RESUMEN
X-ray diffraction imaging is a technique for visualizing the structure of biological cells. In X-ray diffraction imaging experiments using synchrotron radiation, cryogenic conditions are necessary in order to reduce radiation damage in the biological cells. Frozen-hydrated biological specimens kept at cryogenic temperatures are also free from drying and bubbling, which occurs in wet specimens under vacuum conditions. In a previous study, the diffraction apparatus KOTOBUKI-1 [Nakasako et al. (2013), Rev. Sci. Instrum. 84, 093705] was constructed for X-ray diffraction imaging at cryogenic temperatures by utilizing a cryogenic pot, which is a cooling device developed in low-temperature physics. In this study a new cryogenic pot, suitable for tomography experiments, has been developed. The pot can rotate a biological cell over an angular range of ±170° against the direction of the incident X-ray beam. Herein, the details and the performance of the pot and miscellaneous devices are reported, along with established experimental procedures including specimen preparation. The apparatus has been used in tomography experiments for visualizing the three-dimensional structure of a Cyanidioschyzon merolae cell with an approximate size of 5â µm at a resolution of 136â nm. Based on the experimental results, the necessary improvements for future experiments and the resolution limit achievable under experimental conditions within a maximum tolerable dose are discussed.
RESUMEN
We have developed an experimental apparatus named KOTOBUKI-1 for use in coherent X-ray diffraction imaging experiments of frozen-hydrated non-crystalline particles at cryogenic temperature. For cryogenic specimen stage with small positional fluctuation for a long exposure time of more than several minutes, we here use a cryogenic pot cooled by the evaporation cooling effect for liquid nitrogen. In addition, a loading device is developed to bring specimens stored in liquid nitrogen to the specimen stage in vacuum. The apparatus allows diffraction data collection for frozen-hydrated specimens at 66 K with a positional fluctuation of less than 0.4 µm and provides an experimental environment to easily exchange specimens from liquid nitrogen storage to the specimen stage. The apparatus was developed and utilized in diffraction data collection of non-crystalline particles with dimensions of µm from material and biological sciences, such as metal colloid particles and chloroplast, at BL29XU of SPring-8. Recently, it has been applied for single-shot diffraction data collection of non-crystalline particles with dimensions of sub-µm using X-ray free electron laser at BL3 of SACLA.
