High-throughput imaging of heterogeneous cell organelles with an X-ray laser

Hantke, Max F.; Hasse, Dirk; Maia, Filipe R. N. C.; Ekeberg, Tomas; John, Katja; Svenda, Martin; Loh, N. Duane; Martin, Andrew V.; Tı̂mneanu, Nicuşor; Larsson, Daniel; Schot, Gijs van der; Carlsson, Gunilla; Ingelman, Margareta; Andreasson, Jakob; Westphal, Daniel; Liang, Mao; Stellato, Francesco; DePonte, Daniel P.; Hartmann, R.; Kimmel, Nils; Kirian, Richard A.; Seibert, M. Marvin; Mühlig, Kerstin; Schorb, Sebastian; Ferguson, Ken; Bostedt, Christoph; Carron, S.; Bozek, John D.; Rolles, Daniel; Rudenko, Artem; Epp, Sascha W.; Chapman, Henry N.; Barty, Anton; Hajdu, János; Andersson, Inger

doi:10.1038/nphoton.2014.270

Cited by 166 publications

(156 citation statements)

References 41 publications

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“…These organelles consist of interior enzymes that catalyze sequential metabolic reactions (Yeates et al, 2010), surrounded by a single-layer proteinaceous shell (Kerfeld et al, 2005;Tsai et al, 2007;Tanaka et al, 2008;Sutter et al, 2016). The shell facets are composed of hexameric and pentameric proteins, resulting in an overall shell architecture resembling an icosahedral viral capsid (Kinney et al, 2011;Hantke et al, 2014;Kerfeld and Erbilgin, 2015). Interactions between shell proteins are important for the self-assembly of the shell (Sutter et al, 2016).…”

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confidence: 99%

Light Modulates the Biosynthesis and Organization of Cyanobacterial Carbon Fixation Machinery through Photosynthetic Electron Flow

et al. 2016

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Cyanobacteria have evolved effective adaptive mechanisms to improve photosynthesis and CO 2 fixation. The central CO 2 -fixing machinery is the carboxysome, which is composed of an icosahedral proteinaceous shell encapsulating the key carbon fixation enzyme, Rubisco, in the interior. Controlled biosynthesis and ordered organization of carboxysomes are vital to the CO 2 -fixing activity of cyanobacterial cells. However, little is known about how carboxysome biosynthesis and spatial positioning are physiologically regulated to adjust to dynamic changes in the environment. Here, we used fluorescence tagging and live-cell confocal fluorescence imaging to explore the biosynthesis and subcellular localization of b-carboxysomes within a model cyanobacterium, Synechococcus elongatus PCC7942, in response to light variation. We demonstrated that b-carboxysome biosynthesis is accelerated in response to increasing light intensity, thereby enhancing the carbon fixation activity of the cell. Inhibition of photosynthetic electron flow impairs the accumulation of carboxysomes, indicating a close coordination between b-carboxysome biogenesis and photosynthetic electron transport. Likewise, the spatial organization of carboxysomes in the cell correlates with the redox state of photosynthetic electron transport chain. This study provides essential knowledge for us to modulate the b-carboxysome biosynthesis and function in cyanobacteria. In translational terms, the knowledge is instrumental for design and synthetic engineering of functional carboxysomes into higher plants to improve photosynthesis performance and CO 2 fixation.

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confidence: 99%

Light Modulates the Biosynthesis and Organization of Cyanobacterial Carbon Fixation Machinery through Photosynthetic Electron Flow

et al. 2016

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“…Particles are delivered to the beam at random orientations through either a liquid medium (1) or aerosolization (2). Large-scale experimental facilities, such as the Linac Coherent Light Source (3), have been developed to perform these experiments, with initial results indicating the feasibility of single-particle diffraction as a viable technique to address challenges in the health, material, and energy sciences (4,5).…”

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confidence: 99%

Reconstruction from limited single-particle diffraction data via simultaneous determination of state, orientation, intensity, and phase

Donatelli

Sethian

Zwart

2017

Proc. Natl. Acad. Sci. U.S.A.

