Single-particle Diffraction Imaging Research Articles

New aerodynamic lens injector for single particle diffractive imaging

An aerodynamic lens injector was developed specifically for the needs of single-particle diffractive imaging experiments at free-electron lasers. Its design allows for quick changes of injector geometries and focusing properties in order to optimize injection for specific individual samples. Here, we present results of its first use at the FLASH free-electron-laser facility. Recorded diffraction patterns of polystyrene spheres are modeled using Mie scattering, which allowed for the characterization of the particle beam under diffractive-imaging conditions and yielded good agreement with particle-trajectory simulations.The complex refractive index of polystyrene at λ=4.5nm was determined as m=0.976−0.001i.

DNA-Origami-Assisted Flow-Aligned Single-Particle Diffractive Imaging using XFEL Pulses

Outrunning radiation damage, highly intense femtosecond pulses of X-ray free-electron lasers (XFELs) open up the possibility of structure determination of macromolecules to viruses at room temperature, by aggregating diffraction patterns recorded from uncrystallized single-particles. A key challenge in XFEL single-particle diffractive imaging (SPI) is to either constrain the orientation of the particle or to determine it from each of the very noisy weak diffraction patterns. Here we report a unique approach to address these challenges using structural DNA nanotechnology. A DNA-origami “rigid tail” is site-specifically attached to the macromolecule to flow-align it in a thin liquid jet, and also provides a strong holographic reference. In a proof-of-principle study, the computational design and production of the DNA-origami-target construct has been achieved and the experimental results obtained from the Linac Coherent Light Source (LCLS), USA show the alignment of the target single-particle, consistent with simulations, at extremely low concentrations approaching single-molecule in the interaction region with the nanofocus hard X-ray laser beam, in a sum of as few as a thousand single-shots. The results open up the possibility of single-molecule diffractive imaging in solution with XFEL pulses. Acknowledgements: The Human Frontier Science Program (RGP0010/2017).

Low-signal limit of X-ray single particle diffractive imaging.

An outstanding question in X-ray single particle imaging experiments has been the feasibility of imaging sub 10-nm-sized biomolecules under realistic experimental conditions where very few photons are expected to be measured in a single snapshot and instrument background may be significant relative to particle scattering. While analyses of simulated data have shown that the determination of an average image should be feasible using Bayesian methods such as the EMC algorithm, this has yet to be demonstrated using experimental data containing realistic non-isotropic instrument background, sample variability and other experimental factors. In this work, we show that the orientation and phase retrieval steps work at photon counts diluted to the signal levels one expects from smaller molecules or with weaker pulses, using data from experimental measurements of 60-nm PR772 viruses. Even when the signal is reduced to a fraction as little as 1/256, the virus electron density determined using ab initio phasing is of almost the same quality as the high-signal data. However, we are still limited by the total number of patterns collected, which may soon be mitigated by the advent of high repetition-rate sources like the European XFEL and LCLS-II.

Supervised classification methods for flash X-ray single particle diffraction imaging.

Current Flash X-ray single-particle diffraction Imaging (FXI) experiments, which operate on modern X-ray Free Electron Lasers (XFELs), can record millions of interpretable diffraction patterns from individual biomolecules per day. Due to the practical limitations with the FXI technology, those patterns will to a varying degree include scatterings from contaminated samples. Also, the heterogeneity of the sample biomolecules is unavoidable and complicates data processing. Reducing the data volumes and selecting high-quality single-molecule patterns are therefore critical steps in the experimental setup. In this paper, we present two supervised template-based learning methods for classifying FXI patterns. Our Eigen-Image and Log-Likelihood classifier can find the best-matched template for a single-molecule pattern within a few milliseconds. It is also straightforward to parallelize them so as to match the XFEL repetition rate fully, thereby enabling processing at site. The methods perform in a stable way on various kinds of synthetic data. As a practical example we tested our methods on a real mimivirus dataset, obtaining a convincing classification accuracy of 0.9.

Optimizing aerodynamic lenses for single-particle imaging

A numerical simulation infrastructure capable of calculating the flow of gas and the trajectories of particles through an aerodynamic lens injector is presented. The simulations increase the fundamental understanding and predict optimized injection geometries and parameters. Our simulation results were compared to previous reports and also validated against experimental data for 500 nm polystyrene spheres from an aerosol-beam-characterization setup. The simulations yielded a detailed understanding of the radial phase-space distribution and highlighted weaknesses of current aerosol injectors for single-particle diffractive imaging. With the aid of these simulations we developed new experimental implementations to overcome current limitations.

