Multi-scale time-resolved electron diffraction: A case study in moiré materials.

Ultrafast-optical-pump - structural-probe measurements, including ultrafast electron and x-ray scattering, provide direct experimental access to the fundamental timescales of atomic motion, and are thus foundational techniques for studying matter out of equilibrium. High-performance detectors are needed in scattering experiments to obtain maximum scientific value from every probe particle. We deploy a hybrid pixel array direct electron detector to perform ultrafast electron diffraction experiments on a WSe2/MoSe2 2D heterobilayer, resolving the weak features of diffuse scattering and moiré superlattice structure without saturating the zero order peak. Enabled by the detector's high frame rate, we show that a chopping technique provides diffraction difference images with signal-to-noise at the shot noise limit. Finally, we demonstrate that a fast detector frame rate coupled with a high repetition rate probe can provide continuous time resolution from femtoseconds to seconds, enabling us to perform a scanning ultrafast electron diffraction experiment that maps thermal transport in WSe2/MoSe2 and resolves distinct diffusion mechanisms in space and time.

Experimental 3D coherent diffractive imaging from photon-sparse random projections.

The routine atomic resolution structure determination of single particles is expected to have profound implications for probing structure-function relationships in systems ranging from energy-storage materials to biological molecules. Extremely bright ultrashort-pulse X-ray sources - X-ray free-electron lasers (XFELs) - provide X-rays that can be used to probe ensembles of nearly identical nanoscale particles. When combined with coherent diffractive imaging, these objects can be imaged; however, as the resolution of the images approaches the atomic scale, the measured data are increasingly difficult to obtain and, during an X-ray pulse, the number of photons incident on the 2D detector is much smaller than the number of pixels. This latter concern, the signal 'sparsity', materially impedes the application of the method. An experimental analog using a conventional X-ray source is demonstrated and yields signal levels comparable with those expected from single biomolecules illuminated by focused XFEL pulses. The analog experiment provides an invaluable cross check on the fidelity of the reconstructed data that is not available during XFEL experiments. Using these experimental data, it is established that a sparsity of order 1.3 × 10-3 photons per pixel per frame can be overcome, lending vital insight to the solution of the atomic resolution XFEL single-particle imaging problem by experimentally demonstrating 3D coherent diffractive imaging from photon-sparse random projections.

Intermittent plasticity in individual grains: A study using high energy x-ray diffraction.

Long-standing evidence suggests that plasticity in metals may proceed in an intermittent fashion. While the documentation of intermittency in plastically deforming materials has been achieved in several experimental settings, efforts to draw connections from dislocation motion and structure development to stress relaxation have been limited, especially in the bulk of deforming polycrystals. This work uses high energy x-ray diffraction measurements to build these links by characterizing plastic deformation events inside individual deforming grains in both the titanium alloy, Ti-7Al, and the magnesium alloy, AZ31. This analysis is performed by combining macroscopic stress relaxation data, complete grain stress states found using far-field high energy diffraction microscopy, and rapid x-ray diffraction spot measurements made using a Mixed-Mode Pixel Array Detector. Changes in the dislocation content within the deforming grains are monitored using the evolution of the full 3-D shapes of the diffraction spot intensity distributions in reciprocal space. The results for the Ti-7Al alloy show the presence of large stress fluctuations in contrast to AZ31, which shows a lesser degree of intermittent plastic flow.

Time-resolved x-ray diffraction techniques for bulk polycrystalline materials under dynamic loading.

We have developed two techniques for time-resolved x-ray diffraction from bulk polycrystalline materials during dynamic loading. In the first technique, we synchronize a fast detector with loading of samples at strain rates of ~10(3)-10(4) s(-1) in a compression Kolsky bar (split Hopkinson pressure bar) apparatus to obtain in situ diffraction patterns with exposures as short as 70 ns. This approach employs moderate x-ray energies (10-20 keV) and is well suited to weakly absorbing materials such as magnesium alloys. The second technique is useful for more strongly absorbing materials, and uses high-energy x-rays (86 keV) and a fast shutter synchronized with the Kolsky bar to produce short (~40 µs) pulses timed with the arrival of the strain pulse at the specimen, recording the diffraction pattern on a large-format amorphous silicon detector. For both techniques we present sample data demonstrating the ability of these techniques to characterize elastic strains and polycrystalline texture as a function of time during high-rate deformation.

