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

Abstract

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.

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

“…2a; Takayama & Nakasako, 2012;Takayama, Inui et al, 2015). For sample support, we prepared a $20 nm-thick carbon membrane adhered onto a custommade metal disc with multiple measurement windows or a 100 nm-thick silicon nitride membrane (Norcada, Canada) with carbon deposition.…”

“…For sample support, we prepared a $20 nm-thick carbon membrane adhered onto a custommade metal disc with multiple measurement windows or a 100 nm-thick silicon nitride membrane (Norcada, Canada) with carbon deposition. The membrane surface was treated with 0.1%(w/v) polylysine solution (Sigma-Aldrich, USA), which increases the affinity of the surface for biological samples (Takayama, Inui et al, 2015). Then, we used a custom-made humidity-controlling device with a light microscope ( Fig.…”

“…This advantage is clearly demonstrated in a study of damage-free structures up to at least $2 Å resolution by XFEL protein crystallography (Hirata et al, 2014). XFEL-coherent diffraction imaging (CDI) has so far revealed 30-100 nm resolution structures of non-crystalline biological samples such as viruses (Seibert et al, 2011;Kassemeyer et al, 2012), cellular components (Hantke et al, 2014;Takayama, Inui et al, 2015;Oroguchi et al, 2015) and bacterial cells (Kimura et al, 2014;van der Schot et al, 2015;Oroguchi et al, 2015). In addition, the high repetition rate of XFEL pulses yields much diffraction data in a short time, allowing reconstruction of three-dimensional structures of homogeneous samples (Ekeberg et al, 2015) and statistical analysis of both the shape and internal structures of inhomogeneous samples .…”

“…Living cell morphologies, however, may change during the experiment or isolated cell organelles are often unstable and deteriorate. Cryo-XFEL-CDI can help solve this (Takayama & Nakasako, 2012;Nakasako et al, 2013;Takayama, Inui et al, 2015;Oroguchi et al, 2015). Flashcooling of samples can retain hydrated states and the integrity of cellular and sub-cellular structures (McDowall et al, 1983) even in a vacuum.…”

Three-dimensional reconstruction for coherent diffraction patterns obtained by XFEL

“…XFELs can significantly relax the resolution barrier imposed by radiation damage by recording the diffraction pattern before specimen destruction, due to femtosecond-short pulse duration (Neutze et al, 2000;Chapman et al, 2011;Hirata et al, 2014;Suga et al, 2014). XFEL experimental data are becoming increasingly available and several low-resolution structures from single-particle approaches have been reported Gallagher-Jones et al, 2014;Kimura et al, 2014;Xu et al, 2014;Ekeberg et al, 2015;Takayama et al, 2015;. It has also been shown, theoretically, that high-resolution 3D structures could be obtained using millions of diffraction patterns (Loh & Elser, 2009;Tegze & Bortel, 2012;Tokuhisa et al, 2012;Hosseinizadeh et al, 2014) and that dynamics could be directly extracted from two-dimensional (2D) data (Tokuhisa et al, 2016).…”