Studying crystallisation processes using electron microscopy: The importance of sample preparation.

We present a comparison of common electron microscopy sample preparation methods for studying crystallisation processes from solution using both scanning and transmission electron microscopy (SEM and TEM). We focus on two widely studied inorganic systems: calcium sulphate, gypsum (CaSO4·2H2O) and calcium carbonate (CaCO3). We find significant differences in crystallisation kinetics and polymorph selection between the different sample preparation methods, which indicate that drying and chemical quenching can induce severe artefacts that are capable of masking the true native state of the crystallising solution. Overall, these results highlight the importance of cryogenic (cryo)-quenching crystallising solutions and the use of full cryo-TEM as the most reliable method for studying the early stages of crystallisation.

Confinement generates single-crystal aragonite rods at room temperature.

The topic of calcite and aragonite polymorphism attracts enormous interest from fields including biomineralization and paleogeochemistry. While aragonite is only slightly less thermodynamically stable than calcite under ambient conditions, it typically only forms as a minor product in additive-free solutions at room temperature. However, aragonite is an abundant biomineral, and certain organisms can selectively generate calcite and aragonite. This fascinating behavior has been the focus of decades of research, where this has been driven by a search for specific organic macromolecules that can generate these polymorphs. However, despite these efforts, we still have a poor understanding of how organisms achieve such selectivity. In this work, we consider an alternative possibility and explore whether the confined volumes in which all biomineralization occurs could also influence polymorph. Calcium carbonate was precipitated within the cylindrical pores of track-etched membranes, where these enabled us to systematically investigate the relationship between the membrane pore diameter and polymorph formation. Aragonite was obtained in increasing quantities as the pore size was reduced, such that oriented single crystals of aragonite were the sole product from additive-free solutions in 25-nm pores and significant quantities of aragonite formed in pores as large as 200 nm in the presence of low concentrations of magnesium and sulfate ions. This effect can be attributed to the effect of the pore size on the ion distribution, which becomes of increasing importance in small pores. These intriguing results suggest that organisms may exploit confinement effects to gain control over crystal polymorph.

Amino Acid Assisted Incorporation of Dye Molecules within Calcite Crystals.

Biomineralisation processes invariably occur in the presence of multiple organic additives, which act in combination to give exceptional control over structures and properties. However, few synthetic studies have investigated the cooperative effects of soluble additives. This work addresses this challenge and focuses on the combined effects of amino acids and coloured dye molecules. The experiments demonstrate that strongly coloured calcite crystals only form in the presence of Brilliant Blue R (BBR) and four of the seventeen soluble amino acids, as compared with almost colourless crystals using the dye alone. The active amino acids are identified as those which themselves effectively occlude in calcite, suggesting a mechanism where they can act as chaperones for individual molecules or even aggregates of dyes molecules. These results provide new insight into crystal-additive interactions and suggest a novel strategy for generating materials with target properties.

Tuning hardness in calcite by incorporation of amino acids.

Structural biominerals are inorganic/organic composites that exhibit remarkable mechanical properties. However, the structure-property relationships of even the simplest building unit-mineral single crystals containing embedded macromolecules-remain poorly understood. Here, by means of a model biomineral made from calcite single crystals containing glycine (0-7 mol%) or aspartic acid (0-4 mol%), we elucidate the origin of the superior hardness of biogenic calcite. We analysed lattice distortions in these model crystals by using X-ray diffraction and molecular dynamics simulations, and by means of solid-state nuclear magnetic resonance show that the amino acids are incorporated as individual molecules. We also demonstrate that nanoindentation hardness increased with amino acid content, reaching values equivalent to their biogenic counterparts. A dislocation pinning model reveals that the enhanced hardness is determined by the force required to cut covalent bonds in the molecules.

The role of phase separation and related topography in the exceptional ice-nucleating ability of alkali feldspars.

Our understanding of crystal nucleation is a limiting factor in many fields, not least in the atmospheric sciences. It was recently found that feldspar, a component of airborne desert dust, plays a dominant role in triggering ice formation in clouds, but the origin of this effect was unclear. By investigating the structure/property relationships of a wide range of feldspars, we demonstrate that alkali feldspars with certain microtextures, related to phase separation into Na and K-rich regions, show exceptional ice-nucleating abilities in supercooled water. We found no correlation between ice-nucleating efficiency and the crystal structures or the chemical compositions of these active feldspars, which suggests that specific topographical features associated with these microtextures are key in the activity of these feldspars. That topography likely acts to promote ice nucleation, improves our understanding of ice formation in clouds, and may also enable the design and manufacture of bespoke nucleating materials for uses such as cloud seeding and cryopreservation.

