Phh Paul Bomans

The initial stages of template-controlled CaCO3 formation revealed by Cryo-TEM

Biogenic calcium carbonate forms the inorganic component of seashells, otoliths, and many marine skeletons, and its formation is directed by an ordered template of macromolecules. Classical nucleation theory considers crystal formation to occur from a critical nucleus formed by the assembly of ions from solution. Using cryotransmission electron microscopy, we found that template-directed calcium carbonate formation starts with the formation of prenucleation clusters. Their aggregation leads to the nucleation of amorphous nanoparticles in solution. These nanoparticles assemble at the template and, after reaching a critical size, develop dynamic crystalline domains, one of which is selectively stabilized by the template. Our findings have implications for template-directed mineral formation in biological as well as in synthetic systems.

The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors

Bone is a composite material in which collagen fibrils form a scaffold for a highly organized arrangement of uniaxially oriented apatite crystals. In the periodic 67 nm cross-striated pattern of the collagen fibril, the less dense 40-nm-long gap zone has been implicated as the place where apatite crystals nucleate from an amorphous phase, and subsequently grow. This process is believed to be directed by highly acidic non-collagenous proteins; however, the role of the collagen matrix during bone apatite mineralization remains unknown. Here, combining nanometre-scale resolution cryogenic transmission electron microscopy and cryogenic electron tomography with molecular modelling, we show that collagen functions in synergy with inhibitors of hydroxyapatite nucleation to actively control mineralization. The positive net charge close to the C-terminal end of the collagen molecules promotes the infiltration of the fibrils with amorphous calcium phosphate (ACP). Furthermore, the clusters of charged amino acids, both in gap and overlap regions, form nucleation sites controlling the conversion of ACP into a parallel array of oriented apatite crystals. We developed a model describing the mechanisms through which the structure, supramolecular assembly and charge distribution of collagen can control mineralization in the presence of inhibitors of hydroxyapatite nucleation.

The development of morphology and structure in hexagonal vaterite

Inspired by the remarkable shapes and properties of CaCO(3) biominerals, many studies have investigated biomimetic routes aiming at synthetic equivalents with similar morphological and structural complexity. Control over the morphology of CaCO(3) crystals has been demonstrated, among other methods, by the use of additives that selectively allow the development of specific crystal faces, while inhibiting others. Both for biogenic and biomimetic CaCO(3), the crystalline state is often preceded by an amorphous precursor phase, but still limited information is available on the details of the amorphous-to-crystalline transition. By using a combination of cryoTEM techniques (bright field imaging, cryo-tomography, low dose electron diffraction and cryo-darkfield imaging), we show for the first time the details of this transition during the formation of hexagonal vaterite crystals grown in the presence of NH(4)(+) ions. The formation of hexagonal plate-like vaterite occurs via an amorphous precursor phase. This amorphous phase converts into the crystalline state through a solid state transformation in which order and morphology develop simultaneously. The mineral initially develops as polycrystalline vaterite which transforms into a single crystal directed by an NH(4)(+)-induced crystal plane that acts as a templating surface.

A quasi-time-resolved CryoTEM study of the nucleation of CaCO3 under langmuir monolayers.

Calcium carbonate biomineralization uses complex assemblies of macromolecules that control the nucleation, growth, and positioning of the mineral with great detail. To investigate the mechanisms involved in these processes, for many years Langmuir monolayers have been used as model systems. Here, we descibe the use of cryogenic transmission electron microscopy in combination with selected area electron diffraction as a quasi-time-resolved technique to study the very early stages of this process. In this way, we assess the evolution of morphology, polymorphic type, and crystallographic orientation of the calcium carbonate formed. For this, we used a self-assembled Langmuir monolayer of a valine-based bisureido surfactant (1) spread on a CaCl2-containing subphase and deposited on a holey carbon TEM grid. In a controlled environment, the grid is exposed to an atmosphere containing NH3 and CO2 (the (NH4)2CO3 diffusion method) for precisely determined periods of time (reaction times 30-1800 s) before it was plunged into melting ethane. This procedure allows us to observe amorphous calcium carbonate (ACC) particles growing from a few tens of nanometers to hundreds of nanometers and then crystallizing to form [00.1] oriented vaterite. The vaterite in turn transforms to yield [10.0] oriented calcite. We also performed the reaction in the absence of monolayer or in the presence of a nondirective monolayer of surfactant containing an oligo(ethylene oxide) 2 head group. Both experiments also showed the formation of a transient amorphous phase followed by a direct conversion into randomly oriented calcite crystals. These results imply the specific though temporary stabilization of the (00.1) vaterite by the monolayer. However, experiments performed at higher CaCl2 concentrations show the direct conversion of ACC into [10.0] oriented calcite. Moreover, prolonged exposure to the electron beam shows that this transformation can take place as a topotactic process. The formation of the (100) calcite as final product under different conditions shows that the surfactant is very effective in directing the formation of this crystal plane. In addition, we present evidence that more than one type of ACC is involved in the processes described.