Characterizing gas flow from aerosol particle injectors

A novel methodology for measuring gas flow from small orifices or nozzles into vacuum is presented. It utilizes a high-intensity femtosecond laser pulse to create a plasma within the gas plume produced by the nozzle, which is imaged by a microscope. Calibration of the imaging system allows for the extraction of absolute number densities. We show detection down to helium densities of 4×1016 cm−3 with a spatial resolution of a few micrometers. The technique is used to characterize the gas flow from a convergent-nozzle aerosol injector [Kirian et al., Struct. Dyn. 2, 041717 (2015)] as used in single-particle diffractive imaging experiments at free-electron laser sources. Based on the measured gas-density profile, we estimate the scattering background signal under typical operating conditions of single-particle imaging experiments and estimate that fewer than 50 photons per shot can be expected on the detector.

CCP-FEL: a collection of computer programs for free-electron laser research

The latest virtual special issue ofJournal of Applied Crystallography(http://journals.iucr.org/special_issues/2016/ccpfel) collects software for free-electron laser research and presents tools for a range of topics such as simulation of experiments, online monitoring of data collection, selection of hits, diagnostics of data quality, data management, data analysis and structure determination for both nanocrystallography and single-particle diffractive imaging. This article provides an introduction to the special issue.

Investigation of Structural Dynamics in Lithium Layered Oxide Positive Electrodes Via Coherent Diffraction Imaging

The need to increase energy density in the layered oxide positive electrode materials has lead to a push to higher voltages.1 However, as a consequence of these higher voltages, the cycle stability drops considerably due to lattice contraction, electrode surface reactions with the electrolyte, and related strain. The lithium-excess layered oxides, a subset of layered oxide materials, exhibit one of the highest capacities in positive electrode materials due to the addition of anion redox pairs to compensate the charge mechanism2. While promising, this material also suffers from thermodynamic instabilities and surface transformations when charged to higher voltages, necessary to utilize its full capacity3. Operando coherent X-ray diffraction is an eloquent technique that has provided information in the nonequilibrium structural dynamics at the single particle level in lithium battery spinel positive electrode materials4. In addition, through phase retrieval algorithms, three-dimensional defect dynamics can also be determined to understand the nanoscale mechanisms during high voltage cycling5,6. In this study, we apply the same principles to the lithium excess layered oxide, Li1.2Ni0.133Mn0.533Co0.133O2where a plateau can be observed for the Li-rich material at 4.5 V that correspond to a simultaneous extraction of lithium and oxygen. Figure (Left) illustrates powder diffraction at the (003) bragg peak during electrochemical cycling illuminating ~ 20 individual particles where they can be found at relatively high and low angles during the plateau region. Figure 2(right) is the corresponding single particle diffractive imaging for two different primary particles of the same material. Interestingly, an inhomogeneous structural evolution during the first charge occurs where some particles shows a delay before moving to lower angles. Acknowledgements This work was supported by U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract DE-SC0001805. Y.S.M., S.H. and H.L. acknowledge the support by the Assistant Secretary for Energy Efficiency and Renewable Energy, Office of Vehicle Technologies of the U.S. Department of Energy (DOE) under Contract No. DE-AC02-05CH11231, Subcontract No. 7073923, under the Advanced Battery Materials Research (BMR) Program. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. (1) Liu, W.; Oh, P.; Liu, X.; Lee, M.-J.; Cho, W.; Chae, S.; Kim, Y.; Cho, J. Angew. Chem. Int. Ed. 2015, n/a. (2) Yu, H.; Zhou, H. J. Phys. Chem. Lett. 2013, 4, 1268. (3) Xu, B.; Fell, C. R.; Chi, M.; Meng, Y. S. Energy & Environmental Science 2011, 4, 2223. (4) Singer, A.; Ulvestad, A.; Cho, H. M.; Kim, J. W.; Maser, J.; Harder, R.; Meng, Y. S.; Shpyrko, O. G. Nano Lett. 2014, 14, 5295. (5) Ulvestad, A.; Singer, A.; Cho, H. M.; Clark, J. N.; Harder, R.; Maser, J.; Meng, Y. S.; Shpyrko, O. G. Nano Lett. 2014, 14, 5123. (6) Ulvestad, A.; Singer, A.; Clark, J. N.; Cho, H. M.; Kim, J. W.; Harder, R.; Maser, J.; Meng, Y. S.; Shpyrko, O. G. Science 2015, 348, 1344. Figure 1

Simple convergent-nozzle aerosol injector for single-particle diffractive imaging with X-ray free-electron lasers.

A major challenge in high-resolution x-ray free-electron laser-based coherent diffractive imaging is the development of aerosol injectors that can efficiently deliver particles to the peak intensity of the focused X-ray beam. Here, we consider the use of a simple convergent-orifice nozzle for producing tightly focused beams of particles. Through optical imaging we show that 0.5 μm particles can be focused to a full-width at half maximum diameter of 4.2 μm, and we demonstrate the use of such a nozzle for injecting viruses into a micro-focused soft-X-ray FEL beam.

