| 1. |
- Daurer, Benedikt J., et al.
(author)
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Experimental strategies for imaging bioparticles with femtosecond hard X-ray pulses
- 2017
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In: IUCrJ. - : INT UNION CRYSTALLOGRAPHY. - 2052-2525. ; 4, s. 251-262
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Journal article (peer-reviewed)abstract
- This study explores the capabilities of the Coherent X-ray Imaging Instrument at the Linac Coherent Light Source to image small biological samples. The weak signal from small samples puts a significant demand on the experiment. Aerosolized Omono River virus particles of similar to 40 nm in diameter were injected into the submicrometre X-ray focus at a reduced pressure. Diffraction patterns were recorded on two area detectors. The statistical nature of the measurements from many individual particles provided information about the intensity profile of the X-ray beam, phase variations in the wavefront and the size distribution of the injected particles. The results point to a wider than expected size distribution (from similar to 35 to similar to 300 nm in diameter). This is likely to be owing to nonvolatile contaminants from larger droplets during aerosolization and droplet evaporation. The results suggest that the concentration of nonvolatile contaminants and the ratio between the volumes of the initial droplet and the sample particles is critical in such studies. The maximum beam intensity in the focus was found to be 1.9 * 10(12) photons per mu m(2) per pulse. The full-width of the focus at half-maximum was estimated to be 500 nm (assuming 20% beamline transmission), and this width is larger than expected. Under these conditions, the diffraction signal from a sample-sized particle remained above the average background to a resolution of 4.25 nm. The results suggest that reducing the size of the initial droplets during aerosolization is necessary to bring small particles into the scope of detailed structural studies with X-ray lasers.
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| 2. |
- Gorkhover, Tais, et al.
(author)
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Femtosecond X-ray Fourier holography imaging of free-flying nanoparticles
- 2018
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In: Nature Photonics. - : Springer Science and Business Media LLC. - 1749-4885 .- 1749-4893. ; 12:3, s. 150-153
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Journal article (peer-reviewed)abstract
- Ultrafast X-ray imaging on individual fragile specimens such as aerosols 1 , metastable particles 2 , superfluid quantum systems 3 and live biospecimens 4 provides high-resolution information that is inaccessible with conventional imaging techniques. Coherent X-ray diffractive imaging, however, suffers from intrinsic loss of phase, and therefore structure recovery is often complicated and not always uniquely defined 4,5 . Here, we introduce the method of in-flight holography, where we use nanoclusters as reference X-ray scatterers to encode relative phase information into diffraction patterns of a virus. The resulting hologram contains an unambiguous three-dimensional map of a virus and two nanoclusters with the highest lateral resolution so far achieved via single shot X-ray holography. Our approach unlocks the benefits of holography for ultrafast X-ray imaging of nanoscale, non-periodic systems and paves the way to direct observation of complex electron dynamics down to the attosecond timescale.
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| 3. |
- Hantke, Max F., et al.
(author)
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A data set from flash X-ray imaging of carboxysomes
- 2016
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In: Scientific Data. - : Springer Science and Business Media LLC. - 2052-4463. ; 3
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Journal article (peer-reviewed)abstract
- Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth’s carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26 nm from Halothiobacillus neapolitanus. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows sampling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere.
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| 4. |
- Hantke, Max F., et al.
(author)
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High-throughput imaging of heterogeneous cell organelles with an X-ray laser
- 2014
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In: Nature Photonics. - : Springer Science and Business Media LLC. - 1749-4885 .- 1749-4893. ; 8:12, s. 943-949
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Journal article (peer-reviewed)abstract
- We overcome two of the most daunting challenges in single-particle diffractive imaging: collecting many high-quality diffraction patterns on a small amount of sample and separating components from mixed samples. We demonstrate this on carboxysomes, which are polyhedral cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min with the Linac Coherent Light Source running at 120 Hz. We separate different structures directly from the diffraction data and show that the size distribution is preserved during sample delivery. We automate phase retrieval and avoid reconstruction artefacts caused by missing modes. We attain the highest-resolution reconstructions on the smallest single biological objects imaged with an X-ray laser to date. These advances lay the foundations for accurate, high-throughput structure determination by flash-diffractive imaging and offer a means to study structure and structural heterogeneity in biology and elsewhere.
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| 5. |
- Hedegaard, Jakob, et al.
(author)
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Comprehensive Transcriptional Analysis of Early-Stage Urothelial Carcinoma
- 2016
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In: Cancer Cell. - : Elsevier BV. - 1535-6108 .- 1878-3686. ; 30:1, s. 27-42
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Journal article (peer-reviewed)abstract
- Non-muscle-invasive bladder cancer (NMIBC) is a heterogeneous disease with widely different outcomes. We performed a comprehensive transcriptional analysis of 460 early-stage urothelial carcinomas and showed that NMIBC can be subgrouped into three major classes with basal-and luminal-like characteristics and different clinical outcomes. Large differences in biological processes such as the cell cycle, epithelial-mesenchymal transition, and differentiation were observed. Analysis of transcript variants revealed frequent mutations in genes encoding proteins involved in chromatin organization and cytoskeletal functions. Furthermore, mutations in well-known cancer driver genes (e.g., TP53 and ERBB2) were primarily found in high-risk tumors, together with APOBEC-related mutational signatures. The identification of subclasses in NMIBC may offer better prognostication and treatment selection based on subclass assignment.
