Diffraction data from aerosolized Coliphage PR772 virus particles imaged with the Linac Coherent Light Source.

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 1.7 keV and the pulse energy was between 2 and 4 mJ in a focal spot of about 1.3 µm x 1.7 µm 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.

Electrospray sample injection for single-particle imaging with x-ray lasers.

The possibility of imaging single proteins constitutes an exciting challenge for x-ray lasers. Despite encouraging results on large particles, imaging small particles has proven to be difficult for two reasons: not quite high enough pulse intensity from currently available x-ray lasers and, as we demonstrate here, contamination of the aerosolized molecules by nonvolatile contaminants in the solution. The amount of contamination on the sample depends on the initial droplet size during aerosolization. Here, we show that, with our electrospray injector, we can decrease the size of aerosol droplets and demonstrate virtually contaminant-free sample delivery of organelles, small virions, and proteins. The results presented here, together with the increased performance of next-generation x-ray lasers, constitute an important stepping stone toward the ultimate goal of protein structure determination from imaging at room temperature and high temporal resolution.

Erratum: Experimental strategies for imaging bioparticles with femtosecond hard X-ray pulses. Corrigendum.

[This corrects the article DOI: 10.1107/S2052252517003591.].

Using convex optimization of autocorrelation with constrained support and windowing for improved phase retrieval accuracy.

In imaging modalities recording diffraction data, such as the imaging of viruses at X-ray free electron laser facilities, the original image can be reconstructed assuming known phases. When phases are unknown, oversampling and a constraint on the support region in the original object can be used to solve a non-convex optimization problem using iterative alternating-projection methods. Such schemes are ill-suited for finding the optimum solution for sparse data, since the recorded pattern does not correspond exactly to the original wave function. Different iteration starting points can give rise to different solutions. We construct a convex optimization problem, where the only local optimum is also the global optimum. This is achieved using a modified support constraint and a maximum-likelihood treatment of the recorded data as a sample from the underlying wave function. This relaxed problem is solved in order to provide a new set of most probable "healed" signal intensities, without sparseness and missing data. For these new intensities, it should be possible to satisfy the support constraint and intensity constraint exactly, without conflicts between them. By making both constraints satisfiable, traditional phase retrieval with superior results is made possible. On simulated data, we demonstrate the benefits of our approach visually, and quantify the improvement in terms of the crystallographic R factor for the recovered scalar amplitudes relative to true simulations from .405 to .097, as well as the mean-squared error in the reconstructed image from .233 to .139. We also compare our approach, with regards to theory and simulation results, to other approaches for healing as well as noise-tolerant phase retrieval. These tests indicate that the COACS pre-processing allows for best-in-class results.

Considerations for three-dimensional image reconstruction from experimental data in coherent diffractive imaging.

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.

Artifact reduction in the CSPAD detectors used for LCLS experiments.

The existence of noise and column-wise artifacts in the CSPAD-140K detector and in a module of the CSPAD-2.3M large camera, respectively, is reported for the L730 and L867 experiments performed at the CXI Instrument at the Linac Coherent Light Source (LCLS), in low-flux and low signal-to-noise ratio regime. Possible remedies are discussed and an additional step in the preprocessing of data is introduced, which consists of performing a median subtraction along the columns of the detector modules. Thus, we reduce the overall variation in the photon count distribution, lowering the mean false-positive photon detection rate by about 4% (from 5.57 × 10-5 to 5.32 × 10-5 photon counts pixel-1 frame-1 in L867, cxi86715) and 7% (from 1.70 × 10-3 to 1.58 × 10-3 photon counts pixel-1 frame-1 in L730, cxi73013), and the standard deviation in false-positive photon count per shot by 15% and 35%, while not making our average photon detection threshold more stringent. Such improvements in detector noise reduction and artifact removal constitute a step forward in the development of flash X-ray imaging techniques for high-resolution, low-signal and in serial nano-crystallography experiments at X-ray free-electron laser facilities.

Experimental strategies for imaging bioparticles with femtosecond hard X-ray pulses.

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 ∼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 × 1012 photons per µm2 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.

Melting of DNA nonoriented fibers: a wide-angle X-ray diffraction study.

The melting transition of A- and B-DNA has been investigated by wide-angle X-ray diffraction. A significant crystalline phase is present in both the systems, even if the fibers have not been artificially aligned. The behavior of the intramolecular Bragg peaks of both A- and B-DNA as a function of the temperature clearly reveals the unfolding structural transition of the double helix. This transition occurs at the same temperature as the melting of the crystalline phase. The trends of the intramolecular correlations and the index of crystallinity are nicely described by the Peyrard-Bishop-Dauxois model for DNA melting. A description of the processes taking place at a microscopic level, i.e., double-helix deformation, crystalline dilation, and collapse, on approaching and during thermal melting is proposed.