The Protein Crystallisation Diagnostics Facility (PCDF) on Board ESA Columbus Laboratory

Crystals of proteins or macromolecules are at the basis of X-ray crystallography to reveal structural information necessary for the understanding of their likely mode of action. However, the structural resolution is strongly dependent on the crystalline quality, which is known to be related to gravity dependent processes. The facilities and instruments used so far to grow crystals in space have mostly focused on the growing of crystals for detailed post-flight analysis on ground, and less on the understanding of phenomena associated to the crystallisation processes. The Protein Crystallisation Diagnostics Facility (PCDF), developed by Astrium under contract of the European Space Agency (ESA), allows to study with several diagnostics means in situ the crystallisation of macromolecules over long periods in microgravity. In addition, several ground models with PCDF similar capabilities were developed to allow scientists to prepare their experiments. The PCDF is installed in the European Drawer Rack (EDR), on board ESA’s Columbus Laboratory module launched in February 2008 to the International Space Station (ISS) for research in microgravity on protein nucleation and assembling sequences. The PCDF configuration for this first mission accommodates four reactors, using the batch crystallization technique. Individual process control for temperature and concentration will allow several crystallizations of solutions to be performed. Each reactor will be observed by several optical diagnostics, including video microscopy, dynamic light scattering, and Mach–Zehnder interferometry. This paper presents the overall PCDF design and details the different diagnostics allowing the scientific community to use the PCDF in orbit for microgravity research on molecule assemblies grown from solutions.     

Handling of particulate solids on the International Space Station

We present the principles of a particle-handling system for the International Space Station (ISS) with which experiments with astrophysical and planetological applications will be performed. The principle of dust deagglomeration and dispersion was successfully tested in short-duration microgravity experiments. The flight of the systems on the ISS is planned for 2012.     

Polymorphism in molecular crystals

Reports concerning polymorphism in molecular crystals published in the period 2007–2008 are reviewed. A general survey is given of structural and crystallisation studies in polymorphic systems, and selected advances are described in more detail. These include application of contemporary crystallographic techniques to obtain comprehensive structural information for established polymorphic compounds, multi-disciplinary experimental and simulation studies to probe the mechanisms of polymorph crystallisation, exploration of crystallisation confined within nanopores and characterisation of single crystals containing distinct polymorphic domains.     

The crystallisation of Er-Si-Al-O-N B-phase glass-ceramics

The development of microstructure during crystallisation of a glass with composition (e/o) 35Er:45Si:20Al:83O:17N has been studied by analytical transmission electron microscopy, including electron spectroscopic imaging. The crystals take up a wide range of composition after crystallisation heat treatment at 1050 and 1150°C; the erbium cation percentage varies around that of the expected B-phase composition (Er₂SiAlO₅N), but the aluminium content is slightly lower and the silicon content higher than that. In addition, the erbium content is strongly anti-correlated with the silicon content. A comparison of B-phase composition after crystallisation of equivalent glasses formed with either yttrium, erbium or ytterbium showed that the B-phase solid solution range depends on the particular cation radius. As a consequence of this, the degree of crystallisation and the composition of the residual glass will, for equivalent glasses, also depend on the cation radius. The crystal shape is lenticular after growth at 1050°C, while heat treatment at 1150°C results in crystals with an irregular shape. In addition to crystal growth, the crystallisation heat treatment results in a phase separation of the residual glass whereby smaller silicon and nitrogen rich amorphous features form. These features are effectively pinning boundaries of growing crystals.     

Growth striations in vertically pulled oxide and fluoride single crystals

Temperature oscillations have been detected in oxide and fluoride melts which can be directly correlated to the movement of convection patterns visible on the melt surface. It is shown that such oscillations cause growth striations in single crystals grown from these melts. Additional experiments are reported in which the effect of changes in growth parameters upon the oscillations and melt temperatures have been investigated.     

Crystallization of the EGF receptor ectodomain on US space mission STS-47

Although biochemists working in the field of biological signal transduction have characterized cell surface receptors for numerous growth factors within the past ten years, none of the three-dimensional structures could be obtained for these important proteins which represent major components of the cells' growth control system. Now, the extracellular ligand binding domain of the EGF receptor was crystallized in the presence of EGF under microgravity on US Shuttle mission STS-47. In 8 out of 9 experiments prepared under different conditions crystal growth was observed. One of these space-grown crystals showed higher diffraction quality than all crystals previously obtained in the laboratory. It allowed, for the first time, evaluation of the real space group by partial data collection.     

