title: 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.
abstract: 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.
authors.affiliation: Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
authors.forename: Amane
authors.initials: A
authors.lastname: Kobayashi
authors.affiliation: Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sekiguchi
authors.affiliation: Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
authors.forename: Tomotaka
authors.initials: T
authors.lastname: Oroguchi
authors.affiliation: Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Okajima
authors.affiliation: Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
authors.forename: Asahi
authors.initials: A
authors.lastname: Fukuda
authors.affiliation: Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
authors.forename: Mao
authors.initials: M
authors.lastname: Oide
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yamamoto
authors.affiliation: Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Nakasako
mesh_terms: D004583:Electrons; D007834:Lasers; D015388:Organelles; D014961:X-Ray Diffraction; D014965:X-Rays
keywords: X-ray free-electron laser; coherent X-ray diffraction imaging; cryogenic X-ray diffraction experiment; frozen-hydrated non-crystalline specimens; structures of cells and cellular organelles