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.

Ecofriendly alkali metal cations diffusion improves fabrication of mixed-phase titania polymorphs on fixed substrate by chemical vapor deposition (CVD) for photocatalytic degradation of azo dye.

Controlling the nanoscale synthesis of semiconductor TiO2 on a fixed substrate has fascinated the curiosity of academics for decades. Synthesis development is required to give an easy-to-control technique and parameters for TiO2 manufacture, leading to advancements in prospective applications such as photocatalysts. This study, mixed-phase TiO2(B)/other titania thin films were synthesized on a fused quartz substrate utilizing a modified Chemical vapor depodition involving alkali-metal ions (Li+, Na+, and K+) solution pre-treatment. It was discovered that different cations promote dramatically varied phases and compositions of thin films. The films had a columnar structure with agglomerated irregular-shaped particles with a mean thickness of 800-2000 nm. Na+ ions can promote TiO2(B) more effectively than K+ ions, however Li+ ions cannot synthesize TiO2(B). The amounts of TiO2(B) in thin films increase with increasing alkali metal (K+ and Na+) concentration. According to experimental and DFT calculations, the hypothesized TiO2(B) production mechanism happened via the meta-stable intermediate alkaline titanate transformation caused by alkali-metal ion diffusion. The mixed phase of TiO2(B) and anatase TiO2 on the fixed substrate (1 × 1 cm2) obtained from Na+ pre-treated procedures showed significant photocatalytic activity for the degradation of methylene blue. K2Ti6O12, Li2TiO3, Rutile TiO2, and Brookite TiO2 phase formations produced by K+ and Li + pretreatment are low activity photocatalysts. Photocatalytic activities were more prevalent in NaOH pre-treated samples (59.1% dye degradation) than in LiOH and KOH pre-treated samples (49.6% and 34.2%, respectively). This revealed that our developed CVD might generate good photocatalytic thin films of mixed-phase TiO2(B)/anatase TiO2 on any substrate, accelerating progress in future applications.

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.

Mapping nanocrystalline disorder within an amorphous metal-organic framework.

Intentionally disordered metal-organic frameworks (MOFs) display rich functional behaviour. However, the characterisation of their atomic structures remains incredibly challenging. X-ray pair distribution function techniques have been pivotal in determining their average local structure but are largely insensitive to spatial variations in the structure. Fe-BTC (BTC = 1,3,5-benzenetricarboxylate) is a nanocomposite MOF, known for its catalytic properties, comprising crystalline nanoparticles and an amorphous matrix. Here, we use scanning electron diffraction to first map the crystalline and amorphous components to evaluate domain size and then to carry out electron pair distribution function analysis to probe the spatially separated atomic structure of the amorphous matrix. Further Bragg scattering analysis reveals systematic orientational disorder within Fe-BTC's nanocrystallites, showing over 10° of continuous lattice rotation across single particles. Finally, we identify candidate unit cells for the crystalline component. These independent structural analyses quantify disorder in Fe-BTC at the critical length scale for engineering composite MOF materials.

Surface Site Density of Synthetic Goethites and Its Relationship to Atomic Surface Roughness and Crystal Size.

The capacity for crystals to adsorb elements and molecules is a function of the structures of their crystal faces and the relative proportions of those faces. More importantly, this study shows that the surface structure of crystal faces is affected by their surface roughness and is the dominant factor controlling the absorption site density. In a continuation of the study of synthetic goethites with varying single crystal size distributions, two more synthetic goethites with intermediate sizes were analyzed by Brunauer-Emmett-Teller (BET) and atomic-resolution scanning transmission electron microscopy (STEM) to determine the effects of crystal size on their shape, atomic-scale surface roughness, and ultimately on their total surface site density. Results show that surface roughness scales directly with the size [or inversely with the specific surface area (SSA)] of synthetic goethites in the SSA range of 40-75 m2/g. This surface roughness, in turn, increases the total site density over ideal atomically smooth crystals. The total site density of synthetic goethite increases from a combination of decreasing crystal length/width ratio and increasing surface roughness.

Heterogeneous Rate Constant for Amorphous Silica Nanoparticle Adsorption on Phospholipid Monolayers.

