下的信息传输。此外，可以收集数据的倾斜范围受到各种样品环境约束的组合的限制，包括物镜极片中的有限空间以及可能使用固定导电编织物来冷却样品。因此，大多数倾斜级数限制为最大 ±70°，导致傅里叶空间中存在楔形缺失。因此，在不丢失楔形的情况下采集冷冻电子断层扫描数据（例如使用圆柱形样品几何形状）对于低对称性结构（例如细胞器或囊泡、裂解事件、孔形成或丝状体）的体积分析很有吸引力，而丢失的信息无法对其进行分析。通过平均技术进行补偿。无论几何形状如何，电子束对样本的损坏都是一个问题，并且第一批获取的图像将比最后获取的图像传输更多的高分辨率信息。傅里叶空间中的更高采样率和避免光束损坏样品之间也存在固有的权衡。最后，需要使用足够的电子注量来对齐倾斜图像，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需要考虑的因素。 这里，描述并模拟了 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柱几何形状，从而允许将整个柱体积重建为单个单元。还评估了三种相关的倾斜方案，将冷冻电子断层扫描的连续和经典剂量对称倾斜方案扩展到柱样品，从而能够收集所有空间频率的各向同性信息。提出了四重剂量对称方案，该方案在均匀信息传输和数据采集的复杂性之间提供了实际的折衷。