3D atomic structure from a single XFEL pulse

X-ray Free Electron Lasers (XFEL) are the most advanced pulsed x-ray sources. Their extraordinary pulse parameters promise unique applications. Indeed, several new methods have been developed at XFEL-s. However, no methods are known, which would allow ab initio atomic level structure determination using only a single XFEL pulse. Here, we present experimental results, demonstrating the determination of the 3D atomic structure from data obtained during a single 25 fs XFEL pulse. Parallel measurement of hundreds of Bragg reflections was done by collecting Kossel line patterns of GaAs and GaP. With these measurements, we reached the ultimate temporal limit of the x-ray structure solution possible today. These measurements open the way for studying non-repeatable fast processes and structural transformations in crystals for example measuring the atomic structure of matter at extremely non-ambient conditions or transient structures formed in irreversible physical, chemical, or biological processes. It would also facilitate time resolved pump-probe structural studies making them significantly shorter than traditional serial crystallography.Comment: 16 pages of manuscript followed by 15 pages of supplementary informatio

Expected resolution limits of x-ray free-electron laser single-particle imaging for realistic source and detector properties

The unprecedented intensity of x-ray free-electron laser sources has enabled single-particle x-ray diffraction imaging (SPI) of various biological specimens in both two-dimensional projection and three dimensions (3D). The potential of studying protein dynamics in their native conditions, without crystallization or chemical staining, has encouraged researchers to aim for increasingly higher resolutions with this technique. The currently achievable resolution of SPI is limited to the sub-10 nanometer range, mainly due to background effects, such as instrumental noise and parasitic scattering from the carrier gas used for sample delivery. Recent theoretical studies have quantified the effects of x-ray pulse parameters, as well as the required number of diffraction patterns to achieve a certain resolution, in a 3D reconstruction, although the effects of detector noise and the random particle orientation in each diffraction snapshot were not taken into account. In this work, we show these shortcomings and address limitations on achievable image resolution imposed by the adaptive gain integrating pixel detector noise

Online dynamic flat-field correction for MHz Microscopy data at European XFEL

The X-ray microscopy technique at the European X-ray free-electron laser (EuXFEL), operating at a MHz repetition rate, provides superior contrast and spatial-temporal resolution compared to typical microscopy techniques at other X-ray sources. In both online visualization and offline data analysis for microscopy experiments, baseline normalization is essential for further processing steps such as phase retrieval and modal decomposition. In addition, access to normalized projections during data acquisition can play an important role in decision-making and improve the quality of the data. However, the stochastic nature of XFEL sources hinders the use of existing flat-flied normalization methods during MHz X-ray microscopy experiments. Here, we present an online dynamic flat-field correction method based on principal component analysis of dynamically evolving flat-field images. The method is used for the normalization of individual X-ray projections and has been implemented as an online analysis tool at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of EuXFEL.Comment: 14 pages, 7 figure

Observation of a single protein by ultrafast X-ray diffraction

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 theory1, 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 explodes2. This was first demonstrated on biological samples a decade ago on the giant mimivirus3. Since then a large collaboration4 has been pushing the limit of the smallest sample that can be imaged5,6. 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 diameter7 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

3D diffractive imaging of nanoparticle ensembles using an X-ray laser

We report the 3D structure determination of gold nanoparticles (AuNPs) by X-ray single particle imaging (SPI). Around 10 million diffraction patterns from gold nanoparticles were measured in less than 100 hours of beam time, more than 100 times the amount of data in any single prior SPI experiment, using the new capabilities of the European X-ray free electron laser which allow measurements of 1500 frames per second. A classification and structural sorting method was developed to disentangle the heterogeneity of the particles and to obtain a resolution of better than 3 nm. With these new experimental and analytical developments, we have entered a new era for the SPI method and the path towards close-to-atomic resolution imaging of biomolecules is apparent

Anomalous temperature dependence of the experimental x-ray structure factor of supercooled water

