Radiation damage in single-particle cryo-electron microscopy: effects of dose and dose rate
Radiation damage in single-particle cryo-electron microscopy: effects of dose and dose rate
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DOI：
10.1107/s090904951100820x
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发表时间：
2011-05-01
影响因子：
2.5
通讯作者：
Ravelli, Raimond B. G.
中科院分区：
文献类型：
--
作者：
Karuppasamy, Manikandan;Nejadasl, Fatemeh Karimi;Ravelli, Raimond B. G.
辐射损伤在大分子X射线晶体学和冷冻电子显微镜中都是一个重要的分辨率限制因素。大分子X射线晶体学中的系统研究极大地受益于剂量的使用，剂量表示为每质量单位沉积的能量，它由包括入射通量、束流能量、束斑大小、样品成分和样品大小等参数推导得出。在此，将剂量的使用重新引入电子显微镜，考虑了电子能量、入射通量以及测量的样品厚度和成分。对沉积能量数量的了解使我们能够将剂量与大分子X射线晶体学中的实验极限进行比较，获得在玻璃态样品中积累的自由基浓度的上限估计，并将针对大分子X射线晶体学进行的热传递模拟转换到冷冻电子显微镜中。从陆正蚓细胞外血红蛋白中收集了50 - 250张图像的频闪曝光系列，针对不同的入射通量密度和积分时间。每个系列中的图像通过计算进行对齐，并使用诸如傅里叶环相关、傅里叶环相位残差和品质因数等相似性度量进行分析。在气泡形成之前，图像随着剂量线性变亮，速率约为每10兆戈瑞0.1%。在一系列高达5500兆戈瑞的剂量下，可以观察到玻璃化血红蛋白样品的逐渐分解，到这个剂量时样品升华了。对用不同入射通量密度收集的等剂量系列的比较显示出有利于较低通量密度的剂量率效应。热模拟预测，只有在非常大的剂量率（50电子/埃²·秒⁻¹或更高）以及网格和冷冻支架之间热接触不良的情况下，样品加热才会成为一个问题。二次辐射分解效应可能在剂量率效应中起作用。频闪数据收集结合对剂量和剂量率效应的更好理解，将有助于单颗粒冷冻电子显微镜学家更好地控制其实验结果。
Radiation damage is an important resolution limiting factor both in macromolecular X-ray crystallography and cryo-electron microscopy. Systematic studies in macromolecular X-ray crystallography greatly benefited from the use of dose, expressed as energy deposited per mass unit, which is derived from parameters including incident flux, beam energy, beam size, sample composition and sample size. In here, the use of dose is reintroduced for electron microscopy, accounting for the electron energy, incident flux and measured sample thickness and composition. Knowledge of the amount of energy deposited allowed us to compare doses with experimental limits in macromolecular X-ray crystallography, to obtain an upper estimate of radical concentrations that build up in the vitreous sample, and to translate heat-transfer simulations carried out for macromolecular X-ray crystallography to cryo-electron microscopy. Stroboscopic exposure series of 50-250 images were collected for different incident flux densities and integration times from Lumbricus terrestris extracellular hemoglobin. The images within each series were computationally aligned and analyzed with similarity metrics such as Fourier ring correlation, Fourier ring phase residual and figure of merit. Prior to gas bubble formation, the images become linearly brighter with dose, at a rate of approximately 0.1% per 10 MGy. The gradual decomposition of a vitrified hemoglobin sample could be visualized at a series of doses up to 5500 MGy, by which dose the sample was sublimed. Comparison of equal-dose series collected with different incident flux densities showed a dose-rate effect favoring lower flux densities. Heat simulations predict that sample heating will only become an issue for very large dose rates (50 e-A-2 s-1 or higher) combined with poor thermal contact between the grid and cryo-holder. Secondary radiolytic effects are likely to play a role in dose-rate effects. Stroboscopic data collection combined with an improved understanding of the effects of dose and dose rate will aid single-particle cryo-electron microscopists to have better control of the outcome of their experiments.