Coherent X-Ray Diffraction Imaging of Chloroplasts from Cyanidioschyzon merolae by Using X-Ray Free Electron Laser.
Coherent X-ray diffraction imaging (CXDI) is a lens-less technique for visualizing the structures of non-crystalline particles with the dimensions of submicrometer to micrometer at a resolution of several tens of nanometers. We conducted cryogenic CXDI experiments at 66 K to visualize the internal structures of frozen-hydrated chloroplasts of Cyanidioschyzon merolae using X-ray free electron laser (XFEL) as a coherent X-ray source. Chloroplast dispersed specimen disks at a number density of 7/(10×10 µm(2)) were flash-cooled with liquid ethane without staining, sectioning or chemical labeling. Chloroplasts are destroyed at atomic level immediately after the diffraction by XFEL pulses. Thus, diffraction patterns with a good signal-to-noise ratio from single chloroplasts were selected from many diffraction patterns collected through scanning specimen disks to provide fresh specimens into the irradiation area. The electron density maps of single chloroplasts projected along the direction of the incident X-ray beam were reconstructed by using the iterative phase-retrieval method and multivariate analyses. The electron density map at a resolution of 70 nm appeared as a C-shape. In addition, the fluorescence image of proteins stained with Flamingo™ dye also appeared as a C-shape as did the autofluorescence from Chl. The similar images suggest that the thylakoid membranes with an abundance of proteins distribute along the outer membranes of chloroplasts. To confirm the present results statistically, a number of projection structures must be accumulated through high-throughput data collection in the near future. Based on the results, we discuss the feasibility of XFEL-CXDI experiments in the structural analyses of cellular organelles.
Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522 Japan RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo. 679-5148 Japan These authors contributed equally to this work.
Yuki
Y
Takayama
Department of Applied Biological Science Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510 Japan These authors contributed equally to this work.
Yayoi
Y
Inui
Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522 Japan RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo. 679-5148 Japan These authors contributed equally to this work.
Yuki
Y
Sekiguchi
Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522 Japan RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo. 679-5148 Japan.
Amane
A
Kobayashi
Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522 Japan RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo. 679-5148 Japan.
Tomotaka
T
Oroguchi
RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo. 679-5148 Japan.
Masaki
M
Yamamoto
Department of Applied Biological Science Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510 Japan nakasako@phys.keio.ac.jp sachi@rs.tus.ac.jp.
Sachihiro
S
Matsunaga
Department of Physics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522 Japan RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo. 679-5148 Japan nakasako@phys.keio.ac.jp sachi@rs.tus.ac.jp.
Masayoshi
M
Nakasako
D000465:Algorithms; D060365:Chloroplast Proteins / Q000737:chemistry / Q000378:metabolism; D002736:Chloroplasts / Q000737:chemistry* / Q000378:metabolism; D007834:Lasers; D018613:Microscopy, Confocal; D008962:Models, Theoretical; D000461:Rhodophyta / Q000737:chemistry* / Q000378:metabolism; D020524:Thylakoids / Q000737:chemistry / Q000378:metabolism; D014961:X-Ray Diffraction / Q000379:methods*; D014965:X-Rays
D060365:Chloroplast Proteins
Chloroplast; Coherent X-ray diffraction imaging; Cyanidioschyzon merolae; Fluorescence optical microscopy