We use extremely bright and ultrashort pulses from an x-ray free-electron laser (XFEL) to measure correlations in x rays scattered from individual bioparticles. This allows us to go beyond the ...traditional crystallography and single-particle imaging approaches for structure investigations. We employ angular correlations to recover the three-dimensional (3D) structure of nanoscale viruses from x-ray diffraction data measured at the Linac Coherent Light Source. Correlations provide us with a comprehensive structural fingerprint of a 3D virus, which we use both for model-based and ab initio structure recovery. The analyses reveal a clear indication that the structure of the viruses deviates from the expected perfect icosahedral symmetry. Our results anticipate exciting opportunities for XFEL studies of the structure and dynamics of nanoscale objects by means of angular correlations.
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.
Ewald sphere curvature correction, which extends beyond the projection approximation, stretches the shallow depth of field in cryo-EM reconstructions of thick particles. Here we show that even for ...previously assumed thin particles, reconstruction artifacts which we refer to as ghosts can appear. By retrieving the lost phases of the electron exitwaves and accounting for the first Born approximation scattering within the particle, we show that these ghosts can be effectively eliminated. Our simulations demonstrate how such ghostbusting can improve reconstructions as compared to existing state-of-the-art software. Like ptychographic cryo-EM, our Ghostbuster algorithm uses phase retrieval to improve reconstructions, but unlike the former, we do not need to modify the existing data acquisition pipelines.
•Ewald sphere curvature correction methods for cryo-EM have had limited success due to lost phase information in measured micrographs.•Phase retrieval algorithms can recover these phases.•Backpropagating these phase-retrieved exitwaves through the sample can computationally stretch a cryo-EM reconstruction’s depth of field.
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.
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.
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.
Nanoparticles, exhibiting functionally relevant structural heterogeneity, are at the forefront of cutting-edge research. Now, high-throughput single-particle imaging (SPI) with X-ray free-electron ...lasers (XFELs) creates opportunities for recovering the shape distributions of millions of particles that exhibit functionally relevant structural heterogeneity. To realize this potential, three challenges have to be overcome: (1) simultaneous parametrization of structural variability in real and reciprocal spaces; (2) efficiently inferring the latent parameters of each SPI measurement; (3) scaling up comparisons between 105 structural models and 106 XFEL-SPI measurements. Here, we describe how we overcame these three challenges to resolve the nonequilibrium shape distributions within millions of gold nanoparticles imaged at the European XFEL. These shape distributions allowed us to quantify the degree of asymmetry in these particles, discover a relatively stable “shape envelope” among nanoparticles, discern finite-size effects related to shape-controlling surfactants, and extrapolate nanoparticles’ shapes to their idealized thermodynamic limit. Ultimately, these demonstrations show that XFEL SPI can help transform nanoparticle shape characterization from anecdotally interesting to statistically meaningful.
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.Diffractive imaging of single-particle nanoscale systems has so far been hindered by low hit probabilities and repetition rates. Here, single-particle imaging of nanospheres and viruses at megahertz repetition rates is demonstrated at the European X-ray Free-Electron Laser (XFEL) for the first time.
The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. ...According to theory, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes. This was first demonstrated on biological samples a decade ago on the giant mimivirus. Since then, a large collaboration has been pushing the limit of the smallest sample that can be imaged. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.
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