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IUCrJ 5(1), 103 - 117 (2018). doi:10.1107/S2052252517017043, Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals., Published by IUCr, Chester

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introduction via a translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Z plastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low-Z fixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

Bragg diffraction achieved from two-dimensional protein crystals using femtosecond X-ray laser snapshots is presented., X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

Lasers have long played a critical role in the advancement of aerosol science. A new regime of ultrafast laser technology has recently be realized, the world's first soft x-ray free electron laser. The Free electron LASer in Hamburg, FLASH, user facility produces a steady source of 10 femtosecond pulses of 7–32 nm x-rays with 1012 photons per pulse. The high brightness, short wavelength, and high repetition rate (> 500 pulses per second) of this laser offers unique capabilities for aerosol characterization. Here we use FLASH to perform the highest resolution imaging of single PM2.5 aerosol particles in flight to date. We resolve to 35 nm the morphology of fibrous and aggregated spherical carbonaceous nanoparticles that existed for less than two milliseconds in vacuum. Our result opens the possibility for high spatial- and time-resolved single particle aerosol dynamics studies, filling a critical technological need in aerosol science.

Extremely intense and ultrafast X-ray pulses from free-electron lasers offer unique opportunities to study fundamental aspects of complex transient phenomena in materials. Ultrafast time-resolved methods usually require highly synchronized pulses to initiate a transition and then probe it after a precisely defined time delay. In the X-ray regime, these methods are challenging because they require complex optical systems and diagnostics. Here we propose and apply a simple holographic measurement scheme, inspired by Newton's 'dusty mirror' experiment, to monitor the X-ray-induced explosion of microscopic objects. The sample is placed near an X-ray mirror; after the pulse traverses the sample, triggering the reaction, it is reflected back onto the sample by the mirror to probe this reaction. The delay is encoded in the resulting diffraction pattern to an accuracy of one femtosecond, and the structural change is holographically recorded with high resolution. We apply the technique to monitor the dynamics of polystyrene spheres in intense free-electron-laser pulses, and observe an explosion occurring well after the initial pulse. Our results support the notion that X-ray flash imaging can be used to achieve high resolution, beyond radiation damage limits for biological samples. With upcoming ultrafast X-ray sources we will be able to explore the three-dimensional dynamics of materials at the timescale of atomic motion.

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.

Optics express 21(23), 28729(2013). doi:10.1364/OE.21.028729, Single shot diffraction imaging experiments via X-ray free- electron lasers can generate as many as hundreds of thousands of diffraction patterns of scattering objects. Recovering the real space contrast of a scat- tering object from these patterns currently requires a reconstruction process with user guidance in a number of steps, introducing severe bottlenecks in data processing. We present a series of measures that replace user guidance with algorithms that reconstruct contrasts in an unsupervised fashion. We demonstrate the feasibility of automating the reconstruction process by generating hundreds of contrasts obtained from soot particle diffraction experiments., Published by Soc., Washington, DC

Profiling structured beams produced by X-ray free-electron lasers (FELs) is crucial to both maximizing signal intensity for weakly scattering targets and interpreting their scattering patterns. Earlier ablative imprint studies describe how to infer the X-ray beam profile from the damage that an attenuated beam inflicts on a substrate. However, the beams in-situ profile is not directly accessible with imprint studies because the damage profile could be different from the actual beam profile. On the other hand, although a Shack-Hartmann sensor is capable of in-situ profiling, its lenses may be quickly damaged at the intense focus of hard X-ray FEL beams. We describe a new approach that probes the in-situ morphology of the intense FEL focus. By studying the translations in diffraction patterns from an ensemble of randomly injected sub-micron latex spheres, we were able to determine the non-Gaussian nature of the intense FEL beam at the Linac Coherent Light Source (SLAC National Laboratory) near the FEL focus. We discuss an experimental application of such a beam-profiling technique, and the limitations we need to overcome before it can be widely applied. © (2012) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Lasers have long played a critical role in the advancement of aerosol science. A new regime of ultrafast laser technology has recently be realized, the world's first soft xray free electron laser. The Free electron LASer in Hamburg, FLASH, user facility produces a steady source of 10 femtosecond pulses of 7-32 nm x-rays with 10{sub 12} photons per pulse. The high brightness, short wavelength, and high repetition rate (>500 pulses per second) of this laser offers unique capabilities for aerosol characterization. Here we use FLASH to perform the highest resolution imaging of single PM2.5 aerosol particles in flight to date. We resolve to 35 nm the morphology of fibrous and aggregated spherical carbonaceous nanoparticles that existed for less than two milliseconds in vacuum. Our result opens the possibility for high spatialand time-resolved single particle aerosol dynamics studies, filling a critical technological need in aerosol science.

Theory predicts that with an ultrashort and extremely bright coherent X-ray pulse, a single diffraction pattern may be recorded from a large macromolecule, a virus, or a cell before the sample explodes and turns into a plasma. Here we report the first experimental demonstration of this principle using the FLASH soft X-ray free-electron laser. An intense 25 fs, 4 10^13 W/cm^2 pulse, containing 10^12 photons at 32 nm wavelength, produced a coherent diffraction pattern from a nano-structured non-periodic object, before destroying it at 60,000 K. A novel X-ray camera assured single photon detection sensitivity by filtering out parasitic scattering and plasma radiation. The reconstructed image, obtained directly from the coherent pattern by phase retrieval through oversampling, shows no measurable damage, and extends to diffraction-limited resolution. A three-dimensional data set may be assembled from such images when copies of a reproducible sample are exposed to the beam one by one., revtex, 6 pages, 4 figures

The ultrafast, ultrabright X-ray pulses offered by a new generation of free-electron lasers is ushering in extraordinary new capabilities in X-ray science, with a wide range of applications in fundamental atomic-physics, ultrafast-chemistry and materials science.