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The outstanding opto-electronic properties of lead halide perovskites have been related to the formation of polarons. Nevertheless, the observation of the atomistic deformation brought about by one electron-hole pair in these materials has remained elusive. Here, we measure the diffraction patterns of single CsPbBr$_3$ quantum dots (QDs) with and without resonant excitation in the single exciton limit using serial femtosecond crystallography (SFX). By reconstructing the 3D differential diffraction pattern, we observe small shifts of the Bragg peaks indicative of a crystal-wide deformation field. Building on DFT calculations, we show that these shifts are consistent with the lattice distortion induced by a delocalized electron and a localized hole, forming a mixed large/small exciton polaron. This result creates a clear picture of the polaronic deformation in CsPbBr$_3$ QDs, highlights the exceptional sensitivity of SFX to lattice distortions in few-nanometer crystallites, and establishes an experimental platform for future studies of electron-lattice interactions., Comment: Main: 12 pages, 5 figures; Supplemental: 21 pages, 11 figures

Coherent Diffraction Imaging (CDI) is an experimental technique to gain images of isolated structures by recording the light scattered off the sample. In principle, the sample density can be recovered from the scattered light field through a straightforward Fourier Transform operation. However, only the amplitude of the field is recorded, while the phase is lost during the measurement process and has to be retrieved by means of suitable, well-established, phase retrieval algorithms. In this work we present SPRING, an analysis framework tailored on X-ray Free Electron Laser (XFEL) diffraction data that implements the Memetic Phase Retrieval method to mitigate the shortcomings of conventional algorithms. We benchmark the approach on experimental data acquired in two experimental campaigns at SwissFEL and European XFEL. Imaging results on isolated nanostructures reveal unprecedented stability and resilience of the algorithm's behavior on the input parameters, as well as the capability of identifying the solution in conditions hardly treatable so far with conventional methods. A user-friendly implementation of SPRING is released as open-source software, aiming at being a reference tool for the coherent diffraction imaging community at XFEL and synchrotron facilities., Comment: 30 pages, 13 figures. Authors list updated

Single-stranded RNA viruses co-assemble their capsid with the genome and variations in capsid structures can have significant functional relevance. In particular, viruses need to respond to a dehydrating environment to prevent genomic degradation and remain active upon rehydration. Theoretical work has predicted low-energy buckling transitions in icosahedral capsids which could protect the virus from further dehydration. However, there has been no direct experimental evidence, nor molecular mechanism, for such behaviour. Here we observe this transition using X-ray single particle imaging of MS2 bacteriophages after aerosolization. Using a combination of machine learning tools, we classify hundreds of thousands of single particle diffraction patterns to learn the structural landscape of the capsid morphology as a function of time spent in the aerosol phase. We found a previously unreported compact conformation as well as intermediate structures which suggest an incoherent buckling transition which does not preserve icosahedral symmetry. Finally, we propose a mechanism of this buckling, where a single 19-residue loop is destabilised, leading to the large observed morphology change. Our results provide experimental evidence for a mechanism by which viral capsids protect themselves from dehydration. In the process, these findings also demonstrate the power of single particle X-ray imaging and machine learning methods in studying biomolecular structural dynamics., Comment: 10 pages, 4 figures plus 9 pages supplementary information

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 unprecedented 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 $10^5$ structural models and $10^6$ XFEL-SPI measurements. Here, we describe how we overcame these three challenges to resolve the non-equilibrium 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' amongst 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.

This paper introduces Spectral Incoherent Diffractive Imaging (SIDI) as a novel method for achieving dark-field imaging of nanostructures with heterogeneous oxidation states. With SIDI, shifts in photoemission profiles can be spatially resolved, enabling the independent imaging of the underlying emitter distributions contributing to each spectral line. In the X-ray domain, this approach offers unique insights beyond the conventional combination of diffraction and X-ray Emission Spectroscopy (XES). When applied at X-ray Free-Electron Lasers (XFELs), SIDI promises to be a versatile tool for investigating a broad range of systems, offering unprecedented opportunities for detailed characterization of heterogeneous nanostructures for catalysis and energy storage, including of their ultrafast dynamics., Comment: 6 pages, 4 figures