The interaction of amorphous silica nanoparticles with phospholipid monolayers and bilayers has received a great deal of interest in recent years and is of importance for assessing potential cellular toxicity of such species, whether natural or synthesized for the purpose of nanomedical drug delivery and other applications. This present communication studies the rate of silica nanoparticle adsorption on to phospholipid monolayers in order to extract a heterogeneous rate constant from the data. This rate constant relates to the initial rate of growth of an adsorbed layer of nanoparticles as SiO2 on a unit area of the monolayer surface from unit concentration in dispersion. Experiments were carried out using the system of dioleoyl phosphatidylcholine (DOPC) monolayers deposited on Pt/Hg electrodes in a flow cell. Additional studies were carried out on the interaction of soluble silica with these layers. Results show that the rate constant is effectively constant with respect to silica nanoparticle size. This is interpreted as indicating that the interaction of hydrated SiO2 molecular species with phospholipid polar groups is the molecular initiating event (MIE) defined as the initial interaction of the silica particle surface with the phospholipid layer surface promoting the adsorption of silica nanoparticles on DOPC. The conclusion is consistent with the observed significant interaction of soluble SiO2 with the DOPC layer and the established properties of the silica-water interface.

Understanding stress-induced disorder and breakage in organic crystals: beyond crystal structure anisotropy.

Crystal engineering has advanced the strategies for design and synthesis of organic solids with the main focus being on customising the properties of the materials. Research in this area has a significant impact on large-scale manufacturing, as industrial processes may lead to the deterioration of such properties due to stress-induced transformations and breakage. In this work, we investigate the mechanical properties of structurally related labile multicomponent solids of carbamazepine (CBZ), namely the dihydrate (CBZ·2H2O), a cocrystal of CBZ with 1,4-benzoquinone (2CBZ·BZQ) and the solvates with formamide and 1,4-dioxane (CBZ·FORM and 2CBZ·DIOX, respectively). The effect of factors that are external (e.g. impact stressing) and/or internal (e.g. phase transformations and thermal motion) to the crystals are evaluated. In comparison to the other CBZ multicomponent crystal forms, CBZ·2H2O crystals tolerate less stress and are more susceptible to breakage. It is shown that this poor resistance to fracture may be a consequence of the packing of CBZ molecules and the orientation of the principal molecular axes in the structure relative to the cleavage plane. It is concluded, however, that the CBZ lattice alone is not accountable for the formation of cracks in the crystals of CBZ·2H2O. The strength and the temperature-dependence of electrostatic interactions, such as hydrogen bonds between CBZ and coformer, appear to influence the levels of stress to which the crystals are subjected that lead to fracture. Our findings show that the appropriate selection of coformer in multicomponent crystal forms, targetting superior mechanical properties, needs to account for the intrinsic stress generated by molecular vibrations and not solely by crystal anisotropy. Structural defects within the crystal lattice, although highly influenced by the crystallisation conditions and which are especially difficult to control in organic solids, may also affect breakage.

Engineering of Microcage Carbon Nanotube Architectures with Decoupled Multimodal Porosity and Amplified Catalytic Performance.

New approaches for the engineering of the 3D microstructure, pore modality, and chemical functionality of hierarchically porous nanocarbon assemblies are key to develop the next generation of functional aerogel and membrane materials. Here, interfacially driven assembly of carbon nanotubes (CNT) is exploited to fabricate structurally directed aerogels with highly controlled internal architectures, composed of pseudo-monolayer, CNT microcages. CNT Pickering emulsions enable engineering at fundamentally different length scales, whereby the microporosity, mesoporosity, and macroporosity are decoupled and individually controlled through CNT type, CNT number density, and process energy, respectively. In addition, metal nanocatalysts (Cu, Pd, and Ru) are embedded within the architectures through an elegant sublimation and shock-decomposition approach; introducing the first approach that enables through-volume functionalization of intricate, pre-designed aerogels without microstructural degradation. Catalytic structure-function relationships are explored in a pharma-important amidation reaction; providing insights on how the engineered frameworks enhance catalyst activity. A sophisticated array of advanced tomographic, spectroscopic, and microscopic techniques reveal an intricate 3D assembly of CNT building-blocks and their influence on the functional properties of the enhanced nanocatalysts. These advances set a basis to modulate structure and chemistry of functional aerogel materials independently in a controlled fashion for a variety of applications, including energy conversion and storage, smart electronics, and (electro)catalysis.