The Effect of Additives on the Early Stages of Growth of Calcite Single Crystals.

As crystallization processes are often rapid, it can be difficult to monitor their growth mechanisms. In this study, we made use of the fact that crystallization proceeds more slowly in small volumes than in bulk solution to investigate the effects of the soluble additives Mg2+ and poly(styrene sulfonate) (PSS) on the early stages of growth of calcite crystals. Using a "Crystal Hotel" microfluidic device to provide well-defined, nanoliter volumes, we observed that calcite crystals form via an amorphous precursor phase. Surprisingly, the first calcite crystals formed are perfect rhombohedra, and the soluble additives have no influence on the morphology until the crystals reach sizes of 0.1-0.5 µm for Mg2+ and 1-2 µm for PSS. The crystals then continue to grow to develop morphologies characteristic of these additives. These results can be rationalized by considering additive binding to kink sites, which is consistent with crystal growth by a classical mechanism.

Three-dimensional imaging of dislocation propagation during crystal growth and dissolution.

Atomic-level defects such as dislocations play key roles in determining the macroscopic properties of crystalline materials. Their effects range from increased chemical reactivity to enhanced mechanical properties. Dislocations have been widely studied using traditional techniques such as X-ray diffraction and optical imaging. Recent advances have enabled atomic force microscopy to study single dislocations in two dimensions, while transmission electron microscopy (TEM) can now visualize strain fields in three dimensions with near-atomic resolution. However, these techniques cannot offer three-dimensional imaging of the formation or movement of dislocations during dynamic processes. Here, we describe how Bragg coherent diffraction imaging (BCDI; refs 11, 12) can be used to visualize in three dimensions, the entire network of dislocations present within an individual calcite crystal during repeated growth and dissolution cycles. These investigations demonstrate the potential of BCDI for studying the mechanisms underlying the response of crystalline materials to external stimuli.

Structure-property relationships of a biological mesocrystal in the adult sea urchin spine.

Structuring over many length scales is a design strategy widely used in Nature to create materials with unique functional properties. We here present a comprehensive analysis of an adult sea urchin spine, and in revealing a complex, hierarchical structure, show how Nature fabricates a material which diffracts as a single crystal of calcite and yet fractures as a glassy material. Each spine comprises a highly oriented array of Mg-calcite nanocrystals in which amorphous regions and macromolecules are embedded. It is postulated that this mesocrystalline structure forms via the crystallization of a dense array of amorphous calcium carbonate (ACC) precursor particles. A residual surface layer of ACC and/or macromolecules remains around the nanoparticle units which creates the mesocrystal structure and contributes to the conchoidal fracture behavior. Nature's demonstration of how crystallization of an amorphous precursor phase can create a crystalline material with remarkable properties therefore provides inspiration for a novel approach to the design and synthesis of synthetic composite materials.

Correlation between anisotropy and lattice distortions in single crystal calcite nanowires grown in confinement.

Growing nanostructures in confinement allows for the control of their shape, size and structure, as required in many technological applications. We investigated the crystal structure and morphology of calcite nanowires, precipitated in the pores of track-etch membranes, by employing transmission electron microscopy and selected area electron diffraction (SAED). The data showed that the nanowires show no preferred growth orientation and that the crystallographic orientation rotated along the length of the nanowire, with lattice rotation angles of several degrees per micrometer. Finite element calculations indicated that the rotation is caused by the anisotropic crystallographic nature of the calcite mineral, the nanoscale diameter of the wires and the confined space provided by the membrane pore. This phenomenon should also be observed in other single crystal nanowires made from anisotropic materials, which could offer the potential of generating nanostructures with tailored optical, electronic and mechanical properties.

Simple photosystem II water oxidation centre analogues in visible light oxygen and H+ generation.

