Microstructure and crystal order during freezing of supercooled water drops.

Supercooled water droplets are widely used to study supercooled water1,2, ice nucleation3-5 and droplet freezing6-11. Their freezing in the atmosphere affects the dynamics and climate feedback of clouds12,13 and can accelerate cloud freezing through secondary ice production14-17. Droplet freezing occurs at several timescales and length scales14,18 and is sufficiently stochastic to make it unlikely that two frozen drops are identical. Here we use optical microscopy and X-ray laser diffraction to investigate the freezing of tens of thousands of water microdrops in vacuum after homogeneous ice nucleation around 234-235 K. On the basis of drop images, we developed a seven-stage model of freezing and used it to time the diffraction data. Diffraction from ice crystals showed that long-range crystalline order formed in less than 1 ms after freezing, whereas diffraction from the remaining liquid became similar to that from quasi-liquid layers on premelted ice19,20. The ice had a strained hexagonal crystal structure just after freezing, which is an early metastable state that probably precedes the formation of ice with stacking defects8,9,18. The techniques reported here could help determine the dynamics of freezing in other conditions, such as drop freezing in clouds, or help understand rapid solidification in other materials.

Rapid sample delivery for megahertz serial crystallography at X-ray FELs.

Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse. This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using 4.3 nm radiation. The operation of gas-dynamic nozzles that produce liquid microjets with velocities greater than 80 m s-1 was demonstrated. Furthermore, this article provides optical images of X-ray-induced explosions together with Bragg diffraction from protein microcrystals exposed to trains of X-ray pulses repeating at rates of up to 4.5 MHz. The results indicate the feasibility for megahertz serial crystallography measurements with hard X-rays and give guidance for the design of such experiments.

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.

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.

Drop-on-demand sample delivery for studying biocatalysts in action at X-ray free-electron lasers.

X-ray crystallography at X-ray free-electron laser sources is a powerful method for studying macromolecules at biologically relevant temperatures. Moreover, when combined with complementary techniques like X-ray emission spectroscopy, both global structures and chemical properties of metalloenzymes can be obtained concurrently, providing insights into the interplay between the protein structure and dynamics and the chemistry at an active site. The implementation of such a multimodal approach can be compromised by conflicting requirements to optimize each individual method. In particular, the method used for sample delivery greatly affects the data quality. We present here a robust way of delivering controlled sample amounts on demand using acoustic droplet ejection coupled with a conveyor belt drive that is optimized for crystallography and spectroscopy measurements of photochemical and chemical reactions over a wide range of time scales. Studies with photosystem II, the phytochrome photoreceptor, and ribonucleotide reductase R2 illustrate the power and versatility of this method.

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

Correction for 'The magnitude of lift forces acting on drops and bubbles in liquids flowing inside microchannels' by Claudiu A. Stan et al., Lab Chip, 2013, 13, 365-376.

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.

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.

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).

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

The dioxygen we breathe is formed by light-induced oxidation of water in photosystem II. O2 formation takes place at a catalytic manganese cluster within milliseconds after the photosystem II reaction centre 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.

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.

Sheathless hydrodynamic positioning of buoyant drops and bubbles inside microchannels.

Particles, bubbles, and drops carried by a fluid in a confined environment such as a pipe can be subjected to hydrodynamic lift forces, i.e., forces that are perpendicular to the direction of the flow. We investigated the positioning effect of lift forces acting on buoyant drops and bubbles suspended in a carrier fluid and flowing in a horizontal microchannel. We report experiments on drops of water in fluorocarbon liquid, and on bubbles of nitrogen in hydrocarbon liquid and silicone oil, inside microchannels with widths on the order of 0.1-1 mm. Despite their buoyancy, drops and bubbles could travel without contacting with the walls of channels; the most important parameters for reaching this flow regime in our experiments were the viscosity and the velocity of the carrier fluid, and the sizes of drops and bubbles. The dependencies of the transverse position of drops and bubbles on these parameters were investigated. At steady state, the trajectories of drops and bubbles approached the center of the channel for drops and bubbles almost as large as the channel, carried by rapidly flowing viscous liquids; among our experiments, these flow conditions were characterized by larger capillary numbers and smaller Reynolds numbers. Analytical models of lift forces developed for the flow of drops much smaller than the width of the channel failed to predict their transverse position, while computational fluid dynamic simulations of the experiments agreed better with the experimental measurements. The degrees of success of these predictions indicate the importance of confinement on generating strong hydrodynamic lift forces. We conclude that, inside microfluidic channels, it is possible to support and position buoyant drops and bubbles simply by flowing a single-stream (i.e., "sheathless") carrier liquid that has appropriate velocity and hydrodynamic properties.

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.

A microfluidic apparatus for the study of ice nucleation in supercooled water drops.

This paper describes a microfluidic instrument that produces drops of supercooled water suspended in a moving stream of liquid fluorocarbon, and measures the temperatures at which ice nucleates in the drops. A microfluidic chip containing a monodisperse drop generator and a straight channel with 38 embedded resistance thermometers was placed in contact with a seven-zone temperature-control plate and imaged under a microscope with a high-speed camera. This instrument can record the freezing temperatures of tens of thousands of drops within minutes, with an accuracy of 0.4 degrees C. The ice-nucleation temperatures in approximately 80-microm drops were reported for the freezing of 37 061 drops of pure water, and of 8898 drops of water seeded with silver iodide. Nucleation of ice in pure water was homogenous and occurred at temperatures between -36 and -37.8 degrees C, while water containing silver iodide froze between -10 and -19 degrees C. The instrument recorded the largest sets of individual freezing temperatures (37 061), had the fastest data acquisition rate (75 measurements/s), and the best optical (3 microm) and temporal (70 micros) resolutions among instruments designed to study nucleation of ice. The dendritic growth of ice in 150-microm drops of supercooled water at -35 degrees C was observed and imaged at a rate of 16 000 frames/s.

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.

Dynamically reconfigurable liquid-core liquid-cladding lens in a microfluidic channel.

This paper describes the design and operation of a liquid-core liquid-cladding (L(2)) lens formed by the laminar flow of three streams of liquids in a microchannel whose width expands laterally in the region where the lens forms. Two streams of liquid with a lower refractive index (the cladding) sandwich a stream of liquid with a higher refractive index (the core). As the core stream enters the expansion chamber, it widens and becomes biconvex in shape, for some rates of flow. This biconvex fluidic element focuses light. Manipulating the relative rates of flow of the streams reconfigures the shape, and therefore the focal distance, of the L(2) lens. This paper also describes a technique for beam tracing, and for characterization of a lens in an enclosed micro-scale optical system. The use of a cladding liquid with refractive index matched to that of the material used in the fabrication of the microfluidic system (here, poly(dimethylsiloxane)) improves the quality of the focused beam.