Intense x-ray free-electron laser (XFEL) pulses hold great promise for imaging function in nanoscale and biological systems with atomic resolution. So far, however, the spatial resolution obtained ...from single shot experiments lags averaging static experiments. Here we report on a combined computational and experimental study about ultrafast diffractive imaging of sucrose clusters which are benchmark organic samples. Our theoretical model matches the experimental data from the water window to the keV x-ray regime. The large-scale dynamic scattering calculations reveal that transient phenomena driven by non-linear x-ray interaction are decisive for ultrafast imaging applications. Our study illuminates the complex interplay of the imaging process with the rapidly changing transient electronic structures in XFEL experiments and shows how computational models allow optimization of the parameters for ultrafast imaging experiments.
Ultrafast X-ray imaging on individual fragile specimens such as aerosols1, metastable particles2, superfluid quantum systems3 and live biospecimens4 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 defined4,5. 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.
Flash X‐ray imaging has the potential to determine structures down to molecular resolution without the need for crystallization. The ability to accurately predict the diffraction signal and to ...identify the optimal experimental configuration within the limits of the instrument is important for successful data collection. This article introduces Condor, an open‐source simulation tool to predict X‐ray far‐field scattering amplitudes of isolated particles for customized experimental designs and samples, which the user defines by an atomic or a refractive index model. The software enables researchers to test whether their envisaged imaging experiment is feasible, and to optimize critical parameters for reaching the best possible result. It also aims to support researchers who intend to create or advance reconstruction algorithms by simulating realistic test data. Condor is designed to be easy to use and can be either installed as a Python package or used from its web interface (http://lmb.icm.uu.se/condor). X‐ray free‐electron lasers have high running costs and beam time at these facilities is precious. Data quality can be substantially improved by using simulations to guide the experimental design and simplify data analysis.
Condor, an open‐source simulation tool to predict X‐ray scattering amplitudes for flash X‐ray imaging experiments, is introduced.
Advances in X‐ray detectors and increases in the brightness of X‐ray sources combined with more efficient sample delivery techniques have brought about tremendous increases in the speed of data ...collection in diffraction experiments. Using X‐ray free‐electron lasers such as the Linac Coherent Light Source (LCLS), more than 100 diffraction patterns can be collected in a second. These high data rates are invaluable for flash X‐ray imaging (FXI), where aerosolized samples are exposed to the X‐ray beam and the resulting diffraction patterns are used to reconstruct a three‐dimensional image of the sample. Such experiments require immediate feedback on the quality of the data collected to adjust or validate experimental parameters, such as aerosol injector settings, beamline geometry or sample composition. The scarcity of available beamtime at the laser facilities makes any delay extremely costly. This paper presents Hummingbird, an open‐source scalable Python‐based software tool for real‐time analysis of diffraction data with the purpose of giving users immediate feedback during their experiments. Hummingbird provides a fast, flexible and easy‐to‐use framework. It has already proven to be of great value in numerous FXI experiments at the LCLS.
Hummingbird is an open‐source scalable Python‐based software tool for real‐time analysis of diffraction data with the purpose of giving users immediate feedback during their experiments.
Nucleocytoplasmic large DNA viruses (NCLDVs) blur the line between viruses and cells. Melbournevirus (MelV, family Marseilleviridae) belongs to a new family of NCLDVs. Here we present an electron ...cryo-microscopy structure of the MelV particle, with the large triangulation number T = 309 constructed by 3080 pseudo-hexagonal capsomers. The most distinct feature of the particle is a large and dense body (LDB) consistently found inside all particles. Electron cryo-tomography of 147 particles shows that the LDB is preferentially located in proximity to the probable lipid bilayer. The LDB is 30 nm in size and its density matches that of a genome/protein complex. The observed LDB reinforces the structural complexity of MelV, setting it apart from other NCLDVs.
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•The first interpretable capsid lattice of a Marseilleviridae family member.•The Melbournevirus particle forms a large T = 309 capsid lattice.•The Melbournevirus particle encapsulates a unique large and dense body (LDB).•The LDB reinforces the structural complexity, setting it apart from other NCLDVs.•The LDB is loosely constrained to the proximity of the capsid/membrane.
Ultra-bright femtosecond X-ray pulses generated by X-ray free-electron lasers (XFELs) can be used to image high-resolution structures without the need for crystallization. For this approach, aerosol ...injection has been a successful method to deliver 70-2000 nm particles into the XFEL beam efficiently and at low noise. Improving the technique of aerosol sample delivery and extending it to single proteins necessitates quantitative aerosol diagnostics. Here a lab-based technique is introduced for Rayleigh-scattering microscopy allowing us to track and size aerosolized particles down to 40 nm in diameter as they exit the injector. This technique was used to characterize the 'Uppsala injector', which is a pioneering and frequently used aerosol sample injector for XFEL single-particle imaging. The particle-beam focus, particle velocities, particle density and injection yield were measured at different operating conditions. It is also shown how high particle densities and good injection yields can be reached for large particles (100-500 nm). It is found that with decreasing particle size, particle densities and injection yields deteriorate, indicating the need for different injection strategies to extend XFEL imaging to smaller targets, such as single proteins. This work demonstrates the power of Rayleigh-scattering microscopy for studying focused aerosol beams quantitatively. It lays the foundation for lab-based injector development and online injection diagnostics for XFEL research. In the future, the technique may also find application in other fields that employ focused aerosol beams, such as mass spectrometry, particle deposition, fuel injection and three-dimensional printing techniques.
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.
The pressing need for knowledge of the detailed wavefront properties of ultra‐bright and ultra‐short pulses produced by free‐electron lasers has spurred the development of several complementary ...characterization approaches. Here a method based on ptychography is presented that can retrieve high‐resolution complex‐valued wavefunctions of individual pulses without strong constraints on the illumination or sample object used. The technique is demonstrated within experimental conditions suited for diffraction experiments and exploiting Kirkpatrick–Baez focusing optics. This lensless technique, applicable to many other short‐pulse instruments, can achieve diffraction‐limited resolution.
Understanding the wavefront of ultra‐bright and ultra‐short pulses of X‐ray free‐electron lasers is both important and challenging. A method based on ptychography that can retrieve full high‐resolution complex‐valued wave functions of individual pulses is presented.
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.
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
particles of ∼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 ∼35 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
photons per µm
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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