Barium yttrium fluoride based upconversion nanoparticles as dual mode image contrast agents.

Dual labeled contrast agents could provide better complementary information for bioimaging than available solely from a single modality. In this paper we investigate the suitability of Yb3+ and Er3+-doped BaYF5 upconversion nanoparticles (UCNPs) as both optical and X-ray micro computed tomography (µCT) contrast agents. Stable, aqueous UCNP dispersions were synthesised using a hydrothermal method with the addition of polyethyleneimine (PEI). UCNPs were single crystal and had a truncated cuboidal and/or truncated octahedral morphology, with average particle size of 47 ±9 nm from transmission electron microscopy which was further used to characterize the structure and composition in detail. A zeta potential value of +51 mV was measured for the aqueous nanoparticle dispersions which is beneficial for cell permeability. The outer hydrated PEI layer is also advantageous for the attachment of proteins for targeted delivery in biological systems. The prepared UCNPs were proven to be non-toxic to endothelial cells up to a concentration of 3.5 mg/mL, when assessed using an MTT assay. The particles showed intense green upconversion photoluminescence when excited at a wavelength of 976 nm using a diode laser. Quantitative X-ray µCT contrast imaging confirmed the potential of these UCNPs as X-ray contrast agents and confirming their dual modality for bioimaging.

Characterization of Amorphous Solid Dispersions and Identification of Low Levels of Crystallinity by Transmission Electron Microscopy.

Amorphous solid dispersions (ASDs) are used to increase the solubility of oral medicines by kinetically stabilizing the more soluble amorphous phase of an active pharmaceutical ingredient with a suitable amorphous polymer. Low levels of a crystalline material in an ASD can negatively impact the desired dissolution properties of the drug. Characterization techniques such as powder X-ray diffraction (pXRD), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR) are often used to detect and measure any crystallinity within ASDs. These techniques are unable to detect or quantify very low levels because they have limits of detection typically in the order of 1-5%. Herein, an ASD of felodipine (FEL) and polyvinylpyrrolidone/vinyl acetate copolymer (PVP/VA) prepared via a hot melt extrusion (HME) in a mass ratio of 30:70 was characterized using a range of techniques. No signs of residual crystallinity were found by pXRD, DSC, or FTIR. However, transmission electron microscopy (TEM) did identify two areas containing crystals at the edges of milled particles from a total of 55 examined. Both crystalline areas contained Cl Kα X-ray peaks when measured by energy-dispersive X-ray spectroscopy, confirming the presence of FEL (due to the presence of Cl atoms in FEL and not in PVP/VA). Further analysis was carried out by TEM using conical dark field (DF) imaging of a HME ASD of 50:50 FEL-PVP/VA to provide insights into the recrystallization process that occurs at the edges of particles during accelerated ageing conditions in an atmosphere of 75% relative humidity. Multiple metastable polymorphs of recrystallized FEL could be identified by selected area electron diffraction (SAED), predominately form II and the more stable form I. Conical DF imaging was also successful in spatially resolving and sizing crystals. This work highlights the potential for TEM-based techniques to improve the limit of detection of crystallinity in ASDs, while also providing insights into transformation pathways by identifying the location, size, and form of any crystallization that might occur on storage. This opens up the possibility of providing an enhanced understanding of a drug product's stability and performance.

Tuning stable noble metal nanoparticles dispersions to moderate their interaction with model membranes.

HYPOTHESIS: The properties of stable gold (Au) nanoparticle dispersions can be tuned to alter their activity towards biomembrane models. EXPERIMENTS: Au nanoparticle coating techniques together with rapid electrochemical screens of a phospholipid layer on fabricated mercury (Hg) on platinum (Pt) electrode have been used to moderate the phospholipid layer activity of Au nanoparticle dispersions. Screening results for Au nanoparticle dispersions were intercalibrated with phospholipid large unilamellar vesicle (LUV) interactions using a carboxyfluorescein (CF) leakage assay. All nanoparticle dispersions were characterised for size, by dynamic light scattering (DLS) and transmission electron microscopy (TEM). FINDINGS: Commercial and high quality home synthesised Au nanoparticle dispersions are phospholipid monolayer active whereas Ag nanoparticle dispersions are not. If Au nanoparticles are coated with a thin layer of Ag then the particle/lipid interaction is suppressed. The electrochemical assays of the lipid layer activity of Au nanoparticle dispersions align with LUV leakage assays of the same. Au nanoparticles of decreasing size and increasing dispersion concentration showed a stronger phospholipid monolayer/bilayer interaction. Treating Au nanoparticles with cell culture medium and incubation of Au nanoparticle dispersions in phosphate buffered saline (PBS) solutions removes their phospholipid layer interaction.