A high-speed area detector for novel imaging techniques in a scanning transmission electron microscope.

A scanning transmission electron microscope (STEM) produces a convergent beam electron diffraction pattern at each position of a raster scan with a focused electron beam, but recording this information poses major challenges for gathering and storing such large data sets in a timely manner and with sufficient dynamic range. To investigate the crystalline structure of materials, a 16x16 analog pixel array detector (PAD) is used to replace the traditional detectors and retain the diffraction information at every STEM raster position. The PAD, unlike a charge-coupled device (CCD) or photomultiplier tube (PMT), directly images 120-200keV electrons with relatively little radiation damage, exhibits no afterglow and limits crosstalk between adjacent pixels. Traditional STEM imaging modes can still be performed by the PAD with a 1.1kHz frame rate, which allows post-acquisition control over imaging conditions and enables novel imaging techniques based on the retained crystalline information. Techniques for rapid, semi-automatic crystal grain segmentation with sub-nanometer resolution are described using cross-correlation, sub-region integration, and other post-processing methods.

Analog pixel array detectors.

X-ray pixel array detectors (PADs) are generally thought of as either digital photon counters (DPADs) or X-ray analog-integrating pixel array detectors (APADs). Experiences with APADs, which are especially well suited for X-ray imaging experiments where transient or high instantaneous flux events must be recorded, are reported. The design, characterization and experimental applications of several APAD designs developed at Cornell University are discussed. The simplest design is a ;flash' architecture, wherein successive integrated X-ray images, as short as several hundred nanoseconds in duration, are stored in the detector chips for later off-chip digitization. Radiography experiments using a prototype flash APAD are summarized. Another design has been implemented that combines flash capability with the ability to continuously stream X-ray images at slower (e.g. milliseconds) rates. Progress is described towards radiation-hardened APADs that can be tiled to cover a large area. A mixed-mode PAD, design by combining many of the attractive features of both APADs and DPADs, is also described.

Modeling liquid crystal bilayer structures with minimal surfaces.

This paper describes a new convenient and accurate method of calculating x-ray diffraction integrated intensities from detailed cubic bilayer structures. The method is employed to investigate the structure of a particular surfactant system (didodecyldimethylammonium bromide in a solution of oil and heavy water), for which single-crystal experimental data have recently been collected. The diffracted peak intensities correlate well with theoretical structures based on mathematical minimal surfaces. Optimized electron density profiles of the bilayer are presented, providing new insight into key features of the bilayer structure.

Time resolved collapse of a folding protein observed with small angle x-ray scattering.

High-intensity, "pink" beam from an undulator was used in conjunction with microfabricated rapid-fluid mixing devices to monitor the early events in protein folding with time resolved small angle x-ray scattering. This Letter describes recent work on the protein bovine beta-lactoglobulin where collapse from an expanded to a compact set of states was directly observed on the millisecond time scale. The role of chain collapse, one of the initial stages of protein folding, is not currently understood. The characterization of transient, compact states is vital in assessing the validity of theories and models of the folding process.

Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle x-ray scattering.

Time-resolved small-angle x-ray scattering was used to measure the radius of gyration of cytochrome c after initiation of folding by a pH jump. Submillisecond time resolution was obtained with a microfabricated diffusional mixer and synchrotron radiation. The results show that the protein first collapses to compact denatured structures before folding very fast to the native state.

A Pixel-Array Detector for Time-Resolved X-ray Diffraction.

An integrating pixel-array detector for recording time-resolved X-ray diffraction measurements on microsecond timescales has been designed and tested as a 4 x 4 pixel prototype. Operational characteristics and radiation tolerance are discussed. A 100 x 92 array with 151.2 micro m square pixels is currently under construction.

High-resolution macromolecular structure determination using CCD detectors and synchrotron radiation.