Calcium manganese oxide nanoparticles for application in water oxidation are synthesized by combination with a carboxylated biopolymer stabilizing agent to form very simple but effective analogues of the photosynthetic PSII oxygen evolving complex. The relative efficiency of these materials for production of O(2) and protons under visible light-promoted reactions is evaluated and prolonged reaction lifetimes are observed.

Electron transparent nanotubes reveal crystallization pathways in confinement.

The cylindrical pores of track-etched membranes offer excellent environments for studying the effects of confinement on crystallization as the pore diameter is readily varied and the anisotropic morphologies can direct crystal orientation. However, the inability to image individual crystals in situ within the pores in this system has prevented many of the underlying mechanisms from being characterized. Here, we study the crystallization of calcium sulfate within track-etched membranes and reveal that oriented gypsum forms in 200 nm diameter pores, bassanite in 25-100 nm pores and anhydrite in 10 nm pores. The crystallization pathways are then studied by coating the membranes with an amorphous titania layer prior to mineralization to create electron transparent nanotubes that protect fragile precursor materials. By visualizing the evolutionary pathways of the crystals within the pores we show that the product single crystals derive from multiple nucleation events and that orientation is determined at early reaction times. Finally, the transformation of bassanite to gypsum within the membrane pores is studied using experiment and potential mean force calculations and is shown to proceed by localized dissolution/reprecipitation. This work provides insight into the effects of confinement on crystallization processes, which is relevant to mineral formation in many real-world environments.

An artificial biomineral formed by incorporation of copolymer micelles in calcite crystals.

Biominerals exhibit morphologies, hierarchical ordering and properties that invariably surpass those of their synthetic counterparts. A key feature of these materials, which sets them apart from synthetic crystals, is their nanocomposite structure, which derives from intimate association of organic molecules with the mineral host. We here demonstrate the production of artificial biominerals where single crystals of calcite occlude a remarkable 13 wt% of 20 nm anionic diblock copolymer micelles, which act as 'pseudo-proteins'. The synthetic crystals exhibit analogous texture and defect structures to biogenic calcite crystals and are harder than pure calcite. Further, the micelles are specifically adsorbed on {104} faces and undergo a change in shape on incorporation within the crystal lattice. This system provides a unique model for understanding biomineral formation, giving insight into both the mechanism of occlusion of biomacromolecules within single crystals, and the relationship between the macroscopic mechanical properties of a crystal and its microscopic structure.

Calcite Kinetics for Spiral Growth and Two-Dimensional Nucleation.

Calcite crystals grow by means of molecular steps that develop on {10.4} faces. These steps can arise stochastically via two-dimensional (2D) nucleation or emerge steadily from dislocations to form spiral hillocks. Here, we determine the kinetics of these two growth mechanisms as a function of supersaturation. We show that calcite crystals larger than ∼1 µm favor spiral growth over 2D nucleation, irrespective of the supersaturation. Spirals prevail beyond this length scale because slow boundary layer diffusion creates a low surface supersaturation that favors the spiral mechanism. Sub-micron crystals favor 2D nucleation at high supersaturations, although diffusion can still limit the growth of nanoscopic crystals. Additives can change the dominant mechanism by impeding spiral growth or by directly promoting 2D nucleation.

Early stages of crystallization of calcium carbonate revealed in picoliter droplets.

In this work, we studied the heterogeneous nucleation and growth of CaCO(3) within regular arrays of picoliter droplets created on patterned self-assembled monolayers (SAMs). The SAMs provide well-defined substrates that offer control over CaCO(3) nucleation, and we used these impurity-free droplet arrays to study crystal growth in spatially and chemically controlled, finite-reservoir environments. The results demonstrate a number of remarkable features of precipitation within these confined volumes. CaCO(3) crystallization proceeds significantly more slowly in the droplets than in the bulk, allowing the mechanism of crystallization, which progresses via amorphous calcium carbonate, to be easily observed. In addition, the precipitation reaction terminates at an earlier stage than in the bulk solution, revealing intermediate growth forms. Confinement can therefore be used as a straightforward method for studying the mechanisms of crystallization on a substrate without the requirement for specialized analytical techniques. The results are also of significance to biomineralization processes, where crystallization typically occurs in confinement and in association with organic matrices, and it is envisaged that the method is applicable to many crystallizing systems.

Capillarity creates single-crystal calcite nanowires from amorphous calcium carbonate.