Analysis of complex, beam-sensitive materials by transmission electron microscopy and associated techniques.

We review the use of transmission electron microscopy (TEM) and associated techniques for the analysis of beam-sensitive materials and complex, multiphase systems in-situ or close to their native state. We focus on materials prone to damage by radiolysis and explain that this process cannot be eliminated or switched off, requiring TEM analysis to be done within a dose budget to achieve an optimum dose-limited resolution. We highlight the importance of determining the damage sensitivity of a particular system in terms of characteristic changes that occur on irradiation under both an electron fluence and flux by presenting results from a series of molecular crystals. We discuss the choice of electron beam accelerating voltage and detectors for optimizing resolution and outline the different strategies employed for low-dose microscopy in relation to the damage processes in operation. In particular, we discuss the use of scanning TEM (STEM) techniques for maximizing information content from high-resolution imaging and spectroscopy of minerals and molecular crystals. We suggest how this understanding can then be carried forward for in-situ analysis of samples interacting with liquids and gases, provided any electron beam-induced alteration of a specimen is controlled or used to drive a chosen reaction. Finally, we demonstrate that cryo-TEM of nanoparticle samples snap-frozen in vitreous ice can play a significant role in benchmarking dynamic processes at higher resolution. This article is part of a discussion meeting issue 'Dynamic in situ microscopy relating structure and function'.

Nanoparticle corona artefacts derived from specimen preparation of particle suspensions.

Progress in the implementation of nanoparticles for therapeutic applications will accelerate with an improved understanding of the interface between nanoparticle surfaces and the media they are dispersed in. We examine this interface by analytical scanning transmission electron microscopy and show that incorrect specimen preparation or analysis can induce an artefactual, nanoscale, calcium phosphate-rich, amorphous coating on nanoparticles dispersed in cell culture media. We report that this ionic coating can be induced on five different types of nanoparticles (Au, BaTiO3, ZnO, TiO2 and Fe2O3) when specimen preparation causes a significant rise in pH above physiological levels. Such a pH change reduces ionic solubility in the suspending media to permit precipitation of calcium phosphate. Finally, we demonstrate that there is no indication of a calcium-phosphorus-rich coating on BaTiO3 nanoparticles suspended in culture media when prepared without alteration of the pH of the suspending media and imaged by cryo-STEM. Therefore we recommend that future reports utilising nanoparticles dispersed in cell culture media monitor and report the pH of suspensions during sample preparation.

Sub-Nanometer Thick Gold Nanosheets as Highly Efficient Catalysts.

2D metal nanomaterials offer exciting prospects in terms of their properties and functions. However, the ambient aqueous synthesis of atomically-thin, 2D metallic nanomaterials represents a significant challenge. Herein, freestanding and atomically-thin gold nanosheets with a thickness of only 0.47 nm (two atomic layers thick) are synthesized via a one-step aqueous approach at 20 °C, using methyl orange as a confining agent. Owing to the high surface-area-to-volume ratio, abundance of unsaturated atoms exposed on the surface and large interfacial areas arising from their ultrathin 2D nature, the as-prepared Au nanosheets demonstrate excellent catalysis performance in the model reaction of 4-nitrophenol reduction, and remarkable peroxidase-mimicking activity, which enables a highly sensitive colorimetric sensing of H2O2 with a detection limit of 0.11 × 10-6 m. This work represents the first fabrication of freestanding 2D gold with a sub-nanometer thickness, opens up an innovative pathway toward atomically-thin metal nanomaterials that can serve as model systems for inspiring fundamental advances in materials science, and holds potential across a wide region of applications.

