X-ray reflectivity measurement of interdiffusion in metallic multilayers during rapid heating.

A technique for measuring interdiffusion in multilayer materials during rapid heating using X-ray reflectivity is described. In this technique the sample is bent to achieve a range of incident angles simultaneously, and the scattered intensity is recorded on a fast high-dynamic-range mixed-mode pixel array detector. Heating of the multilayer is achieved by electrical resistive heating of the silicon substrate, monitored by an infrared pyrometer. As an example, reflectivity data from Al/Ni heated at rates up to 200 K s-1 are presented. At short times the interdiffusion coefficient can be determined from the rate of decay of the reflectivity peaks, and it is shown that the activation energy for interdiffusion is consistent with a grain boundary diffusion mechanism. At longer times the simple analysis no longer applies because the evolution of the reflectivity pattern is complicated by other processes, such as nucleation and growth of intermetallic phases.

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.

Time-resolved x-ray diffraction of biological materials.

Instrumental and specimen considerations pertinent to performing time-resolved x-ray diffraction on biological materials are discussed. Existing synchrotron x-ray sources, in conjunction with integrating x-ray detectors, have made millisecond diffraction experiments feasible; exposure times several orders of magnitude shorter than this will be possible with synchrotron sources now on the drawing boards. Experience gained from time-resolved studies together with order-of-magnitude estimates of specimen requirements can be used to determine the instrumental capabilities needed for various time-resolved experiments. Existing instrumental capabilities and methods of dealing with time-resolved specimens are reviewed.

Role of lipid polymorphism in pulmonary surfactant.

The development of artificial surfactants for the treatment of respiratory distress syndrome (RDS) requires lipid systems that can spread rapidly from solution to the air-water interface. Because hydration-repulsion forces stabilize liposomal bilayers and oppose spreading, liposome systems that undergo geometric rearrangement from the bilayer (lamellar) phase to the hexagonal II (HII) phase could hasten lipid transfer to the air-water interface through unstable transition intermediates. A liposome system containing dipalmitoylphosphatidylcholine was designed; the system is stable at 23 degrees C but undergoes transformation to the HII phase as the temperature increases to 37 degrees C. The spreading of lipid from this system to the air-water interface was rapid at 37 degrees C but slow at 23 degrees C. When tested in vivo in a neonatal rabbit model, such systems elicited an onset of action equal to that of native human surfactant. These findings suggest that lipid polymorphic phase behavior may have a crucial role in the effective functioning of pulmonary surfactant.

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.

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.

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.

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.

X-ray diffraction demonstrates that phosphatidyldiacylglycerol and phosphatidylcholesterol are not lamellar above the main transition temperature.

X-ray diffraction was used to investigate the lattice structure of aqueous dispersions of two phosphatidyldiacylglycerols and of a phosphatidylcholesterol above and below the chain melting transition temperature. Previously, Noggle et al. (Biochim. Biophys. Acta (1982) 691, 240-248) had investigated these lipids and had concluded on the basis of electron microscopy that the lipids were in a lamellar state above the transition temperature. However, they found the 31P-NMR signals were not characteristic of lamellar phases. It was, therefore, concluded that these lipids were yielding unexpected 31P-NMR spectra. The present X-ray results demonstrate that, in fact, the lipids are not in a lamellar state above the transition temperature and that the 31P-NMR and X-ray data are not necessarily in disagreement. Characteristics of the phases both above and below the chain melt temperature are discussed.

A 12 A resolution X-ray diffraction study of the profile structure of isolated bovine retinal rod outer segment disk membranes.

Electron density profiles of disk membranes isolated from bovine retinal rod outer segments have been determined to 12 A resolution by analysis of the X-ray diffraction from oriented multilayers, in the absence of lipid phase separation. Data were collected on both film and a two-dimensional TV-detector; both detectors yielded identical patterns consisting of relatively sharp lamellar reflections of small mosaic spread. The unit cell repeat was reversibly varied over the range of 143 to 183 A. The diffraction patterns changed dramatically at 150 A; consequently, the low (less than 150 A) and high (greater than 150 A) periodicity data were independently analyzed via a swelling algorithm. The high periodicity data yielded two statistically equivalent phase choices corresponding to two symmetric, but different membrane profiles. The low periodicity data yielded essentially one, characteristically asymmetric profile. These profiles have been modeled with regard to the separate profiles of rhodopsin, lipid and water, subject to the known composition of the isolated disk membranes.