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| 6. |
- Li, Haoyuan, et al.
(author)
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Diffraction data from aerosolized Coliphage PR772 virus particles imaged with the Linac Coherent Light Source
- 2020
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In: Scientific Data. - : NATURE RESEARCH. - 2052-4463. ; 7:1
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Journal article (peer-reviewed)abstract
- Single Particle Imaging (SPI) with intense coherent X-ray pulses from X-ray free-electron lasers (XFELs) has the potential to produce molecular structures without the need for crystallization or freezing. Here we present a dataset of 285,944 diffraction patterns from aerosolized Coliphage PR772 virus particles injected into the femtosecond X-ray pulses of the Linac Coherent Light Source (LCLS). Additional exposures with background information are also deposited. The diffraction data were collected at the Atomic, Molecular and Optical Science Instrument (AMO) of the LCLS in 4 experimental beam times during a period of four years. The photon energy was either 1.2 or 1.7keV and the pulse energy was between 2 and 4 mJ in a focal spot of about 1.3 mu m x 1.7 mu m full width at half maximum (FWHM). The X-ray laser pulses captured the particles in random orientations. The data offer insight into aerosolised virus particles in the gas phase, contain information relevant to improving experimental parameters, and provide a basis for developing algorithms for image analysis and reconstruction.
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| 7. |
- Lundholm, Ida V., et al.
(author)
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Considerations for three-dimensional image reconstruction from experimental data in coherent diffractive imaging
- 2018
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In: IUCrJ. - : International Union of Crystallography. - 2052-2525. ; 5, s. 531-541
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Journal article (peer-reviewed)abstract
- Diffraction before destruction using X-ray free-electron lasers (XFELs) has the potential to determine radiation-damage-free structures without the need for crystallization. This article presents the three-dimensional reconstruction of the Melbournevirus from single-particle X-ray diffraction patterns collected at the LINAC Coherent Light Source (LCLS) as well as reconstructions from simulated data exploring the consequences of different kinds of experimental sources of noise. The reconstruction from experimental data suffers from a strong artifact in the center of the particle. This could be reproduced with simulated data by adding experimental background to the diffraction patterns. In those simulations, the relative density of the artifact increases linearly with background strength. This suggests that the artifact originates from the Fourier transform of the relatively flat background, concentrating all power in a central feature of limited extent. We support these findings by significantly reducing the artifact through background removal before the phase-retrieval step. Large amounts of blurring in the diffraction patterns were also found to introduce diffuse artifacts, which could easily be mistaken as biologically relevant features. Other sources of noise such as sample heterogeneity and variation of pulse energy did not significantly degrade the quality of the reconstructions. Larger data volumes, made possible by the recent inauguration of high repetition-rate XFELs, allow for increased signal-to-background ratio and provide a way to minimize these artifacts. The anticipated development of three-dimensional Fourier-volume-assembly algorithms which are background aware is an alternative and complementary solution, which maximizes the use of data.
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| 8. |
- Mühlig, Kerstin, et al.
(author)
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Nanometre-sized droplets from a gas dynamic virtual nozzle
- 2019
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In: Journal of Applied Crystallography. - : INT UNION CRYSTALLOGRAPHY. - 1600-5767 .- 0021-8898. ; 52, s. 800-808
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Journal article (peer-reviewed)abstract
- This paper reports on improved techniques to create and characterize nanometre-sized droplets from dilute aqueous solutions by using a gas dynamic virtual nozzle (GDVN). It describes a method to measure the size distribution of uncharged droplets, using an environmental scanning electron microscope, and provides theoretical models for the droplet sizes created. The results show that droplet sizes can be tuned by adjusting the gas and liquid flow rates in the GDVN, and at the lowest liquid flow rates, the size of the water droplets peaks at about 120 nm. This droplet size is similar to droplet sizes produced by electrospray ionization but requires neither electrolytes nor charging of the solution. The results presented here identify a new operational regime for GDVNs and show that predictable droplet sizes, comparable to those obtained by electrospray ionization, can be produced by purely mechanical means in GDVNs.
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| 9. |
- Munke, Anna, et al.
(author)
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Data Descriptor : Coherent diffraction of single Rice Dwarf virus particles using hard X-rays at the Linac Coherent Light Source
- 2016
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In: Scientific Data. - : Nature Publishing Group. - 2052-4463. ; 3
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Journal article (peer-reviewed)abstract
- Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a wellcharacterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 mu m diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 angstrom ngstrom were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.
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| 10. |
- Oelze, Tim, et al.