Single particle imaging (SPI) at X-ray free electron lasers (XFELs) is a technique to determine the 3D structure of nanoscale objects like biomolecules from a large number of diffraction patterns of copies of these objects in random orientations. Millions of low signal-to-noise diffraction patterns with unknown orientation are collected during an X-ray SPI experiment. The patterns are then analyzed and merged using a reconstruction algorithm to retrieve the full 3D-structure of particle. The resolution of reconstruction is limited by background noise, signal-to-noise ratio in diffraction patterns and total amount of data collected. We recently introduced a reference-enhanced holographic single particle imaging methodology [Optica 7,593-601(2020)] to collect high enough signal-to-noise and background tolerant patterns and a reconstruction algorithm to recover missing parameters beyond orientation and then directly retrieve the full Fourier model of the sample of interest. Here we describe a phase retrieval algorithm based on maximum likelihood estimation using pattern search dubbed as MaxLP, with better scalability for fine sampling of latent parameters and much better performance in the low signal limit. Furthermore, we show that structural variations within the target particle are averaged in real space, significantly improving robustness to conformational heterogeneity in comparison to conventional SPI. With these computational improvements, we believe reference-enhanced SPI is capable of reaching sub-nm resolution biomolecule imaging., Comment: 9 pages, 6 figures

Second-order intensity correlations from incoherent emitters can reveal the Fourier transform modulus of their spatial distribution, but retrieving the phase to enable completely general Fourier inversion to real space remains challenging. Phase retrieval via the third-order intensity correlations has relied on special emitter configurations which simplified an unaddressed sign problem in the computation. Without a complete treatment of this sign problem, the general case of retrieving the Fourier phase from a truly arbitrary configuration of emitters is not possible. In this paper, a general method for ab initio phase retrieval via the intensity triple correlations is described. Simulations demonstrate accurate phase retrieval for clusters of incoherent emitters which could be applied to imaging stars or fluorescent atoms and molecules. With this work, it is now finally tractable to perform Fourier inversion directly and reconstruct images of arbitrary arrays of independent emitters via far-field intensity correlations alone.

Multilayer Laue lenses were used for the first time to focus x-rays from an X-ray Free Electron Laser (XFEL). In an experiment, which was performed at the European XFEL, we demonstrated focusing to a spot size of a few tens of nanometers. A series of runs in which the number of pulses per train was increased from 1 to 2, 3, 4, 5, 6, 7, 10, 20 and 30 pulses per train, all with a pulse separation of 3.55 us, was done using the same set of lenses. The increase in the number of pulses per train was accompanied with an increase of x-ray intensity (transmission) from 9% to 92% at 5 pulses per train, and then the transmission was reduced to 23.5 % when the pulses were increased further. The final working condition was 30 pulses per train and 23.5% transmission. Only at this condition we saw that the diffraction efficiency of the MLLs changed over the course of a pulse train, and this variation was reproducible from train to train. We present the procedure to align and characterize these lenses and discuss challenges working with the pulse trains from this unique x-ray source.

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 that even identical objects can yield patterns that vary greatly when orientation is taken into consideration. We propose 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. We implement both these methods in combination with the noise-tolerant expand-maximize-compress (EMC) algorithm and demonstrate its utility by applying it to an experimental dataset from gold nanoparticles with only a few thousand photons per pattern and recover both discrete structural classes as well as continuous deformations. These developments diverge from previous approaches of extracting reproducible subsets of patterns from a dataset and open up the possibility to move beyond studying homogeneous sample sets and study open questions on topics such as nanocrystal growth and dynamics as well as phase transitions which have not been externally triggered., Comment: 29 pages, 9 figures

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 yield good agreement with particle-trajectory simulations.

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., Comment: 25 pages, 5 main figures, 6 supplementary figures, 2 supplementary movies (link in document)

We propose an encryption-decryption framework for validating diffraction intensity volumes reconstructed using single-particle imaging (SPI) with x-ray free-electron lasers (XFELs) when the ground truth volume is absent. This framework exploits each reconstructed volumes' ability to decipher latent variables (e.g. orientations) of unseen sentinel diffraction patterns. Using this framework, we quantify novel measures of orientation disconcurrence, inconsistency, and disagreement between the decryptions by two independently reconstructed volumes. We also study how these measures can be used to define data sufficiency and its relation to spatial resolution, and the practical consequences of focusing XFEL pulses to smaller foci. This framework overcomes critical ambiguities in using Fourier Shell Correlation (FSC) as a validation measure for SPI. Finally, we show how this encryption-decryption framework naturally leads to an information-theoretic reformulation of the resolving power of XFEL-SPI, which we hope will lead to principled frameworks for experiment and instrument design.

Intensity interferometry is a well known method in astronomy. Recently, a related method called incoherent diffractive imaging (IDI) was proposed to apply intensity correlations of x-ray fluorescence radiation to determine the 3D arrangement of the emitting atoms in a sample. Here we discuss inherent sources of noise affecting IDI and derive a model to estimate the dependence of the signal to noise ratio (SNR) on the photon counts per pixel, the temporal coherence (or number of modes), and the shape of the imaged object. Simulations in two- and three-dimensions have been performed to validate the predictions of the model. We find that contrary to coherent imaging methods, higher intensities and higher detected counts do not always correspond to a larger SNR. Also, larger and more complex objects generally yield a poorer SNR despite the higher measured counts. The framework developed here should be a valuable guide to future experimental design.