Small concentrations of alamethicin induce a cubic phase in bulk phosphatidylethanolamine mixtures.

Under normal conditions, excess water dispersions of liquid crystalline 1,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (DEPE) are known to convert from a liquid crystalline lamellar (L alpha) to inverse hexagonal (HII) phase at about 60 degrees Celsius. The nonlamellar phase behavior of lipid systems is also known to depend on the monolayer spontaneous curvature. The single-channel activity of alamethicin in black lipid bilayer membranes has been shown to be dependent upon the lipid composition of the membrane. Since the monolayer spontaneous curvature properties (e.g., the monolayer spontaneous curvature, curvature coefficients and bilayer thickness) vary with lipid composition, the single-channel activity of alamethicin presumably also correlates with the monolayer spontaneous curvature properties. Accordingly, we reasoned that if alamethicin couples to the curvature properties of a lipid film, then the curvature properties must, in turn, be perturbed by the presence of alamethicin and that this perturbation should be observable in the lipid phase behavior. Here X-ray diffraction and NMR are used to show that the presence of as little as 1% alamethicin introduces a large region of cubic phase into the thermal phase diagram. This suggests that perturbation of the nonlamellar phase behavior of a lipid system may be a method to survey different channel-forming molecules for possible behavior that indicates that the ion channel is sensitive to the monolayer spontaneous curvature properties.

Lipid extracts from membranes of Acholeplasma laidlawii A grown with different fatty acids have a nearly constant spontaneous curvature.

X-ray diffraction methods were used to explore the variation in the spontaneous curvature of lipid extracts from Acholeplasma laidlawii strain A-EF22 grown with different mixtures of palmitic acid and oleic acid. It was shown that the cells respond to the different growing conditions by altering the polar head group compositions in order to keep the phase transition between lamellar and nonlamellar structures within a narrow temperature range. This has been interpreted to mean that the membrane lipids are adjusted toward an optimal packing (Lindblom et al. (1986) Biochemistry 25, 7502). Here it is shown that for these extracts, the membrane curvature is kept within a narrow range (58-73 A), compared to the range in curvatures exhibited by pure lipids extracts from the membrane (17-123 A). These observations support the hypothesis (Gruner (1989) J. Phys. Chem. 93, 7562) that the spontaneous curvature is a functionally important membrane parameter which is regulated by the organism and is likely to be one of the constraints controlling the lipid composition of the bilayer.

X-ray diffraction analysis of wet isolated bovine rod outer segment disks. A dehydration study.

Sequences of X-ray diffraction patterns were obtained from dehydrating, artificially oriented multilayers of isolated, bovine rod outer segment disks. A direct-phase analysis was applied to highly hydrated specimens to determine sequences of low resolution (approx. 30 A) electron density profiles of the disks as dehydration proceeded. The profiles were found to evolve smoothly as the multilayer lattice simultaneously shrank and became increasingly ordered. The bilayer profiles were largely invariant under dehydration and the evolution of the diffraction consistent with simple decreases in fluid spacings. The specimens were observed to phase separate into characteristic primary and a secondary lattices when the multi-layer became too dehydrated. The small unit cell size of the secondary lattice was suggestive of a lipid phase. Large changes in the diffraction patterns from phase separated specimens were observed upon bleaching of the specimen. The changes were consistent with a reversible disordering of the primary lattice.

Thermotropic characterization of the 2-O-acyl,polyprenyl alpha-D-glucopyranoside isolated from palmitate-enriched Acholeplasma laidlawii B membranes.