Temperature-Responsive Nanospheres with Bicontinuous Internal Structures from a Semicrystalline Amphiphilic Block Copolymer

Internally structured self-assembled nanospheres, cubosomes, are formed from a semicrystalline block copolymer, poly(ethylene oxide)-block-poly(octadecyl methacrylate) (PEO(39)-b-PODMA(17)), in aqueous dispersion. The PODMA block provides them with a temperature-responsive structure and morphology. Using cryo-electron tomography, we show that at room temperature these internally bicontinuous aggregates undergo an unprecedented order-disorder transition of the microphase-separated domains that is accompanied by a change in the overall aggregate morphology. This allows switching between spheres with ordered bicontinuous internal structures at temperatures below the transition temperature and more planar oblate spheroids with a disordered microphase-separated state above the transition temperature. The bicontinuous structures offer a number of possibilities for application as templates, e.g., for biomimetic mineralization or polymerization. Furthermore, the unique nature of the thermal transition observed for this system offers up considerable possibilities for their application as temperature-controlled release vessels.

Three-dimensional structure of P3HT assemblies in organic solvents revealed by Cryo-TEM

Poly(3-hexylthiophene) (P3HT) assemblies in vitrified organic solvents were visualized at nanometer scale resolution by cryo-transmission electron microscopy, low dose electron diffraction, and cryo-tomography revealing a three-dimensional lamellar structure formed by the stacking of the conjugated backbones of P3HT with a distance of 1.7 nm and increased order in the bulk of the nanowire. This combination of techniques reveals local structures in dispersion and the condensed state that play a crucial role in the performance of organic electronic devices.

Stabilization of amorphous calcium carbonate by controlling its particle size

Amorphous calcium carbonate (ACC) nanoparticles of different size are prepared using a flow system. Post-synthesis stabilization with a layer of poly[(α,β)-dl-aspartic acid] leads to stabilization of the ACC, but only for particles <100 nm. Larger and uncoated particles readily convert into the crystalline forms of CaCO₃. This shows that ACC is intrinsically stable below 100 nm.

The role of the amorphous phase on the biomimetic mineralization of collagen.

Bone is a hierarchically structured composite material whose basic building block is the mineralized collagen fibril, where the collagen is the scaffold into which the hydroxyapatite (HA) crystals nucleate and grow. Understanding the mechanisms of hydroxyapatite formation inside the collagen is key to unravelling osteogenesis. In this work, we employed a biomimetic in vitro mineralization system to investigate the role of the amorphous precursor calcium phosphate phase in the mineralization of collagen. We observed that the rate of collagen mineralization is highly dependent on the concentration of polyaspartic acid, an inhibitor of hydroxyapatite nucleation and inducer of intrafibrillar mineralization. The lower the concentration of the polymer, the faster the mineralization and crystallization. Addition of the non-collagenous protein C-DMP1, a nucleator of hydroxyapatite, substantially accelerates mineral infiltration as well as HA nucleation. We have also demonstrated that Cu ions interfere with the mineralization process first by inhibiting the entry of the calcium phosphate into the collagen, and secondly by stabilizing the ACP, such that it does not convert into HA. Interestingly, under these conditions mineralization happens preferentially in the overlap regions of the collagen fibril. Our results show that the interactions between the amorphous precursor phase and the collagen fibril play an important role in the control over mineralization.

Complex morphologies of self-assembled block copolymer micelles in binary solvent mixtures: the role of solvent–solvent correlations

The morphologies and sizes of micellar aggregates, composed of the tri-block copolymer P123 (EO20PO70EO20) in a mixture of the aprotic solvent N,N-dimethylformamide (DMF) and water, were investigated by combining Dynamic Light Scattering (DLS) and Cryogenic Transmission Electron Microscopy (cryo-TEM) experiments. At water concentrations between about 27 and 35 wt% bicontinuous micelles with distinct patterns were formed, in coexistence with very long, non-branched, worm-like micelles. Water concentration affects both the size and the morphology of the micellar aggregates. A careful study of the pure binary solvent mixture revealed the presence of dynamic solvent domains of nanometric size, even in the absence of copolymer. Strikingly, the size of these solvent nano-domains closely matched the size of the bicontinuous micelles in a polymer solution for the same water/DMF ratios. We discuss these findings in terms of spinodal decomposition of the polymer solution, in which two-solvent domains contain solvent quality fluctuations that could determine the decomposition. In addition, we suggest another “soft confinement” mechanism that could be responsible for the formation of bicontinuous micelles. The local excess of one of the solvent species in the nano-domains could entrap a metastable morphology.

Visualizing order in dispersions and solid state morphology with CryoTEM and electron tomography: P3HT:PCBM organic solar cells

Building blocks for organic solar cells are made from P3HT in a P3HT:PCBM solution in toluene and used to tune the morphology of the photoactive layer. The approach presented here decouples the structure and morphology formation, providing precise control over both the structures in solution and the morphology of the photoactive layer. For the characterization of the nanostructures in the organic casting solutions, cryo-TEM was successfully employed, and reveals the P3HT crystals and even the 1.7 nm lamellar stacking, which in combination with cryogenic low dose electron diffraction clearly proves the high crystallinity of P3HT aggregates realized. The photoactive layers made from pre-crystallized solutions show a morphology that is closely related to the structures in solution. A clear trend of decreasing open circuit voltage and increasing short circuit current with increasing order in the casting solutions and the devices was observed, which correlates with the evolution of the morphology from very intermixed with small fibrillar structures to phase-separated with large polymer crystals, as evaluated from representative devices, characterized in 3D with electron tomography.