BACKGROUND: Synchrotron radiation sources have made impressive contributions to macromolecular crystallography. The delay in development of appropriate X-ray detectors has, however, been a significant limitation to their efficient use. New technologies, based on charge-coupled devices (CCDs), provide capabilities for faster, more accurate, automated data collection. RESULTS: A CCD-based X-ray detector has been developed for use in macromolecular crystallography and has been in operation for about one and a half years at the Cornell High Energy Synchrotron Source. It has been used for a variety of crystallographic projects, including a number of high-resolution structural studies. The statistical quality of the data, the detector's ease and efficiency of use, and the growing number of structural results illustrate the practical utility of this new detector system. CONCLUSIONS: The new detector has enhanced capabilities for measuring diffraction patterns from crystals of macromolecules, especially at high resolution, when the X-ray intensities are weak. The survey of results described here ranges from virus crystallography to weakly diffracting small-molecule structure determination and demonstrates the potential of CCD detectors when combined with synchrotron radiation sources.

Probability of alamethicin conductance states varies with nonlamellar tendency of bilayer phospholipids.

With few exceptions, membrane lipids are usually regarded as a kind of filler or passive solvent for membrane proteins. Yet, cells exquisitely control membrane composition. Many phospholipids found in plasma membrane bilayers favor packing into inverted hexagonal bulk phases. It was suggested that the strain of forcing such lipids into a bilayer may affect membrane protein function, such as the operation of transmembrane channels. To investigate this, we have inserted the peptide alamethicin into bilayer membranes composed of lipids of empirically determined inverted hexagonal phase "spontaneous radii" Ro, which will have expectably different degrees of strain when forced into bilayer form. We observe a correlation between measured Ro and the relative probabilities of different conductance states. States of higher conductance are more probable in dioleoylphosphatidylethanolamine, the lipid of highest curvature, 1/Ro, than in dioleoylphosphatidylcholine, the lipid of lowest curvature.

Curvature dependent induction of the interdigitated gel phase in DPPC vesicles.

Ethanol causes biphasic melting behavior in saturated lecithins (Rowe (1983) Biochemistry 22, 3299-3305), a consequence of the formation of the stable interdigitated phase (Simon, S.A. and McIntosh, T.J. (1984) Biochim. Biophys. Acta 773, 169-172). The membrane systems studied to date have been large vesicle systems in which the membrane surface can be assumed to be locally planar. An immediate question arises as to whether surfaces of higher curvature interdigitate. To address this question we have prepared DPPC vesicles of varying diameters which we employed to determine the limiting size at which interdigitation occurs using ethanol as the inducer. We find that with decreasing vesicle size the concentration of ethanol necessary for the onset of interdigitation increases. Small isolated vesicles, at inducing concentrations of ethanol, do not stably interdigitate but rupture and coalesce into a viscous gel comprised of interdigitated lipid sheets. As discussed elsewhere (Ahl et al. (1992) Biophys. J. 243a) these sheets can be used as precursors for producing liposomes of large size and high internal volumes useful in drug delivery or modeling applications.

Energetics of a hexagonal-lamellar-hexagonal-phase transition sequence in dioleoylphosphatidylethanolamine membranes.

The phase diagram of DOPE/water dispersions was investigated by NMR and X-ray diffraction in the water concentration range from 2 to 20 water molecules per lipid and in the temperature range from -5 to +50 degrees C. At temperatures above 22 degrees C, the dispersions form an inverse (HII) phase at all water concentrations. Below 25 degrees C, an HII phase occurs at high water concentrations, an L alpha phase is formed at intermediate water concentrations, and finally the system switches back to an HII phase at low water concentrations. The enthalpy of the L alpha-HII-phase transition is +0.3 kcal/mol as measured by differential scanning calorimetry. Using 31P and 2H NMR and X-ray diffraction, we measured the trapped water volumes in HII and L alpha phases as a function of osmotic pressure. The change of the HII-phase free energy as a function of hydration was calculated by integrating the osmotic pressure vs trapped water volume curve. The phase diagram calculated on the basis of the known enthalpy of transition and the osmotic pressure vs water volume curves is in good agreement with the measured one. The HII-L alpha-HII double-phase transition at temperatures below 22 degrees C can be shown to be a consequence of (i) the greater degree of hydration of the HII phase in excess water and (ii) the relative sensitivities with which the lamellar and hexagonal phases dehydrate with increasing osmotic pressure. These results demonstrate the usefulness of osmotic stress measurements to understand lipid-phase diagrams.