The structural changes of water upon deep supercooling were studied through wide-angle x-ray scattering at SwissFEL. The experimental setup had a momentum transfer range of 4.5 angstrom(-1), which covered the principal doublet of the x-ray structure factor of water. The oxygen-oxygen structure factor was obtained for temperatures down to 228.5 +/- 0.6 K. Similar to previous studies, the second diffraction peak increased strongly in amplitude as the structural change accelerated toward a local tetrahedral structure upon deep supercooling. We also observed an anomalous trend for the second peak position of the oxygen-oxygen structure factor (q(2)). We found that q(2) exhibits an unprecedented positive partial derivative with respect to temperature for temperatures below 236 K. Based on Fourier inversion of our experimental data combined with reference data, we propose that the anomalous q(2) shift originates from that a repeat spacing in the tetrahedral network, associated with all peaks in the oxygen-oxygen pair-correlation function, gives rise to a less dense local ordering that resembles that of low-density amorphous ice. The findings are consistent with that liquid water consists of a pentamer-based hydrogen-bonded network with low density upon deep supercooling. (C) 2021 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).11Ysciescopu

Online dynamic flat-field correction for MHz microscopy data at European XFEL

The high pulse intensity and repetition rate of the European X-ray Free-Electron Laser (EuXFEL) provide superior temporal resolution compared with other X-ray sources. In combination with MHz X-ray microscopy techniques, it offers a unique opportunity to achieve superior contrast and spatial resolution in applications demanding high temporal resolution. In both live visualization and offline data analysis for microscopy experiments, baseline normalization is essential for further processing steps such as phase retrieval and modal decomposition. In addition, access to normalized projections during data acquisition can play an important role in decision-making and improve the quality of the data. However, the stochastic nature of X-ray free-electron laser sources hinders the use of standard flat-field normalization methods during MHz X-ray microscopy experiments. Here, an online (i.e. near real-time) dynamic flat-field correction method based on principal component analysis of dynamically evolving flat-field images is presented. The method is used for the normalization of individual X-ray projections and has been implemented as a near real-time analysis tool at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of EuXFEL

Enhancement and maximum in the isobaric specific-heat capacity measurements of deeply supercooled water using ultrafast calorimetry

Knowledge of the temperature dependence of the isobaric specific heat (Cp) upon deep supercooling can give insights regarding the anomalous properties of water. If a maximum in Cp exists at a specific temperature, as in the isothermal compressibility, it would further validate the liquid-liquid critical point model that can explain the anomalous increase in thermodynamic response functions. The challenge is that the relevant temperature range falls in the region where ice crystallization becomes rapid, which has previously excluded experiments. Here, we have utilized a methodology of ultrafast calorimetry by determining the temperature jump from femtosecond X-ray pulses after heating with an infrared laser pulse and with a sufficiently long time delay between the pulses to allow measurements at constant pressure. Evaporative cooling of ∼15-μm diameter droplets in vacuum enabled us to reach a temperature down to ∼228 K with a small fraction of the droplets remaining unfrozen. We observed a sharp increase in Cp, from 88 J/mol/K at 244 K to about 218 J/mol/K at 229 K where a maximum is seen. The Cp maximum is at a similar temperature as the maxima of the isothermal compressibility and correlation length. From the Cp measurement, we estimated the excess entropy and self-diffusion coefficient of water and these properties decrease rapidly below 235 K.QC 20220317</p

Unsupervised learning approaches to characterizing heterogeneous samples using X-ray single-particle imaging

One of the outstanding analytical problems in X-ray single-particle imaging (SPI) is the classification of structural heterogeneity, which is especially difficult given the low signal-to-noise ratios of individual patterns and the fact that even identical objects can yield patterns that vary greatly when orientation is taken into consideration. Proposed here are two methods which explicitly account for this orientation-induced variation and can robustly determine the structural landscape of a sample ensemble. The first, termed common-line principal component analysis (PCA), provides a rough classification which is essentially parameter free and can be run automatically on any SPI dataset. The second method, utilizing variation auto-encoders (VAEs), can generate 3D structures of the objects at any point in the structural landscape. Both these methods are implemented in combination with the noise-tolerant expand–maximize–compress (EMC) algorithm and its utility is demonstrated by applying it to an experimental dataset from gold nanoparticles with only a few thousand photons per pattern. Both discrete structural classes and continuous deformations are recovered. These developments diverge from previous approaches of extracting reproducible subsets of patterns from a dataset and open up the possibility of moving beyond the study of homogeneous sample sets to addressing open questions on topics such as nanocrystal growth and dynamics, as well as phase transitions which have not been externally triggered