(author)
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THz streak camera performance for single-shot characterization of XUV pulses with complex temporal structures
- 2020
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In: Optics Express. - : OPTICAL SOC AMER. - 1094-4087. ; 28:14, s. 20686-20703
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Journal article (peer-reviewed)abstract
- The THz-field-driven streak camera has proven to be a powerful diagnostic-technique that enables the shot-to-shot characterization of the duration and the arrival time jitter of free electron laser (FEL) pulses. Here we investigate the performance of three computational approaches capable to determine the duration of FEL pulses with complex temporal structures from single-shot measurements of up to three simultaneously recorded spectra. We use numerically simulated FEL pulses in order to validate the accuracy of the pulse length retrieval in average as well as in a single-shot mode. We discuss requirements for the THz field strength in order to achieve reliable results and compare our numerical study with the analysis of experimental data that were obtained at the FEL in Hamburg - FLASH. (C) 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
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| 11. |
- Okamoto, Kenta, et al.
(author)
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The infectious particle of insect-borne totivirus-like Omono River virus has raised ridges and lacks fibre complexes
- 2016
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Journal article (peer-reviewed)abstract
- Omono River virus (OmRV) is a double-stranded RNA virus isolated from Culex mosquitos, and it belongs to a group of unassigned insect viruses that appear to be related to Totiviridae. This paper describes electron cryo-microscopy (cryoEM) structures for the intact OmRV virion to 8.9 angstrom resolution and the structure of the empty virus-like-particle, that lacks RNA, to 8.3 angstrom resolution. The icosahedral capsid contains 120-subunits and resembles another closely related arthropod-borne totivirus-like virus, the infectious myonecrosis virus (IMNV) from shrimps. Both viruses have an elevated plateau around their icosahedral 5-fold axes, surrounded by a deep canyon. Sequence and structural analysis suggests that this plateau region is mainly composed of the extended C-terminal region of the capsid proteins. In contrast to IMNV, the infectious form of OmRV lacks extensive fibre complexes at its 5-fold axes as directly confirmed by a contrast-enhancement technique, using Zernike phase-contrast cryo-EM. Instead, these fibre complexes are replaced by a short "plug" structure at the five-fold axes of OmRV. OmRV and IMNV have acquired an extracellular phase, and the structures at the five-fold axes may be significant in adaptation to cell-to-cell transmission in metazoan hosts.
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| 12. |
- Rath, Asawari D., et al.
(author)
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Explosion dynamics of sucrose nanospheres monitored by time of flight spectrometry and coherent diffractive imaging at the split-and-delay beam line of the FLASH soft X-ray laser
- 2014
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In: Optics Express. - 1094-4087. ; 22:23, s. 28914-28925
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Journal article (peer-reviewed)abstract
- We use a Mach-Zehnder type autocorrelator to split and delay XUV pulses from the FLASH soft X-ray laser for triggering and subsequently probing the explosion of aerosolised sugar balls. FLASH was running at 182 eV photon energy with pulses of 70 fs duration. The delay between the pump-probe pulses was varied between zero and 5 ps, and the pulses were focused to reach peak intensities above 1016 W/cm2 with an off-axis parabola. The direct pulse triggered the explosion of single aerosolised sucrose nano-particles, while the delayed pulse probed the exploding structure. The ejected ions were measured by ion time of flight spectrometry, and the particle sizes were measured by coherent diffractive imaging. The results show that sucrose particles of 560-1000 nm diameter retain their size for about 500 fs following the first exposure. Significant sample expansion happens between 500 fs and 1 ps. We present simulations to support these observations.
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| 13. |
- Reddy, Hemanth K. N., et al.
(author)
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Coherent soft X-ray diffraction imaging of coliphage PR772 at the Linac coherent light source
- 2017
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In: Scientia Danica. Series H. Humanistica 4. - : Nature Publishing Group. - 1904-5506 .- 2052-4463. ; 4
-
Journal article (peer-reviewed)abstract
- Single-particle diffraction from X-ray Free Electron Lasers offers the potential for molecular structure determination without the need for crystallization. In an effort to further develop the technique, we present a dataset of coherent soft X-ray diffraction images of Coliphage PR772 virus, collected at the Atomic Molecular Optics (AMO) beamline with pnCCD detectors in the LAMP instrument at the Linac Coherent Light Source. The diameter of PR772 ranges from 65-70 nm, which is considerably smaller than the previously reported similar to 600 nm diameter Mimivirus. This reflects continued progress in XFEL-based single-particle imaging towards the single molecular imaging regime. The data set contains significantly more single particle hits than collected in previous experiments, enabling the development of improved statistical analysis, reconstruction algorithms, and quantitative metrics to determine resolution and self-consistency.
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| 14. |
- Sobolev, Egor, et al.
(author)
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Megahertz single-particle imaging at the European XFEL
- 2020
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In: Communications Physics. - : Springer Science and Business Media LLC. - 2399-3650. ; 3:1
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Journal article (peer-reviewed)abstract
- The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from sample preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate.
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| 15. |
- Wiedorn, Max O., et al.
(author)
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Megahertz serial crystallography
- 2018
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In: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9
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Journal article (peer-reviewed)abstract
- The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a beta-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.
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