Orientation determination in single-particle x-ray coherent diffraction imaging experiments

Single-particle diffraction imaging experiments at free-electron lasers (FELs) have a great potential for the structure determination of reproducible biological specimens that cannot be crystallized. One of the challenges in processing the data from such an experiment is to determine the correct orientation of each diffraction pattern from samples randomly injected in the FEL beam. We propose an algorithm (Yefanov et al 2010 Photon Science—HASYLAB Annual Report) that can solve this problem and can be applied to samples from tens of nanometres to microns in size, measured with sub-nanometre resolution in the presence of noise. This is achieved by the simultaneous analysis of a large number of diffraction patterns corresponding to different orientations of the particles. The algorithm’s efficiency is demonstrated for two biological samples, an artificial protein structure without any symmetry and a virus with icosahedral symmetry. Both structures are a few tens of nanometres in size and consist of more than 100 000 non-hydrogen atoms. More than 10 000 diffraction patterns with Poisson noise were simulated and analysed for each structure. Our simulations indicate the possibility of achieving resolution of about 3.3 Å at 3 Å wavelength and incoming flux of 1012 photons per pulse focused to 100×100 nm2.

PRIME: Probabilistic Initial 3D Model Generation for Single-Particle Cryo-Electron Microscopy

Low-dose electron microscopy of cryo-preserved individual biomolecules (single-particle cryo-EM) is a powerful tool for obtaining information about the structure and dynamics of large macromolecular assemblies. Acquiring images with low dose reduces radiation damage, preserves atomic structural details, but results in low signal-to-noise ratio of the individual images. The projection directions of the two-dimensional images are random and unknown. The grand challenge is to achieve the precise three-dimensional (3D) alignment of many (tens of thousands to millions) noisy projection images, which may then be combined to obtain a faithful 3D map. An accurate initial 3D model is critical for obtaining the precise 3D alignment required for high-resolution (<10Å) map reconstruction. We report a method (PRIME) that, in a single step and without prior structural knowledge, can generate an accurate initial 3D map directly from the noisy images.

Sensing the wavefront of x-ray free-electron lasers using aerosol spheres

Characterizing intense, focused x-ray free electron laser (FEL) pulses is crucial for their use in diffractive imaging. We describe how the distribution of average phase tilts and intensities on hard x-ray pulses with peak intensities of 10(21) W/m(2) can be retrieved from an ensemble of diffraction patterns produced by 70 nm-radius polystyrene spheres, in a manner that mimics wavefront sensors. Besides showing that an adaptive geometric correction may be necessary for diffraction data from randomly injected sample sources, our paper demonstrates the possibility of collecting statistics on structured pulses using only the diffraction patterns they generate and highlights the imperative to study its impact on single-particle diffractive imaging.

Heterogeneous clusters as a model system for the study of ionization dynamics within tampered samples

Tampering of a sample with a layer of another material is a promising technique to slow down the expansion dynamics within laser irradiated samples, with sound implications for single-particle diffraction imaging. Ideally, if an imaged object is covered by a layer of another material, during the irradiation this layer will be primarily ionized and will expand fast due to Coulomb repulsion, whereas the object located within the net neutral core will expand more slowly (hydrodynamically). We investigate the details of the electronic damage within the tampered samples during their irradiation with an intense extreme ultraviolet (EUV) pulse. We study heterogeneous clusters composed of noble gas atoms, Xe and Ar, for which chemical-bond effects can be neglected. Using a fully nonequilibrium kinetic equation code, we demonstrate the influence of cluster composition on ionization dynamics; in particular, on the electronic damage. The results are obtained for the wavelength of 32 nm, which is available at the free-electron laser in Hamburg (FLASH) facility, but our conclusions can also have implications for samples with a more complex structure and irradiated at a much shorter wavelength.

Reconstruction algorithm for single-particle diffraction imaging experiments

We introduce the EMC algorithm for reconstructing a particle's three-dimensional (3D) diffraction intensity from very many photon shot-noise limited two-dimensional measurements, when the particle orientation in each measurement is unknown. The algorithm combines a maximization step (M) of the intensity's likelihood function, with expansion (E) and compression (C) steps that map the 3D intensity model to a redundant tomographic representation and back again. After a few iterations of the EMC update rule, the reconstructed intensity is given to the difference-map algorithm for reconstruction of the particle contrast. We demonstrate reconstructions with simulated data and investigate the effects of particle complexity, number of measurements, and the number of photons per measurement. The relatively transparent scaling behavior of our algorithm provides an estimate of the data processing resources required for future single-particle imaging experiments.

A reconstruction algorithm for single-particle diffraction imaging experiments

A reconstruction algorithm for single-particle diffraction imaging experiments

Effect of the coherence properties of self-amplified-spontaneous-emission x-ray free electron lasers on single-particle diffractive imaging

The longitudinal coherence properties of self-amplified-spontaneous- emission x-ray free electron lasers limit the resolution of single-particle diffraction imaging. We found that for the Linac Coherent Light Source (LCLS) at a wavelength of 1.5 A the particles have to be smaller than 500 nm in diameter to achieve atomic-resolution imaging with a resolution length of less than 2 A, suggesting that the longitudinal coherence is sufficient for imaging most biomolecular samples of interest.