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

Abstract

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 Å ngstrom 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.

The biological crystallography without crystals

“…На рисунке 2 приведены примеры экспериментально полученных картин рассеяния отдельными биологическими частицами [1][2][3]. Во всех проведенных экспериментах результаты были получены для крупных макромолекулярных комплексов, содержащих большое число атомов N , что, в соответствии с формулой (5), облегчает регистрацию рассеяния.…”

“…Съемка на синхротронном ускорителе ALS в Беркли (США). Зарегистрирована дифракция до разрешения 207 Å. в) Хлоропласт (из [3]). Диаметр частицы около 2 мкм.…”

“…б) Дрожжевая клетка (из [1]). Оценка достигнутого разрешения 300 Å; в) Хлоропласт (из [3]). Оценка достигнутого разрешения 192 Å.…”

Cryo-coherent diffractive imaging of biological samples with X-ray free-electron lasers

“…To achieve this goal, two important things are needed: (1) a high-resolution imaging method and (2) an effective method for preparing biological samples that keeps them in their natural state. In this issue, Takayama & Yonekura (2016) report the progress and achievements in cryogenic coherent X-ray diffractive imaging of biological samples such as cells and organelles at the SPring-8 Angstrom Compact free-electron LAser (SACLA). Although cell imaging has been realized before with single-shot coherent diffractive imaging (CDI) (Kimura et al, 2014;van der Schot et al, 2015), the combination of cryogenic techniques and radiation-damage-free single-shot CDI offers the potential for quantitative imaging of biological samples in their natural state with high spatial resolution.…”

“…Takayama and Yonekura (Takayama & Yonekura, 2016;Takayama et al, 2015) report an effective strategy for combining frozen-hydrated sample preparation with XFEL-CDI while maintaining the hydrated state of the sample, using a special experimental setup at the SACLA XFEL light source. Additionally, specific control and data-processing software [the G-SITENNO suite Sekiguchi, Yamamoto et al, 2014)] has been developed, which enables automatic background subtraction, merging of high-and low-resolution diffraction patterns, and display of the diffraction patterns.…”