Single-crystal calcite nanowires are formed by crystallization of morphologically equivalent amorphous calcium carbonate (ACC) particles within the pores of track etch membranes. The polyaspartic acid stabilized ACC is drawn into the membrane pores by capillary action, and the single-crystal nature of the nanowires is attributed to the limited contact of the intramembrane ACC particle with the bulk solution. The reaction environment then supports transformation to a single-crystal product.

Incorporation of nanogels within calcite single crystals for the storage, protection and controlled release of active compounds.

Nanocarriers have tremendous potential for the encapsulation, storage and delivery of active compounds. However, current formulations often employ open structures that achieve efficient loading of active agents, but that suffer undesired leakage and instability of the payloads over time. Here, a straightforward strategy that overcomes these issues is presented, in which protein nanogels are encapsulated within single crystals of calcite (CaCO3). Demonstrating our approach with bovine serum albumin (BSA) nanogels loaded with (bio)active compounds, including doxorubicin (a chemotherapeutic drug) and lysozyme (an antibacterial enzyme), we show that these nanogels can be occluded within calcite host crystals at levels of up to 45 vol%. Encapsulated within the dense mineral, the active compounds are stable against harsh conditions such as high temperature and pH, and controlled release can be triggered by a simple reduction of the pH. Comparisons with analogous systems - amorphous calcium carbonate, mesoporous vaterite (CaCO3) polycrystals, and calcite crystals containing polymer vesicles - demonstrate the superior encapsulation performance of the nanogel/calcite system. This opens the door to encapsulating a broad range of existing nanocarrier systems within single crystal hosts for the efficient storage, transport and controlled release of various active guest species.

A facile method for generating worm-like micelles with controlled lengths and narrow polydispersity.

This work shows that highly uniform worm micelles formed by polymerisation induced self-assembly can be obtained via simple post-synthesis sonication. Importantly, this straightforward and versatile strategy yields exceptionally monodisperse worms with tunable aspect ratios ranging from 7.2 to 17.6 by simply changing the sonication time.

Ptychographic X-ray tomography reveals additive zoning in nanocomposite single crystals.

Single crystals containing nanoparticles represent a unique class of nanocomposites whose properties are defined by both their compositions and the structural organization of the dispersed phase in the crystalline host. Yet, there is still a poor understanding of the relationship between the synthesis conditions and the structures of these materials. Here ptychographic X-ray computed tomography is used to visualize the three-dimensional structures of two nanocomposite crystals - single crystals of calcite occluding diblock copolymer worms and vesicles. This provides unique information about the distribution of the copolymer nano-objects within entire, micron-sized crystals with nanometer spatial resolution and reveals how occlusion is governed by factors including the supersaturation and calcium concentration. Both nanocomposite crystals are seen to exhibit zoning effects that are governed by the solution composition and interactions of the additives with specific steps on the crystal surface. Additionally, the size and shape of the occluded vesicles varies according to their location within the crystal, and therefore the solution composition at the time of occlusion. This work contributes to our understanding of the factors that govern nanoparticle occlusion within crystalline materials, where this will ultimately inform the design of next generation nanocomposite materials with specific structure/property relationships.

Evaluation of microflow configurations for scale inhibition and serial X-ray diffraction analysis of crystallization processes.

The clean and reproducible conditions provided by microfluidic devices are ideal sample environments for in situ analyses of chemical and biochemical reactions and assembly processes. However, the small size of microchannels makes investigating the crystallization of poorly soluble materials on-chip challenging due to crystal nucleation and growth that result in channel fouling and blockage. Here, we demonstrate a reusable insert-based microfluidic platform for serial X-ray diffraction analysis and examine scale formation in response to continuous and segmented flow configurations across a range of temperatures. Under continuous flow, scale formation on the reactor walls begins almost immediately on mixing of the crystallizing species, which over time results in occlusion of the channel. Depletion of ions at the start of the channel results in reduced crystallization towards the end of the channel. Conversely, segmented flow can control crystallization, so it occurs entirely within the droplet. Consequently, the spatial location within the channel represents a temporal point in the crystallization process. Whilst each method can provide useful crystallographic information, time-resolved information is lost when reactor fouling occurs and changes the solution conditions with time. The flow within a single device can be manipulated to give a broad range of information addressing surface interaction or solution crystallization.