Surface Fatigue Behavior of a WC/aC:H Thin-Film and the Tribochemical Impact of Zinc Dialkyldithiophosphate.

In wind turbine gearboxes, (near-)surface initiated fatigue is attributed to be the primary failure mechanism. In this work, the surface fatigue of a hydrogenated tungsten carbide/amorphous carbon (WC/aC:H) thin-film was tested under severe cyclic tribo-contact using polyalphaolefin (PAO) and PAO + zinc dialkyldithiophosphate (ZDDP) lubricants. The film was characterized in terms of its structure and chemistry using X-ray diffraction, analytical transmission electron microscopy, including electron energy loss spectroscopy (EELS), as well as X-ray photoelectron spectroscopy (XPS). The multilayer carbon thin-film exhibited promising surface fatigue performance showing a slight change in the hybridization state of the aC:H matrix. Dehydrogenation of the thin-film and subsequent transformation of cleaved C-H bonds to nonplanar sp2 carbon rings were inferred from EELS and XPS results. While tribo-induced changes to the aC:H matrix were not influenced by a nanometer-thick ZDDP reaction-film, the rate of oxidation of WC and its oxidation state were affected. While accelerating surface fatigue on a steel surface, the ZDDP-tribofilm protected the WC/aC:H film from surface fatigue. In contrast to the formation of polyphosphates from ZDDP molecules on steel surfaces, it appeared that on the WC/aC:H thin film surface, ZDDP molecules decompose to ZnO, suppressing the oxidative degradation of WC.

Low dose scanning transmission electron microscopy of organic crystals by scanning moiré fringes.

In the pharmaceutical industry, it is important to determine the effects of crystallisation and processes, such as milling, on the generation of crystalline defects in formulated products. Conventional transmission electron microscopy and scanning transmission electron microscopy (STEM) can be used to obtain information on length scales unobtainable by other techniques, however, organic crystals are extremely susceptible to electron beam damage. This work demonstrates a bright field (BF) STEM method that can increase the information content per unit specimen damage by the use of scanning moiré fringes (SMFs). SMF imaging essentially provides a magnification of the crystal lattice through the interference between closely aligned lattice fringes and a scanning lattice of similar spacing. The generation of SMFs is shown for three different organic crystals with varying electron beam sensitivity, theophylline, furosemide and felodipine. The electron fluence used to acquire the BF-STEM for the most sensitive material, felodipine was approximately 3.5 e-/Å2. After one additional scan of felodipine (total fluence of approximately 7.0 e-/Å2), the SMFs were no longer visible due to extensive damage caused to the crystal. Irregularity in the SMFs suggested the presence of defects in all the organic crystals. Further effort is required to improve the data analysis and interpretation of the resulting SMF images, allowing more information regarding the crystal structure and defects to be extracted.

Factors affecting electron beam damage in calcite nanoparticles.

We report electron fluence thresholds for the degradation of calcite nanoparticles under electron irradiation by both conventional and scanning TEM (CTEM and STEM), using time resolved phase contrast imaging and EDX spectroscopy at both 80 kV and 300 kV accelerating voltages. We show that the degradation pathway of calcite involves disruption of the crystal lattice with the evolution of pores and transformation to calcium oxide and carbon dioxide. Depending on irradiation conditions (CTEM or STEM), the calcium oxide formed can be either amorphous or crystalline, with the formation of the latter apparently being hindered by hydrocarbon contamination build up in STEM. For a given electron flux, irradiation at 300 kV prolongs the characteristic lifetime of the calcite lattice as compared to irradiation at 80 kV but with a corresponding reduction in both image contrast and energy dispersive X-ray (EDX) signal, consistent with the change in inelastic mean free path for electron scattering. STEM offers significant benefits over CTEM, however only in the presence of hydrocarbon contamination, increasing the fluence threshold for the detection of irradiation induced faults in the calcite lattice from 2.7 × 107 e- nm-2 for 300 kV CTEM to over 1.8 × 108 e- nm-2 for 300 kV STEM. This work forms a framework for reliable identification of discrete particle crystallinity in nominally amorphous, nanoscale calcium carbonate particles which is of importance for fundamental studies of crystallisation and also for the process control during the synthesis of such surfactant stabilised nanoparticles for application as over-based fuel detergents.