Kinetics of the lamellar-inverse hexagonal phase transition determined by time-resolved X-ray diffraction.

The kinetics of the lamellar (L alpha)-inverse hexagonal (HII) phase transition in diacylphosphatidylethanolamine (PE)--water systems were probed with time-resolved X-ray diffraction. Transition kinetics in the fast time regime (approximately 100 ms) were studied by initiating large temperature jumps (up to 30 degrees C) with a 50-ms electrical current pulse passed through a lipid-salt water dispersion, resulting in ohmic heating of the sample. Diffraction with a time resolution to 10 ms was acquired at the National Synchrotron Light Source. The time constant for the phase transition for 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was on the order of 100 ms for the largest temperature jumps recorded. Faster transition behavior was found for a 1,2-dielaidoyl-sn-glycero-3-PE mixture. The HII lattice parameters for both systems were seen to swell from an initial value commensurate with the lamellar lattice to the final equilibrium value. The rate of swelling was seen to be independent of the magnitude of the temperature jump. For small temperature jumps (less than 10 degrees C), the phase transition kinetics slow dramatically, and transition studies can readily be performed on a conventional rotating anode X-ray source. At 4 degrees C, a DOPE sample was observed to slowly convert to the hexagonal phase over the course of a week, with the decay in the lamellar intensity fitting a power law behavior over four decades of time. This power law behavior is shown to have interesting consequences to the determination of the phase transition temperature of lipid-water dispersions by conventional methods such as calorimetry.

Nonbilayer phases of membrane lipids.

Numerous liquid crystalline biomembrane lipids are known to exhibit non-lamellar phases characterized by curvature of their component lipid monolayers. An understanding of the phase stability of these systems begins with analysis of the energy of bending the monolayers, the interactions which lead to the bending energy, and the geometrical constraints which lead to competing energy terms which arise when the monolayers are bent and packed onto lattices with different structures. Diffraction and other techniques suitable for probing lipid phase structure are described. A phenomenological model is reviewed which successfully explains many of the qualitative features of lipid mesomorphic phase behavior. A key result of this model is that lipid bilayer compositions which are close to the non-lamellar phase boundaries of their phase diagrams are characterized by a frustrated elastic stress which may modulate the activity of imbedded membrane proteins and which may provide a rationale for the prevalence of non-lamellar-tending lipid species in biomembrane bilayers. Areas in need of future research are discussed.

Temperature dependence of the structural dimensions of the inverted hexagonal (HII) phase of phosphatidylethanolamine-containing membranes.

The characteristic temperature dependence of the lattice basis vector length d of phospholipid-water systems in the inverted hexagonal (HII) phase has been investigated with X-ray diffraction. For 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), d falls sharply from 78.1 A at 10 degrees C to 62.5 A at 90 degrees C. When used in conjunction with the volume fractions of the constituents, d can be used to determine the dimensions within the lipid and water regions. These data showed that a reduction in the radius of the HII-phase water cylinders Rw accounted for most of the reduction in d. From geometrical relationships between the dimensions in the HII phase, it was shown that both d and Rw are sensitive functions of the thickness of the lipid monolayer dHII. The characteristic shape of d(T) could be parameterized with the small temperature dependence of dHII along with the ratio v/a, which is the ratio of the specific volume to the area per lipid molecule at the polar interface. The ratio v/a was found to be independent of temperature for the fully hydrated HII system. Additional measurements made with a mixture of DOPE and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), mole ratio 5.07:1, produced a similar parameterization of d(T). The larger basis vector lengths for this mixture compared to those for DOPE can be attributed to a smaller ratio of v/a, which was also found to be temperature independent for this mixture. The smaller value of v/a is due to the larger effective headgroup area of DOPC.(ABSTRACT TRUNCATED AT 250 WORDS)

Unusual lipid structures selectively reduce the toxicity of amphotericin B.