The thermotropic phase behavior of a monoacylated neutral glucolipid (2-O-acyl,polyprenyl alpha-D-glucopyranoside), isolated from palmitate-enriched Acholeplasma laidlawii B membranes, was studied by differential scanning calorimetry, infrared spectroscopy and X-ray diffraction. When equilibrated at low temperatures, aqueous dispersions of this lipid form an ordered, crystal-like lamellar gel phase which transforms to an inverted hexagonal phase at temperatures near 65 degrees C upon heating. However, upon cooling from high temperatures, the inverted hexagonal phase remains stable down to temperatures near 45 degrees C. Further cooling first results in the formation of a metastable lamellar liquid crystalline phase at temperatures near 35 degrees C and then a metastable gel phase at lower temperatures. The metastable gel phase, if immediately reheated at a fast scan rate, undergoes a gel/liquid-crystalline phase transition at temperatures near 33 degrees C. These results indicate that this monoacylated glucolipid exhibits its gel/liquid-crystalline phase transition and its lamellar/non-lamellar phase transition at considerably lower temperatures than does the monoglycosyldiacylglycerol formed under the same conditions. When cultured in media enriched in 'high-melting' fatty acids, Acholeplasma laidlawii B synthesizes large quantities of the 2-O-acyl,polyprenyl alpha-D-glucopyranoside (up to 60 mol%) mainly at the expense of the monoglucosyldiacylglycerol (the only other nonbilayer-forming liquid normally found in the cell membrane of this organism). We thus suggest that the biosynthesis of this novel glucolipid, in response to the biosynthetic incorporation of high-melting exogenous fatty acids, is an adaptive response designed to maintain a predominantly liquid-crystalline membrane lipid bilayer at the growth temperature, while retaining the high proportion of nonbilayer-forming glucolipid species characteristic of A. laidlawii B cells cultured under these conditions.

Phase properties of the aqueous dispersions of n-octadecylphosphocholine.

Properties of the aqueous dispersions of n-octadecylphosphocholine are examined by differential scanning calorimetry, fluorescence depolarization, light scattering, 31P-NMR, pig pancreatic phospholipase A2 binding, and X-ray diffraction. On heating, these dispersions exhibit a sharp lamellar to micelle transition at 20.5 degrees C. The lamellar phase consists of frozen (gel-state) alkyl chains which do not bind phospholipase A2. The kinetics of the transition are asymmetric: the micelle to lamellar transition is very slow and the lamellar to micelle transition is fast. It is suggested that the lamellar phase is a frozen chain bilayer in which the chains interdigitate.

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.

Characterization of cholesterol hemisuccinate and alpha-tocopherol hemisuccinate vesicles.

Cholesterol hemisuccinate (CHS) and alpha-tocopherol hemisuccinate (alpha-THS) were found to be capable of forming liposomes of multi- or single lamellar character. Such vesicles formed spontaneously, did not require the use of organic solvents and yielded high trapping efficiencies and captured volumes. Both CHS and alpha-THS systems greatly restricted the motion of intercalated spin labelled fatty acids, yet were more osmotically responsive than similar vesicle types comprised of phosphatidylcholine. Small angle X-ray diffraction measurements were consistent with vesicles possessing extremely weak interlamellar forces. CHS vesicles were found to remain intact in vivo, yet followed a pattern of distribution dissimilar to phosphatidylcholine vesicles.

Solute-induced shift of phase transition temperature in Di-saturated PC liposomes: adoption of ripple phase creates osmotic stress.

We have examined the calorimetric behavior of large liposomes consisting of symmetric saturated chain phosphatidylcholines. Most notably, for systems made in solutions containing solute (e.g., NaCl, glucose, etc.) there was an additional major endotherm just below the main phase transition temperature. The new endotherm was found to represent a population of lipid whose main phase transition was shifted to lower temperature due to an induced osmotic stress across the membrane. Absent for isoosmotic systems, the osmotic stress was created when the liposome internal volume decreased, a consequence of the Lbeta' (gel) to Pbeta' (rippled) phase transition. That is, rippling of the membrane caused vesicle volume to decrease (> or = 28%) and because the free flow of water outward was restricted by solute, an osmotic gradient was created where none had existed before. The distribution of enthalpy between the new shifted Tm and the expected Tm correlated with the percent of lipid in the outer bilayer and it was concluded that only the outer bilayer sensed the induced stress. Internalized liposome structures were shielded, thus explaining the persistence of the expected Tm in preparations made in solute. The shift in Tm (deltaTm) was discrete and linearly dependent upon lipid chain length for the PC series di-17:0 (deltaTm approximately 1.4 degrees C) through di-20:0 (deltaTm approximately 0.6 degrees C), suggesting a structural change (i.e., lipid packing/orientation) was involved. Although freeze-fracture electron microscopy of stressed and unstressed bilayers revealed no differences in ripple periodicity there were differences in surface features and in vesicle shape. The fact that this phenomenon has gone unnoticed for MLVs is probably due to the fact that these systems are known to exclude solute and thus exist under osmotic compression.