Ring-Sheared Drop (RSD): Microgravity Module for Containerless Flow Studies

Microgravity is potentially a powerful tool for investigating processes that are sensitive to the presence of solid walls, since fluid containment can be achieved by surface tension. One such process is the transformation of protein in solution into amyloid fibrils; these are protein aggregates associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s. In addition to solid walls, experiments with gravity are also subject to influences from sedimentation of aggregates and buoyancy-driven convection. The ring-sheared drop (RSD) module is a flow apparatus currently under development to study formation of amyloid fibrils aboard the International Space Station (ISS). A 25 mm diameter drop of protein solution will be contained by surface tension and constrained by a pair of sharp-edged tubes, forming two contact rings. Shear can be imparted by rotating one ring with the other ring kept stationary. Here we report on parabolic flights conducted to test the growth and pinning of 10 mm diameter drops of water in under 10 s of microgravity. Finite element method (FEM) based fluid dynamics computations using a commercial package (COMSOL) assisted in the design of the parabolic flight experiments. Prior to the parabolic flights, the code was validated against experiments in the lab (1 g), on the growth of sessile and pendant droplets. The simulations show good agreement with the experiments. This modeling capability will enable the development of the RSD at the 25 mm scale for the ISS.     

Simulating isothermal crystallisation during phase separation of oxide glasses and the percolation behaviour of these glass-ceramics

We modelled the isothermal crystallisation of oxide glasses during their phase separation using the static Monte Carlo technique as in the site percolation model. Initial compositions such that crystallisation can only occur in the evolving glassy matrix phase were considered. The isothermal crystallisation behaviour in terms of model analogues of crystal nucleation and growth were found to qualitatively correspond to the experimental results. The percolation behaviour of the crystalline phase in the evolving glass-ceramic was then studied. The validity of the scaling assumption for percolation phase transition was first tested for the considered systems and then various critical exponents at and near the percolation threshold was estimated. It was found that scaling hypothesis followed for all cases considered in the present investigation.     

A New Paradigm of Crystallization Arising from Non-standard Nucleation Pathways

There is increasing evidence that large classes of colloid materials crystallize via a non-standard nucleation mechanism involving intermediate metastable phases. In this paper recent work on the microscopic derivation of the phase diagram and free energy barriers in the nucleation of protein crystals, and on the kinetics of growth of solid particles in the post-nucleation regime is reviewed. The extent to which combined structural and density fluctuation give rise to favourable crystallization pathways involving an intermediate fluid phase is assessed and the connection with experiments in microgravity at ISS (PROMISS-2 and NANOSLAB-2) is discussed.     

Non-equilibrium primary crystallisation in silver–germanium alloy under microgravity conditions

Abstract

The influence of convection on the morphology of primary dendrites and on the distribution of germanium in primary crystals of hypoeutectic Ag–Ge alloy was studied. The experiment in microgravity provided conditions for the suppression of melt flow. Both the morphology of the primary dendrites and their chemical composition profiles were found to vary considerably depending on the intensity of convection. Primary crystals in specimens solidified in the space laboratory exhibited strong supersaturation with germanium. This supersaturation is associated with a decrease of solute in the eutectic. The overall segregation of germanium between crystals and the melt during the growth of primary dendrites is strongly promoted by convection. The influence of convection on the non-equilibrium primary crystallisation, particularly on the solute trapping process, the occurrence of which is likely in the initial stage of primary crystallisation (solute rich core), is discussed. A higher solute gradient close to the core in the space sample implies a higher melt stability below the liquidus when convection is suppressed.     

ENEIDE: An experiment of a spaceborne, L1/L2 integrated GPS/WAAS/EGNOS receiver

The ENEIDE mission consisted of 22 scientific experiments that were carried out on the Soyuz and on the International Space Station (ISS) during the flight of the Soyuz 10S to the ISS in April 2005. Among these experiments there was the ENEIDE instrument, which gave the name to the whole mission. ENEIDE is a space-qualified, dual-frequency, integrated GPS/WAASEGNOS receiver aimed to the verification of the tracking of GPS plus the augmentation systems from space. The receiver is built by Thales Alenia Space-Italia, Milan plant (formerly Laben), on the basis of the space-qualified dual-frequency receiver LAGRANGE, that will be a payload of several ESA and Italian Space Agency missions like ESA’s GOCE or the Italian COSMO SKYMED constellation to cite few examples. This paper addresses the first results of the ENEIDE flight data analysis.     

