scholarly journals 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

2016 ◽  
Vol 23 (4) ◽  
pp. 975-989 ◽  
Author(s):  
Amane Kobayashi ◽  
Yuki Sekiguchi ◽  
Tomotaka Oroguchi ◽  
Koji Okajima ◽  
Asahi Fukuda ◽  
...  
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
X Ray Diffraction ◽  
X Ray ◽  
Internal Structures ◽  
Thin Membranes ◽  
Diffraction Imaging ◽  
Diffraction Patterns ◽  
Cellular Organelles

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.

scholarly journals Data processing software suiteSITENNOfor coherent X-ray diffraction imaging using the X-ray free-electron laser SACLA

2014 ◽  
Vol 21 (3) ◽  
pp. 600-612 ◽  
Author(s):  
Yuki Sekiguchi ◽  
Tomotaka Oroguchi ◽  
Yuki Takayama ◽  
Masayoshi Nakasako
Keyword(s):  
Data Processing ◽  
Free Electron ◽  
Free Electron Laser ◽  
Single Shot ◽  
X Ray Diffraction ◽  
X Ray ◽  
Custom Made ◽  
Diffraction Imaging ◽  
Diffraction Patterns ◽  
Ccd Detectors

Coherent X-ray diffraction imaging is a promising technique for visualizing the structures of non-crystalline particles with dimensions of micrometers to sub-micrometers. Recently, X-ray free-electron laser sources have enabled efficient experiments in the `diffraction before destruction' scheme. Diffraction experiments have been conducted at SPring-8 Angstrom Compact free-electron LAser (SACLA) using the custom-made diffraction apparatus KOTOBUKI-1 and two multiport CCD detectors. In the experiments, ten thousands of single-shot diffraction patterns can be collected within several hours. Then, diffraction patterns with significant levels of intensity suitable for structural analysis must be found, direct-beam positions in diffraction patterns determined, diffraction patterns from the two CCD detectors merged, and phase-retrieval calculations for structural analyses performed. A software suite namedSITENNOhas been developed to semi-automatically apply the four-step processing to a huge number of diffraction data. Here, details of the algorithm used in the suite are described and the performance for approximately 9000 diffraction patterns collected from cuboid-shaped copper oxide particles reported. Using theSITENNOsuite, it is possible to conduct experiments with data processing immediately after the data collection, and to characterize the size distribution and internal structures of the non-crystalline particles.

scholarly journals Common architectures in cyanobacteria Prochlorococcus cells visualized by X-ray diffraction imaging using X-ray free electron laser

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Amane Kobayashi ◽  
Yuki Takayama ◽  
Takeshi Hirakawa ◽  
Koji Okajima ◽  
Mao Oide ◽  
...  
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
Single Cells ◽  
Three Dimensional ◽  
Biological Functions ◽  
X Ray Diffraction ◽  
Characteristic Structure ◽  
X Ray ◽  
Diffraction Imaging ◽  
Diffraction Patterns

AbstractVisualization of intracellular structures and their spatial organization inside cells without any modification is essential to understand the mechanisms underlying the biological functions of cells. Here, we investigated the intracellular structure of cyanobacteria Prochlorococcus in the interphase by X-ray diffraction imaging using X-ray free-electron laser. A number of diffraction patterns from single cells smaller than 1 µm in size were collected with high signal-to-noise ratio with a resolution of up to 30 nm. From diffraction patterns, a set of electron density maps projected along the direction of the incident X-ray were retrieved with high reliability. The most characteristic structure found to be common among the cells was a C-shaped arrangement of 100-nm sized high-density spots, which surrounded a low-density area of 100 nm. Furthermore, a three-dimensional map reconstructed from the projection maps of individual cells was non-uniform, indicating the presence of common structures among cyanobacteria cells in the interphase. By referring to the fluorescent images for distributions of thylakoid membranes, nucleoids, and carboxysomes, we inferred and represented their spatial arrangements in the three-dimensional map. The arrangement allowed us to discuss the relevance of the intracellular organization to the biological functions of cyanobacteria.

scholarly journals Cryogenic coherent X-ray diffraction imaging of biological particles at SACLA

2014 ◽  
Vol 70 (a1) ◽  
pp. C292-C292
Author(s):  
Yuki Takayama ◽  
Masayoshi Nakasako ◽  
Tomotaka Oroguchi ◽  
Yuki Sekiguchi ◽  
Masaki Yamamoto ◽  
...  
Keyword(s):  
Thermal Contact ◽  
X Ray Diffraction ◽  
Promising Technique ◽  
Fresh Sample ◽  
X Ray ◽  
Internal Structures ◽  
Diffraction Imaging ◽  
Diffraction Patterns ◽  
Almost All ◽  
Better Than

Coherent X-ray diffraction imaging (CXDI) is a promising technique to visualize internal structures of whole biological cells without sectioning. Utilizing X-ray free-electron laser (XFEL) for CXDI has potential to collect huge number of projected electron densities of such samples at higher resolutions than that limited by radiation damage. For biological application of XFEL-CXDI, sample particles must be kept in a hydrated state to maintain their functional structures, but be placed in vacuum to obtain weak diffraction signals. In addition, we need to deliver fresh sample particles one after another for XFEL exposure because every particle explodes just after X-ray irradiation. To handle these problems, we have been developing a system to fulfill cryogenic XFEL-CXDI of frozen-hydrated specimens. For cryogenic XFEL-CXDI, we prepare frozen-hydrated specimens by plunge-freezing sample particles dispersed onto thin film with humidity-controlling [1]. The diffraction experiments are conducted by using the cryogenic X-ray diffractometer KOTOBUKI-1 [2]. In a vacuum chamber of KOTOBUKI-1, a cryogenic pot equipped on a goniometer is filled with liquid nitrogen, which cools the specimen via thermal contact. Thus, KOTOBUKI-1 allows data collection at a specimen temperature of ~66 K with a positional fluctuation of less than 0.4 μm. A small angle-resolution of better than 500 nm is attainable by using a pair of L-shaped Si-slits placed before the specimen, which eliminate almost all parasite scattering from upstream. Diffraction patterns recorded on two MPCCD detectors in tandem arrangement are automatically processed and phase-retrieved by program suite SITENNO [3]. In our recent experiments performed in Japanese XFEL facility SACLA, we were able to collect a large number of diffraction patterns from biological samples at a resolution of 50 - 30 nm at an XFEL hit-rate of 20 - 100%. We report details of the cryogenic XFEL-CXDI and introduce imaging of chloroplasts as an example.