Doxorubicin physical state in solution and inside liposomes loaded via a pH gradient.

We have examined doxorubicin's (DOX) physical state in solution and inside EPC/cholesterol liposomes that were loaded via a transmembrane pH gradient. Using cryogenic electron microscopy (cryo-EM) we noted that DOX loaded to 200-300 mM internal concentrations in citrate containing liposomes formed linear, curved, and circular bundles of fibers with no significant interaction/perturbation of the vesicle membrane. The individual DOX fibers are putatively comprised of stacked DOX molecules. From end-on views of bundles of fibers it appeared that they are aligned longitudinally in a hexagonal array with a separation between fibers of approx. 3-3.5 nm. Two distinct small angle X-ray diffraction patterns (oblique and simple hexagonal) were observed for DOX-citrate fiber aggregates that had been concentrated from solution at either pH 4 or 5. The doxorubicin fibers were also present in citrate liposomes loaded with only one-tenth the amount of doxorubicin used above (approx. 20 mM internal DOX concentration) indicating that the threshold concentration at which these structures form is relatively low. In fact, from cryo-EM and circular dichroism spectra, we estimate that the DOX-citrate fiber bundles can account for the vast majority (>99%) of DOX loaded via a pH gradient into citrate buffered liposomes. DOX loaded into liposomes containing lactobionic acid (LBA), a monoanionic buffer to control the internal pH, remained disaggregated at internal DOX concentrations of approx. 20 mM but formed uncondensed fibers (no bundles) when the internal DOX concentration was approx. 200 mM. This finding suggests that in the citrate containing liposomes the citrate multianion electrostatically bridged adjacent fibers to form the observed bundles. 13C-NMR measurements of [1,5-13C]citrate inside liposomes suggested that citrate 'bound' to the DOX complex and 'free' citrate rapidly exchange indicating that the citrate-DOX interaction is quite dynamic. DOX release into buffer was relatively slow (<4% at 1 h) from liposomes containing DOX fibers (in citrate loaded to a low or high DOX concentration or in LBA liposomes loaded to a high internal DOX concentration). LBA containing liposomes loaded with disaggregated DOX, where the internal DOX concentration was only approx. 20 mM, experienced an osmotic stress induced vesicle rupture with as much as 18% DOX leakage in less than 10 min. The possible implications for this in vivo are discussed.

Is the mechanism of general anesthesia related to lipid membrane spontaneous curvature?

Lipid bilayers in biomembranes may exist in a state of elastic curvature stress which may couple to the conformation of integral membrane proteins in a logical, and energetically significant, fashion. Many biomembranes contain sufficiently large fractions of nonlamellar-prone lipids to have monolayers under substantial curvature stress. Although very few experiments have been performed that can be used to correlate protein activity with curvature stress, the literature does contain a small number of studies that indicate that some protein function is nonspecifically modulated by the amounts of nonlamellar-prone lipid in the imbedding bilayers. The spontaneous curvature, is altered by the presence of anesthetics in physiologically relevant concentrations. This leads us to suggest that anesthetic action may be coupled to protein function via alteration of the tensions leading to the spontaneous curvature of biomembrane layers. The spontaneous curvature is also sufficiently sensitive to pressure that a mechanism for the pressure reversal of anesthesia follows if the effects of pressure are to counter changes in membrane lateral tension induced by anesthetics. It is emphasized that many more experimental data must be acquired to determine whether the ideas presented in this paper have validity. In particular, there is a need for data on the effects of different anesthetics and pressure on the spontaneous curvature and, more generally, lipid monolayer lateral tensions. Most importantly, experiments must be performed to investigate whether protein function correlates with these quantities.