Columnar-EquiaxedTransition inSolidification processing: The ESA-MAP CETSOL project

Many castings are the result of a competition between the growth of columnar and equiaxed grains. Indeed, microstructures are at the center of materials science and engineering, and solidification is the most important processing route for structural materials, especially metals and alloys. Presently, microstructure models remain mostly based on diffusive transport mechanisms so that there is a need of critical benchmark data to test fundamental theories of microstructure formation, which often necessitates to have recourse to solidification experiments in the reduced-gravity environment of space. Accordingly, the CETSOL (Columnar-Equiaxed Transition in SOLidification processing)-MAP project of ESA is gathering together European groups with complementary skills to carry out experiments and model the processes, in particular in view of the utilization of reduced-gravity environment that will be afforded by the International Space Station (ISS) to get benchmark data. The ultimate objective of the CETSOL research program is to significantly contribute to the improvement of integrated modeling of grain structure in industrially important castings. To reach this goal, the approach is devised to deepen the quantitative understanding of the basic physical principles that, from the microscopic to the macroscopic scales, govern microstructure formation in solidification processing under diffusive conditions and with fluid flow in the melt. Pending questions are attacked by well-defined model experiments on technical alloys and/or on model transparent systems, physical modeling at microstructure and mesoscopic scales (e.g. large columnar front or equiaxed crystals) and numerical simulation at all scales, up to the macroscopic scales of casting with integrated numerical models.     

Polymorphism in polymers; its implications for polymer crystallisation

The aim of this article is to extend the earlier reported observations on the role of transient metastable phases in polymer crystallisation in relation with the initial crystal size. In this article experimental evidence is provided to bridge the gap between single crystal formation in the melt at elevated pressure and temperatures vs. crystallisation at atmospheric pressure using polyethylene as a model substance. During transformation from the hexagonal to the orthorhombic phase it is shown that in the process of growth, a crystal goes through thermodynamically stable and metastable states before transformation to the orthorhombic phase occurs. The crystal growth, on transformation to the thermodynamically stable orthorhombic phase, has been followed with thex help of in-situ optical microscopy and transmission electron microscopy. The observations are that the newly transformed crystal acts as a nucleation centre for many new crystals starting in the hexagonal phase. It is also noticed that with increasing supercooling multilayering dominates. Subsequently, the distinction between primary and secondary thickening has been made and its morphological consequences will be discussed. In its wider generality, the experimental findings indicate that in polyethylene at atmospheric pressure crystallisation occurs via the hexagonal phase. When extended to atmospheric pressure, the morphological features give further insight into spherulite formation. The observations have been extended to other polymers such as nylon, paraffins, poly-di-alkyl siloxanes, trans-1,4 polybutadiene etc. The proposed viewpoint on the crystal size influence in phase transition has been extended to polymer processing as will be illustrated briefly for the case of processing of the intractable polymer ultra-high-molecular-weight polyethylene (UHMW-PE).     

Protein crystallization in space

The microgravity environment of space is an ideal place to study the complicated protein crystallization process and to grow good-quality protein crystals. A series of crystal growth experiments of 10 different proteins was carried out in space on a Chinese re-entry satellite FSW-2 in August, 1992. The experiments were performed for about two weeks at a temperature of 18.5 +/- 0.5 degrees C using a tube-like crystallization apparatus made in the Shanghai Institute of Technical Physics, Academia Sinica. More than half of 48 samples from 6 proteins produced crystals, and the effects of microgravity on protein crystal growth were observed, especially for hen-egg white lysozyme and an acidic phospholipase A2 from the venom of Agkistrodon halys Pallas. Analyses of the crystallization of these two enzymes in this mission showed that the microgravity environment in space may be beneficial to improve size, external perfection, morphology, internal order, and nucleation of protein crystals. Some of these positive microgravity effects were also demonstrated by the growth of protein crystals in gelled solution with the above two enzymes. A structural analysis of the tetragonal lysozyme crystal grown in space is in progress.     

Non-classical crystal growth of inorganic and organic materials

Abstract

Investigation of the early stage morphologies of some organic and inorganic systems has established that their crystal growth does not always follow the classical route established 120 years ago. The classical theory assumes that a highly symmetric crystal develops from the nucleation and repeated attachment of atoms, molecules or ions to a single nucleus. In instances where surfactants or polymers are introduced, the crystal growth is often found, however, to follow a ‘reversed crystal growth’ route. This non-classical growth route is initiated by organic molecules adsorbed onto the surface of the nanocrystallites in the early stages of growth. In the reversed route, these nanocrystallites form large disordered aggregates followed by surface crystallisation to create a thin, highly crystalline, dense shell and, finally, an extension of the crystallisation from the surface to the core. Although systems following a non-classical crystal growth route have been known for many years, their formation mechanisms have a much shorter history. Discovery of the reversed crystal growth route in several hollow, core–shell and twin crystal type morphologies not only assists in the designing of materials for industrial applications but also helps to understand the interactions between organic and inorganic components. This review highlights recent research relating to organic and inorganic systems whose growth follows the reversed crystal growth route.     