scholarly journals 3P287 Approaches to cohherent X-ray diffraction imaging of single virus particle using X-ray free-electron laser(27. Bioimaging,Poster)

2013 ◽  
Vol 53 (supplement1-2) ◽  
pp. S259
Author(s):  
Akifumi Higashiura ◽  
Marina Murakami ◽  
Kenji Iwasaki ◽  
Eiki Yamashita ◽  
Kazuki Takeda ◽  
...  
Keyword(s):  
Virus Particle ◽  
Free Electron ◽  
Free Electron Laser ◽  
X Ray Diffraction ◽  
X Ray ◽  
Diffraction Imaging

scholarly journals Cryogenic coherent X-ray diffraction imaging of biological samples at SACLA: a correlative approach with cryo-electron and light microscopy

2016 ◽  
Vol 72 (2) ◽  
pp. 179-189 ◽  
Author(s):  
Yuki Takayama ◽  
Koji Yonekura
Keyword(s):  
Light Microscopy ◽  
Biological Samples ◽  
Free Electron ◽  
Free Electron Laser ◽  
X Rays ◽  
X Ray Diffraction ◽  
Cell Organelles ◽  
X Ray ◽  
Electron And Light Microscopy ◽  
Diffraction Imaging

Coherent X-ray diffraction imaging at cryogenic temperature (cryo-CXDI) allows the analysis of internal structures of unstained, non-crystalline, whole biological samples in micrometre to sub-micrometre dimensions. Targets include cells and cell organelles. This approach involves preparing frozen-hydrated samples under controlled humidity, transferring the samples to a cryo-stage inside a vacuum chamber of a diffractometer, and then exposing the samples to coherent X-rays. Since 2012, cryo-coherent diffraction imaging (CDI) experiments have been carried out with the X-ray free-electron laser (XFEL) at the SPring-8 Ångstrom Compact free-electron LAser (SACLA) facility in Japan. Complementary use of cryo-electron microscopy and/or light microscopy is highly beneficial for both pre-checking samples and studying the integrity or nature of the sample. This article reports the authors' experience in cryo-XFEL-CDI of biological cells and organelles at SACLA, and describes an attempt towards reliable and higher-resolution reconstructions, including signal enhancement with strong scatterers and Patterson-search phasing.

scholarly journals Coherent X-ray Diffraction Imaging of Non-Crystalline Particles using X-ray Free Electron Laser

2014 ◽  
Vol 56 (1) ◽  
pp. 27-35
Author(s):  
Masayoshi NAKASAKO ◽  
Tomotaka OROGUCHI ◽  
Yuki SEKIGUCHI ◽  
Amane KOBAYASHI ◽  
Saki HASHIMOTO ◽  
...  
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
X Ray Diffraction ◽  
X Ray ◽  
Diffraction Imaging

scholarly journals Coherent X-Ray Diffraction Imaging of Chloroplasts fromCyanidioschyzon merolaeby Using X-Ray Free Electron Laser

2015 ◽  
Vol 56 (7) ◽  
pp. 1272-1286 ◽  
Author(s):  
Yuki Takayama ◽  
Yayoi Inui ◽  
Yuki Sekiguchi ◽  
Amane Kobayashi ◽  
Tomotaka Oroguchi ◽  
...  
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
X Ray Diffraction ◽  
X Ray ◽  
Diffraction Imaging

scholarly journals Simultaneous Multi-Bragg Peak Coherent X-ray Diffraction Imaging

Crystals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 312
Author(s):  
Florian Lauraux ◽  
Stéphane Labat ◽  
Sarah Yehya ◽  
Marie-Ingrid Richard ◽  
Steven J. Leake ◽  
...  
Keyword(s):  
Bragg Reflection ◽  
Volume Ratio ◽  
X Ray Diffraction ◽  
X Ray ◽  
Crystal Substrate ◽  
In Series ◽  
Diffraction Imaging ◽  
Diffraction Patterns ◽  
Lattice Planes ◽  
Oriented Parallel

The simultaneous measurement of two Bragg reflections by Bragg coherent X-ray diffraction is demonstrated on a twinned Au crystal, which was prepared by the solid-state dewetting of a 30 nm thin gold film on a sapphire substrate. The crystal was oriented on a goniometer so that two lattice planes fulfill the Bragg condition at the same time. The Au 111 and Au 200 Bragg peaks were measured simultaneously by scanning the energy of the incident X-ray beam and recording the diffraction patterns with two two-dimensional detectors. While the former Bragg reflection is not sensitive to the twin boundary, which is oriented parallel to the crystal–substrate interface, the latter reflection is only sensitive to one part of the crystal. The volume ratio between the two parts of the twinned crystal is about 1:9, which is also confirmed by Laue microdiffraction of the same crystal. The parallel measurement of multiple Bragg reflections is essential for future in situ and operando studies, which are so far limited to either a single Bragg reflection or several in series, to facilitate the precise monitoring of both the strain field and defects during the application of external stimuli.

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