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Free-electron lasers now have the ability to collect X-ray diffraction patterns from individual molecules; however, each sample is delivered at unknown orientation and may be in one of several conformational states, each with a different molecular structure. Hit rates are often low, typically around 0.1%, limiting the number of useful images that can be collected. Determining accurate structural information requires classifying and orienting each image, accurately assembling them into a 3D diffraction intensity function, and determining missing phase information. Additionally, single particles typically scatter very few photons, leading to high image noise levels. We develop a multitiered iterative phasing algorithm to reconstruct structural information from singleparticle diffraction data by simultaneously determining the states, orientations, intensities, phases, and underlying structure in a single iterative procedure. We leverage real-space constraints on the structure to help guide optimization and reconstruct underlying structure from very few images with excellent global convergence properties. We show that this approach can determine structural resolution beyond what is suggested by standard Shannon sampling arguments for ideal images and is also robust to noise.single-particle imaging | multitiered iterative phasing | structure determination S ingle-particle X-ray diffraction aims to determine the structures of biological molecules from an ensemble of diffraction patterns, each collected from a single particle per shot. Particles are delivered to the beam at random orientations through either a liquid medium (1) or aerosolization (2). Large-scale experimental facilities, such as the Linac Coherent Light Source (3), have been developed to perform these experiments, with initial results indicating the feasibility of single-particle diffraction as a viable technique to address challenges in the health, material, and energy sciences (4, 5).A fundamental challenge in single-particle imaging is that the orientation of each imaged particle is unknown and must be recovered to determine structural information. Additionally, many biological samples display conformational flexibility and may exist in one of many possible structural states. To account for varying structural states and avoid a loss of resolution due to averaging of states, the diffraction patterns may need to be classified to the correct state. Furthermore, single particles scatter very few photons; hence the images are heavily contaminated by shot noise, often with less than a photon per Shannon pixel at high scattering angles.Current approaches typically determine states, orientations, and structures in separate consecutive steps. Classification work includes manifold mapping (6), spectral clustering (7), principal component analysis, and support vector machines (8). Orientation methods include common curve approaches (9-12), expectation maximization (13-15), and manifold embedding (16)(17)(18)(19). Once images are classified, oriented, and assemb...

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“…Many of these were used to test the validity of XFEL-CDI and included measurements of the morphology of complex structures, functional imaging method development, and dynamic changes under a stimulus (pump-probe). On the biological side, viruses, bacteria, organelles and associated biological components have been imaged at various resolution levels [133][134][135][136][137][138][139][140][141][142] and have helped further develop high throughput imaging methods. Figure 2 shows typical results from non-biological particles using XFEL-CDI.…”

Section: Coherent Diffraction Imaging With X-ray Lasersmentioning

confidence: 99%

“…In this big data era, images from populations of genetically identical cells often reveal heterogeneous phenotypes, which can be helpful to group behavior research. Figure 3b [137] shows the high-throughput imaging of heterogeneous cell organelles with 120 Hz X-ray laser pulses from LCLS. Morphology and inner electron density were reconstructed by phase retrieval automatically.…”

Section: Coherent Diffraction Imaging With X-ray Lasersmentioning

confidence: 99%

Current Status of Single Particle Imaging with X-ray Lasers

Sun

Fan

et al. 2018

Applied Sciences

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Abstract:The advent of ultrafast X-ray free-electron lasers (XFELs) opens the tantalizing possibility of the atomic-resolution imaging of reproducible objects such as viruses, nanoparticles, single molecules, clusters, and perhaps biological cells, achieving a resolution for single particle imaging better than a few tens of nanometers. Improving upon this is a significant challenge which has been the focus of a global single particle imaging (SPI) initiative launched in December 2014 at the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, USA. A roadmap was outlined, and significant multi-disciplinary effort has since been devoted to work on the technical challenges of SPI such as radiation damage, beam characterization, beamline instrumentation and optics, sample preparation and delivery and algorithm development at multiple institutions involved in the SPI initiative. Currently, the SPI initiative has achieved 3D imaging of rice dwarf virus (RDV) and coliphage PR772 viruses at~10 nm resolution by using soft X-ray FEL pulses at the Atomic Molecular and Optical (AMO) instrument of LCLS. Meanwhile, diffraction patterns with signal above noise up to the corner of the detector with a resolution of~6 Ångström (Å) were also recorded with hard X-rays at the Coherent X-ray Imaging (CXI) instrument, also at LCLS. Achieving atomic resolution is truly a grand challenge and there is still a long way to go in light of recent developments in electron microscopy. However, the potential for studying dynamics at physiological conditions and capturing ultrafast biological, chemical and physical processes represents a tremendous potential application, attracting continued interest in pursuing further method development. In this paper, we give a brief introduction of SPI developments and look ahead to further method development.

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High-throughput imaging of heterogeneous cell organelles with an X-ray laser

Cited by 166 publications

References 41 publications

Light Modulates the Biosynthesis and Organization of Cyanobacterial Carbon Fixation Machinery through Photosynthetic Electron Flow

Light Modulates the Biosynthesis and Organization of Cyanobacterial Carbon Fixation Machinery through Photosynthetic Electron Flow

Reconstruction from limited single-particle diffraction data via simultaneous determination of state, orientation, intensity, and phase

Current Status of Single Particle Imaging with X-ray Lasers

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