In situ characterization of interface-microstructure dynamics in 3D-Directional Solidification of model transparent alloys

Microstructure plays a central role in determining properties of materials so that the fundamental understanding of the physics of microstructure selection is critical in the design of materials. Under terrestrial conditions fluid flow effects are dominant in bulk samples which preclude precise characterization of fundamental physics of microstructure selection. Experiments in thin samples, carried out to obtain diffusive growth, give microstructures that are neither 2D nor 3D. Rigorous theoretical models, using the phase-field method, have shown that the fundamental physics of pattern selection in 2D and 3D is significantly different. A benchmark experimental study is required in bulk samples under low gravity conditions. Also, the selection of microstructure occurs during the dynamical growth process so that in situ observations of spatio-temporal evolution of the interface shapes are required. Microgravity experiments on ISS are thus planned in a model transparent system by using a new Directional Solidification Insert (DSI), designed for use in the DECLIC facility of CNES and to be adapted to also fit ESA experiments. The critical aspects of hardware design, the key fundamental issues identified through 1g-experiments, the proposed experimental study on ISS, and the results of rigorous theoretical modeling are presented.     

JAXA-GCF project - high-quality protein crystals grown under microgravity environment for better understanding of protein structure

Since 2003, Japan Aerospace Exploration Agency (JAXA, former NASDA) has been conducting a project on a semi-annual basis (JAXA-GCF) to obtain high-quality protein crystals in the microgravity environment using the Russian transportation system. For this project, protein samples were mostly provided by Japanese users for whom JAXA provided technical and clerical support for crystallization experiments in microgravity. For the project, JAXA has constructed a user-friendly support service for microgravity experiments and provided regular and frequent flight opportunities. To simplify and improve technological matters, JAXA devised a gel-tube method crystallization device, which is effective both in space and on ground, based on the counter-diffusion technique. JAXA also provided ground-based techniques for efficient preliminary optimization of crystallization conditions using a 1-dimensional simulation and for harvesting and cryoprotecting crystals before X-ray diffraction experiments. These improvements have significantly increased the success rate of obtaining useful results. In conclusion, JAXA has developed technologies for growing, in microgravity, high-quality protein crystals, which may diffract up to atomic resolution, for a better understanding of 3-dimensional protein structures through X-ray diffraction experiments.     

Experimental investigation of Marangoni convection and vibration-induced crystal motion during protein crystal growth

The occurrence of Marangoni convection during cytochrome c’ crystal growth and vibration-induced motion of lysozyme crystals were investigated using a High Density Protein Crystal Growth (HDPCG) apparatus. Particle image velocimetry was used to visualize fluid motion, but no particle motion was observed, which suggests that under the experimental conditions used, Marangoni convection is not a significant cause of fluid and crystal motion. When horizontal vibrations of controlled amplitude and frequency were applied to the HDPCG apparatus, lysozyme crystals located on the liquid-vapour interface of the HDPCG cell made significant movements up to 0.5mm in amplitude and velocities reaching 0.06mm/s. These results from the Marangoni convection and horizontal vibration experiments suggest that protein crystal movements observed in past space experiments were most likely caused by g-jitter on the spacecraft rather than Marangoni convection.     

Quantum Relaxation of Ensembles of Nanomagnets

Recent theory and experiment in crystals of molecular magnets suggest that fundamental tests of the decoherence mechanisms of macroscopic quantum phenomena may be feasible in these systems (which are also almost ideal quantum spin glasses). We review these, and suggest new experiments.     

Injury severity scoring: a comparison of early clinical versus discharge diagnosis

The Abbreviated Injury Scale (AIS) and the Injury Severity Score (ISS) are objective means of assessing injury. Accepted methodology involves retrospective scoring of injury based on discharge diagnoses. Recently, early clinical scoring, supplemented by review at discharge, has been introduced. A prospective study was instituted to compare these methodologies. Four hundred sixty consecutive victims of blunt trauma were scored using both clinical and retrospective methodologies by independent, blinded observers. Of these, 333 patients had a change in ISS, 174 with a change of greater than four points. The population mean ISS remained unchanged; however, paired values were significantly different (p less than .03). We conclude that either methodology is applicable for studies of large populations of trauma victims. When accurate individual AIS or ISS scoring is required, the clinical method combined with discharge review is most appropriate.