Ribbon-like structures result when amphotericin B interacts with lipid in an aqueous environment. At high ratios of amphotericin to lipid these structures, which are lipid-stabilized amphotericin aggregates, become prevalent resulting in a dramatic attenuation of amphotericin-mediated mammalian cell, but not fungal cell, toxicity. Studies utilizing freeze-etch electron microscopy, differential scanning calorimetry, 31P NMR, x-ray diffraction, and optical spectroscopy revealed that this toxicity attenuation is related to the macromolecular structure of the complexes in a definable fashion. It is likely that amphotericin in this specific form will have a much improved therapeutic utility.

Observation of inverted cubic phase in hydrated dioleoylphosphatidylethanolamine membranes.

We report the observation of an inverted cubic phase in aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by small-angle X-ray diffraction. DOPE is a paradigm in the study of nonlamellar phases in biological systems: it exhibits a well-known phase transition from the lamellar (L alpha) to the inverted hexagonal phase (HII) as the temperature is raised. The transition is observed to occur rapidly when a DOPE dispersion is heated from 2 degrees C, where the L alpha phase is stable, to 15 degrees C, where the HII phase is stable. We report on the induction of a crystallographically well-defined cubic lattice that is slowly formed when the lipid dispersion is rapidly cycled between -5 and 15 degrees C hundreds of times. Once formed, the cubic lattice is stable at 4 degrees C for several weeks and exhibits the same remarkable metastability that characterizes other cubic phases in lipid-water systems. X-ray diffraction indicates that the cubic lattice is most consistent with either the Pn3m or Pn3 space group. Tests of lipid purity after induction of the cubic indicate the lipid is at least 98% pure. The cubic lattice can be destroyed and the system reset by cycling the specimen several times between -30 and 2 degrees C. The kinetics of the formation of the cubic are dependent on the thermal history of the sample, overall water concentration, and the extreme temperatures of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

X-ray diffraction study of the polymorphic behavior of N-methylated dioleoylphosphatidylethanolamine.

The polymorphic phase behavior of aqueous dispersions of dioleoylphosphatidylethanolamine (DOPE) and its N-methylated analogues, DOPE-Me, DOPE-Me2, and DOPC, has been investigated by X-ray diffraction. In the fully hydrated lamellar (L alpha) phase at 2 degrees C, the major structural difference is a large increase in the interlamellar water width from DOPE to DOPE-Me, with minor increases with successive methylation. Consistent with earlier reports, inverted hexagonal (HII) phases are observed upon heating at 5-10 degrees C in DOPE and at 65-75 degrees C in DOPE-Me and are not observed to at least 85 degrees C in DOPE-Me2 or DOPC. In DOPE, the L alpha-HII transition is facile and is characterized by a relatively narrow temperature range of coexistence of L alpha and HII domains, each with long-range order. DOPE-Me exhibits complex nonequilibrium behavior below the occurrence of the HII phase: Upon heating, the L alpha lattice spontaneously disorders on a time scale of days; on cooling from the HII phase, the disorder rises on a time scale of minutes. It is shown that, in copious water, the disordered state transforms very slowly into phases with cubic symmetry. This process is assisted by the generation of small amounts of lipid degradation products. The relative magnitudes of the monolayer spontaneous radius of curvature, R0 [Kirk, G. L., Gruner, S. M., & Stein, D. L. (1984) Biochemistry 23, 1093; Gruner, S. M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3665], are inferred from the HII lattice spacings vs temperature and are shown to increase with increasing methylation. The relative magnitudes of R0 are categorized as small for DOPE, intermediate for DOPE-Me, and large for DOPC. It is suggested, and examples are used to illustrate, that small R0 lipid systems exhibit facile, low-temperature L alpha-HII transitions, intermediate R0 systems exhibit complex nonequilibrium transition behavior and are likely to form cubic phases, and large R0 systems are stable as L alpha phases. The relationship between the cubic phases and minimal periodic surfaces is discussed. It is suggested that minimal periodic surfaces represent geometries in which near constant, intermediate R0 values can be obtained concomitantly with monolayers of near constant thickness, thereby leading to equilibrium cubic phases. Thus, the relative magnitude of the spontaneous radius of curvature may be used to predict mesomorphic behavior.(ABSTRACT TRUNCATED AT 400 WORDS)