Diffraction apparatus and procedure in tomography X-ray diffraction imaging for biological cells at cryogenic temperature using synchrotron X-ray radiation

Abstract

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 , 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 AE 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 threedimensional structure of a Cyanidioschyzon merolae cell with an approximate size of 5 mm at a resolution of 136 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.research papers

Methods and application of coherent X-ray diffraction imaging of noncrystalline particles

“…Then, a map, which is calculated by the deconvolution of the mask from the dark image, is used as an initial in the subsequent PR calculation for the diffraction pattern without mask (Kobayashi et al 2014). The mask technique was also applied when we exactly evaluated spatial coherence of XFEL pulses (Kobayashi et al 2018b).…”

“…Therefore, cXDI experiment at cryogenic temperature for frozen-hydrated specimen is one of the solutions to significantly reduce radiation damage and also to visualize structures of biological specimens without dehydration and chemical modification. Therefore, experimental devices and procedures dedicated to cryogenic cXDI are developed (Huang et al 2009;Lima et al 2009;Takayama and Nakasako 2012;Nakasako et al 2013;Kobayashi et al 2018b). However, we must be careful to the maximum tolerable radiation dose that a biological specimen can accept before the destruction of structures and functions even at cryogenic temperature (Howells et al 2009).…”

“…In cXDI experiment using synchrotron X-rays, we must be careful on the maximum tolerable irradiation dose to a specimen. Figure 4a schematically shows an experimental result regarding the correlation between exposure time and the maximum resolution in diffraction patterns of a biological cell with a diameter of 6 μm (Kobayashi et al 2018b). Longer exposure of spatially coherent X-rays yields a diffraction pattern extended to a higher resolution.…”

“…4 a Experimentally found correlation between the exposure time and maximum resolution of diffraction patterns from a Cyanidioschyzon merolae (Kuroiwa et al. 2007) cell in the G/M phase of the cell cycle (Kobayashi et al 2018b). In this experiment, we used X-rays with a wavelength of 0.225 nm and the photon flux at the specimen position of approximately 5 × 10 9 X-ray photons/s, and the diffraction patterns were recorded by an Eiger pixel array detector.…”

“…5a, we developed a diffraction apparatus named KOTOBUKI-1 for cXDI tomography experiment using synchrotron X-rays ( Fig. 5b) (Nakasako et al 2013;Kobayashi et al 2018b). The apparatus comprises a vacuum chamber equipped with both a cryogenic pot mounted on a high-precision goniometer and a load-lock chamber to deliver specimen to the pot, and an alignment table for the chamber.…”