X-ray single particle imaging involves the measurement of a large number of noisy diffraction patterns of isolated objects in random orientations. The missing information about these patterns is then computationally recovered in order to obtain a three-dimensional structure of the particle. While the method has promised to deliver room temperature structures at near-atomic resolution, there have been significant experimental hurdles in collecting data of sufficient quality and quantity to achieve this goal. This paper describes two ways to modify the conventional methodology which significantly ease the experimental challenges, at the cost of additional computational complexity in the reconstruction procedure. Both these methods involve the use of holographic reference objects in close proximity to the sample of interest, whose structure can be described with only a few parameters. A reconstruction algorithm to recover the unknown degrees of freedom is also proposed and tested with toy-model simulations., Comment: 9 pages, 5 figures

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 will soon provide 27,000 pulses per second, more than 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 obtained results pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate.

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., Comment: 19 pages, 9 figures

Established x-ray diffraction methods allow for high-resolution structure determination of crystals, crystallized protein structures or even single molecules. While these techniques rely on coherent scattering, incoherent processes like Compton scattering or fluorescence emission -- often the predominant scattering mechanisms -- are generally considered detrimental for imaging applications. Here we show that intensity correlations of incoherently scattered x-ray radiation can be used to image the full 3D structure of the scattering atoms with significantly higher resolution compared to conventional coherent diffraction imaging and crystallography, including additional three-dimensional information in Fourier space for a single sample orientation. We present a number of properties of incoherent diffractive imaging that are conceptually superior to those of coherent methods., Comment: 2 figures

The diffraction pattern of a single non-periodic compact object, such as a molecule, is continuous and is proportional to the square modulus of the Fourier transform of that object. When arrayed in a crystal, the coherent sum of the continuous diffracted wave-fields from all objects gives rise to strong Bragg peaks that modulate the single-object transform. Wilson statistics describe the distribution of continuous diffraction intensities to the same extent that they apply to Bragg diffraction. The continuous diffraction obtained from translationally-disordered molecular crystals consists of the incoherent sum of the wave-fields from the individual rigid units (such as molecules) in the crystal, which is proportional to the incoherent sum of the diffraction from the rigid units in each of their crystallographic orientations. This sum over orientations modifies the statistics in a similar way that crystal twinning modifies the distribution of Bragg intensities. These statistics are applied to determine parameters of continuous diffraction such as its scaling, the beam coherence, and the number of independent wave-fields or object orientations contributing. Continuous diffraction is generally much weaker than Bragg diffraction and may be accompanied by a background that far exceeds the strength of the signal. Instead of just relying upon the smallest measured intensities to guide the subtraction of the background it is shown how all measured values can be utilised to estimate the background, noise, and signal, by employing a modified "noisy Wilson" distribution that explicitly includes the background. Parameters relating to the background and signal quantities can be estimated from the moments of the measured intensities. The analysis method is demonstrated on previously-published continuous diffraction data measured from imperfect crystals of photosystem II., Comment: 34 pages, 11 figures, 2 appendices

Schemes for X-ray imaging single protein molecules using new x-ray sources, like x-ray free electron lasers (XFELs), require processing many frames of data that are obtained by taking temporally short snapshots of identical molecules, each with a random and unknown orientation. Due to the small size of the molecules and short exposure times, average signal levels of much less than 1 photon/pixel/frame are expected, much too low to be processed using standard methods. One approach to process the data is to use statistical methods developed in the EMC algorithm (Loh & Elser, Phys. Rev. E, 2009) which processes the data set as a whole. In this paper we apply this method to a real-space tomographic reconstruction using sparse frames of data (below $10^{-2}$ photons/pixel/frame) obtained by performing x-ray transmission measurements of a low-contrast, randomly-oriented object. This extends the work by Philipp et al. (Optics Express, 2012) to three dimensions and is one step closer to the single molecule reconstruction problem., Comment: 10 pages, 5 figures

Single-particle imaging experiments of biomolecules at x-ray free-electron lasers (XFELs) require processing of hundreds of thousands (or more) of images that contain very few x-rays. Each low-flux image of the diffraction pattern is produced by a single, randomly oriented particle, such as a protein. We demonstrate the feasibility of collecting data at these extremes, averaging only 2.5 photons per frame, where it seems doubtful there could be information about the state of rotation, let alone the image contrast. This is accomplished with an expectation maximization algorithm that processes the low-flux data in aggregate, and without any prior knowledge of the object or its orientation. The versatility of the method promises, more generally, to redefine what measurement scenarios can provide useful signal in the high-noise regime.