Aldroubi A, Trus B L, Unser M, Booy F P, Steven A C
Biomedical Engineering and Instrumentation Program, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892.
Ultramicroscopy. 1992 Oct;46(1-4):175-88. doi: 10.1016/0304-3991(92)90013-a.
Quantitative structural analysis from electron micrographs of biological macromolecules inevitably requires the synthesis of data from many parts of the same micrograph and, ultimately, from multiple micrographs. Higher resolutions require the inclusion of progressively more data, and for the particles analyzed to be consistent to within ever more stringent limits. Disparities in magnification between micrographs or even within the field of one micrograph, arising from lens hysteresis or distortions, limit the resolution of such analyses. A quantitative assessment of this effect shows that its severity depends on the size of the particle under study: for particles that are 100 nm in diameter, for example, a 2% discrepancy in magnification restricts the resolution to approximately 5 nm. In this study, we derive and describe the properties of a family of algorithms designed for cross-calibrating the magnifications of particles from different micrographs, or from widely differing parts of the same micrograph. This approach is based on the assumption that all of the particles are of identical size: thus, it is applicable primarily to cryo-electron micrographs in which native dimensions are precisely preserved. As applied to icosahedral virus capsids, this procedure is accurate to within 0.1-0.2%, provided that at least five randomly oriented particles are included in the calculation. The algorithm is stable in the presence of noise levels typical of those encountered in practice, and is readily adaptable to non-isometric particles. It may also be used to discriminate subpopulations of subtly different sizes.
对生物大分子电子显微照片进行定量结构分析不可避免地需要整合来自同一张显微照片多个部分的数据，最终还需整合来自多张显微照片的数据。更高的分辨率要求纳入越来越多的数据，并且所分析的颗粒要在越来越严格的范围内保持一致。由于透镜滞后或畸变，显微照片之间甚至一张显微照片视野内的放大倍数差异会限制此类分析的分辨率。对这种影响的定量评估表明，其严重程度取决于所研究颗粒的大小：例如，对于直径为100纳米的颗粒，放大倍数2%的差异会将分辨率限制在约5纳米。在本研究中，我们推导并描述了一系列算法的特性，这些算法旨在对来自不同显微照片或同一张显微照片不同部位的颗粒放大倍数进行交叉校准。该方法基于所有颗粒大小相同的假设：因此，它主要适用于原生尺寸得到精确保留的冷冻电子显微照片。应用于二十面体病毒衣壳时，只要计算中包含至少五个随机取向的颗粒，该程序的精度可达0.1 - 0.2%以内。该算法在实际中常见的典型噪声水平下很稳定，并且很容易适用于非等轴颗粒。它还可用于区分细微不同大小的亚群。
