Claudiu A. Stan

Taking snapshots of photosynthetic water oxidation using femtosecond X-ray diffraction and spectroscopy

The dioxygen we breathe is formed from water by its light-induced oxidation in photosystem II. O2 formation takes place at a catalytic manganese cluster within milliseconds after the photosystem II reaction center is excited by three single-turnover flashes. Here we present combined X-ray emission spectra and diffraction data of 2 flash (2F) and 3 flash (3F) photosystem II samples, and of a transient 3F′ state (250 μs after the third flash), collected under functional conditions using an X-ray free electron laser. The spectra show that the initial O-O bond formation, coupled to Mn-reduction, does not yet occur within 250 μs after the third flash. Diffraction data of all states studied exhibit an anomalous scattering signal from Mn but show no significant structural changes at the present resolution of 4.5 Å. This study represents the initial frames in a molecular movie of the structural changes during the catalytic reaction in photosystem II.

Structure of photosystem II and substrate binding at room temperature.

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O–O bond formation and O2 evolution. A detailed understanding of the O–O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O–O bond formation mechanisms.

Independent Control of Drop Size and Velocity in Microfluidic Flow-Focusing Generators Using Variable Temperature and Flow Rate

This paper describes a method to control the volume and the velocity of drops generated in a flow-focusing device dynamically and independently. This method involves simultaneous tuning of the temperature of the nozzle of the device and of the flow rate of the continuous phase; the method requires a continuous phase liquid that has a viscosity that varies steeply with temperature. Increasing the temperature of the flow-focusing nozzle from 0 to 80 degrees C increased the volume of the drops by almost 2 orders of magnitude. Tuning both the temperature and the flow rate controlled the drop volume and the drop velocity independently; this feature is not possible in a basic flow-focusing device. This paper also demonstrates a procedure for identifying the range of possible drop volumes and drop velocities for a given flow-focusing device and shows how to generate drops with a specified volume and velocity within this range. This method is easy to implement in on-chip applications where thermal management is already incorporated in the system, such as DNA amplification using the polymerase chain reaction and nanoparticle synthesis.

Externally applied electric fields up to 1.6 × 10(5) V/m do not affect the homogeneous nucleation of ice in supercooled water.

The freezing of water can initiate at electrically conducting electrodes kept at a high electric potential or at charged electrically insulating surfaces. The microscopic mechanisms of these phenomena are unknown, but they must involve interactions between water molecules and electric fields. This paper investigates the effect of uniform electric fields on the homogeneous nucleation of ice in supercooled water. Electric fields were applied across drops of water immersed in a perfluorinated liquid using a parallel-plate capacitor; the drops traveled in a microchannel and were supercooled until they froze due to the homogeneous nucleation of ice. The distribution of freezing temperatures of drops depended on the rate of nucleation of ice, and the sensitivity of measurements allowed detection of changes by a factor of 1.5 in the rate of nucleation. Sinusoidal alternation of the electric field at frequencies from 3 to 100 kHz prevented free ions present in water from screening the electric field in the bulk of drops. Uniform electric fields in water with amplitudes up to (1.6 ± 0.4) × 10(5) V/m neither enhanced nor suppressed the homogeneous nucleation of ice. Estimations based on thermodynamic models suggest that fields in the range of 10(7)-10(8) V/m might cause an observable increase in the rate of nucleation.

Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II

We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

A simple two-dimensional model system to study electrostatic-self-assembly

This paper surveys the variables controlling the lattice structure and charge in macroscopic Coulombic crystals made from electrically charged, millimeter-sized polymer objects (spheres, cubes, and cylinders). Mechanical agitation of these objects inside planar, bounded containers caused them to charge electrically through contact electrification, and to self-assemble. The processes of electrification and self-assembly, and the characteristics of the assemblies, depended on the type of motion used for agitation, on the type of materials used for the objects and the dish, on the size and shape of the objects and the dish, and on the number of objects. Each of the three different materials in the system (of the dish and of the two types of spheres) influenced the electrification. Three classes of structures formed by self-assembly, depending on the experimental conditions: two-dimensional lattices, one-dimensional chains, and zero-dimensional ‘rosettes’. The lattices were characterized by their structure (disordered, square, rhombic, or hexagonal) and by the electrical charges of individual objects; the whole lattices were approximately electrically neutral. The lattices observed in this study were qualitatively different from ionic crystals; the charge of objects had practically continuous values which changed during agitation and self-assembly, and depended on experimental conditions which included the lattice structure itself. The relationship between charge and structure led to the coexistence of regions with different lattice structures within the same assembly, and to transformations between different lattice structures during agitation.

The magnitude of lift forces acting on drops and bubbles in liquids flowing inside microchannels

Hydrodynamic lift forces offer a convenient way to manipulate particles in microfluidic applications, but there is little quantitative information on how non-inertial lift mechanisms act and compete with each other in the confined space of microfluidic channels. This paper reports measurements of lift forces on nearly spherical drops and bubbles, with diameters from one quarter to one half of the width of the channel, flowing in microfluidic channels, under flow conditions characterized by particle capillary numbers Ca(P) = 0.0003-0.3 and particle Reynolds numbers Re(P) = 0.0001-0.1. For Ca(P) < 0.01 and Re(P) < 0.01 the measured lift forces were much larger than predictions of deformation-induced and inertial lift forces found in the literature, probably due to physicochemical hydrodynamic effects at the interface of drops and bubbles, such as the presence of surfactants. The measured forces could be fit with good accuracy using an empirical formula given herein. The empirical formula describes the power-law dependence of the lift force on hydrodynamic parameters (velocity and viscosity of the carrier phase; sizes of channel and drop or bubble), and includes a numerical lift coefficient that depends on the fluids used. The empirical formula using an average lift coefficient of ~500 predicted, within one order of magnitude, all lift force measurements in channels with cross-sectional dimensions below 1 mm.

How Cubic Can Ice Be

Using an X-ray laser, we investigated the crystal structure of ice formed by homogeneous ice nucleation in deeply supercooled water nanodrops (r ≈ 10 nm) at ∼225 K. The nanodrops were formed by condensation of vapor in a supersonic nozzle, and the ice was probed within 100 μs of freezing using femtosecond wide-angle X-ray scattering at the Linac Coherent Light Source free-electron X-ray laser. The X-ray diffraction spectra indicate that this ice has a metastable, predominantly cubic structure; the shape of the first ice diffraction peak suggests stacking-disordered ice with a cubicity value, χ, in the range of 0.78 ± 0.05. The cubicity value determined here is higher than those determined in experiments with micron-sized drops but comparable to those found in molecular dynamics simulations. The high cubicity is most likely caused by the extremely low freezing temperatures and by the rapid freezing, which occurs on a ∼1 μs time scale in single nanodroplets.

Negative Pressures and Spallation in Water Drops Subjected to Nanosecond Shock Waves

Most experimental studies of cavitation in liquid water at negative pressures reported cavitation at tensions significantly smaller than those expected for homogeneous nucleation, suggesting that achievable tensions are limited by heterogeneous cavitation. We generated tension pulses with nanosecond rise times in water by reflecting cylindrical shock waves, produced by X-ray laser pulses, at the internal surface of drops of water. Depending on the X-ray pulse energy, a range of cavitation phenomena occurred, including the rupture and detachment, or spallation, of thin liquid layers at the surface of the drop. When spallation occurred, we evaluated that negative pressures below -100 MPa were reached in the drops. We model the negative pressures from shock reflection experiments using a nucleation-and-growth model that explains how rapid decompression could outrun heterogeneous cavitation in water, and enable the study of stretched water close to homogeneous cavitation pressures.

Erratum: Sheathless hydrodynamic positioning of buoyant drops and bubbles inside microchannels [Phys. Rev. E 84, 036302 (2011)]

This corrects the article DOI: 10.1103/PhysRevE.84.036302.