Specimen preparation for cryogenic coherent X-ray diffraction imaging of biological cells and cellular organelles by using the X-ray free-electron laser at SACLA.
Specimen preparation for cryogenic coherent X-ray diffraction imaging of biological cells and cellular organelles by using the X-ray free-electron laser at SACLA.
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DOI：
10.1107/s1600577516007736
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发表时间：
2016-07
影响因子：
2.5
通讯作者：
Nakasako M
中科院分区：
文献类型：
--
作者：
Kobayashi A;Sekiguchi Y;Oroguchi T;Okajima K;Fukuda A;Oide M;Yamamoto M;Nakasako M
报道了利用X射线自由电子激光高效低温相干X射线衍射成像制备冷冻水化生物样品的详细步骤和要点。相干X射线衍射成像(CXDI)可以分析生物细胞和细胞器的内部结构。CXDI实验在66 K下对冷冻水化的生物样品进行，在Spring8 Angstrom紧凑型自由电子激光设施(SACLA)进行。在这些使用X射线自由电子激光(XFEL)脉冲的低温CXDI实验中，分散在样品盘薄膜上的样品颗粒被转移到衍射仪的真空室中。由于聚焦的单个XFEL脉冲在原子水平上破坏了样品粒子，因此通过对样品盘进行光栅扫描来收集衍射图，以在辐照区域提供新的样品粒子。在低温实验中，衍射数据采集的效率取决于所制备样品的质量。在这里，我们报告了制备冷冻水合生物标本的详细步骤，特别是我们实验室开发的薄膜和装置。此外，通过对采集的衍射图特征的分析，对冷冻水化样品的质量进行了评价。在实验结果的基础上，讨论了冷冻水化样品的内部结构和高效采集衍射数据的未来发展。
Detailed procedures and key points in preparing frozen-hydrated biological specimens are reported for efficient cryogenic coherent X-ray diffraction imaging using an X-ray free-electron laser. Coherent X-ray diffraction imaging (CXDI) allows internal structures of biological cells and cellular organelles to be analyzed. CXDI experiments have been conducted at 66 K for frozen-hydrated biological specimens at the SPring-8 Angstrom Compact Free-Electron Laser facility (SACLA). In these cryogenic CXDI experiments using X-ray free-electron laser (XFEL) pulses, specimen particles dispersed on thin membranes of specimen disks are transferred into the vacuum chamber of a diffraction apparatus. Because focused single XFEL pulses destroy specimen particles at the atomic level, diffraction patterns are collected through raster scanning the specimen disks to provide fresh specimen particles in the irradiation area. The efficiency of diffraction data collection in cryogenic experiments depends on the quality of the prepared specimens. Here, detailed procedures for preparing frozen-hydrated biological specimens, particularly thin membranes and devices developed in our laboratory, are reported. In addition, the quality of the frozen-hydrated specimens are evaluated by analyzing the characteristics of the collected diffraction patterns. Based on the experimental results, the internal structures of the frozen-hydrated specimens and the future development for efficient diffraction data collection are discussed.