New Vesicle-polymer Hybrids: the parachute architecture

The influence of organized media on polymerization reactions results in many cases in interesting morphologies of the polymeric material. In the present study, vesicle bilayers were used as ordered medium for the free radical polymerization of styrene. Cryo-electron microscopy gives evidence that the polymerization induces phase-separation phenomena leading to parachute-like morphologies. On the basis of general knowledge about vesicles and polymerizations in heterogeneous media, explanations for the observed phenomena are given. Bearing in mind that vesicles are outstanding models for membrane mimetic chemistry, it becomes evident that these findings can be relevant to the investigation of, for example, membrane−protein interactions

MICROANALYSIS ON BIOLOGICAL MATERIAL - THE ROLE OF PREPARATION METHODS AT LOW TEMPERATURES AND A POSSIBLE LINK WITH (IMMUNO)CYTOCHEMISTRY

Les coupes à congélation ultrafines sont idéales pour l'analyse et la localisation des éléments dans leur matrice physiologique. Le système de cryotransfert entre microtome et microscope électronique offre la possibilité d'examiner des coupes à l'état hydraté et congelé et d'observer ensuite le processus de sublimation jusqu'au séchage complet. En sublimant les coupes avant l'observation au microscope, on prend un risque de réhydratation avec les artefacts caractéristiques que celle-ci implique. Les artefacts de réhydratation peuvent être prévenus en exposant les coupes lyophilisées à des vapeurs fixatrices (formaldéhyde, le tétroxyde d'osmium par exemple). Ce procédé de fixation sèche par la vapeur a l'avantage de faciliter la conservation des échantillons en vue d'une microanalyse ultérieure et, de plus, il offre une possibilité d'application des techniques immunocytochimiques. Nous présenterons en illustration un exemple d'étude immunocytochimique et microanalytique des pancréas de rat.Frozen thin sections are considered the objects of choice for the study and localization of elements in their physiological matrix. A cryo-transfer system between microtome and microscope offers the possibility of studying fully hydrated sections in the frozen state and subsequently observe the sublimation process up to complete dryness. By freeze-drying sections before microscopic observation one takes the risk of rehydration with its accompanying typical artefacts. Rehydration artefacts are prevented when freeze-dried sections are exposed to vapour fixatives (e.g. formaldehyde, osmium tetroxide). A dry vapour fixation enables the safe conservation of samples for microanalysis and, in addition, offers the possibility for conducting immunocytochemical reactions on serial sections. This will be illustrated by an immunocytochemical and microanalytical study on the pancreas of the rat

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

The development of a glove-box/Vitrobot combination: Air-water interface events visualized by cryo-TEM

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Morphological control and molecular recognition by bis-urea hydrogen bonding in micelles of amphiphilic tri-block copolymers

Hydrogen bonding between urea groups of amphiphilic tri-block copolymers considerably affects their self-assembly in water, which results in a strong modification of morphology and viscosity of aqueous solutions; the hydrogen bonding motif in these amphiphilic copolymers allows molecular recognition of small molecules with complementary hydrogen bonding units

Morphological control and molecular recognition by bis-urea hydrogen bonding in micelles of amphiphilic tri-block copolymers

Hydrogen bonding between urea groups of amphiphilic tri-block copolymers considerably affects their self-assembly in water, which results in a strong modification of morphology and viscosity of aqueous solutions; the hydrogen bonding motif in these amphiphilic copolymers allows molecular recognition of small molecules with complementary hydrogen bonding units

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

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[(a,ß)-DL-aspartic acid] leads to stabilization of the ACC, but only for particle

Collagen targeting using protein-functionalized micelles: The strength of multiple weak interactions

Collagen is an important marker for the assessment of tissue remodeling, both in normal tissue maturation and in a variety of prevalent disease processes. Given the importance of multivalency in the natural interactions of collagen, multivalent ligands provide unique opportunities to target collagen architectures. Here, we explored the use of micelles as dynamic self-assembling multivalent scaffolds for the collagen binding protein CNA35. Despite the increased popularity of micelles as nanosized carriers in targeted drug delivery and molecular imaging, few studies have actually directly addressed the importance of multivalent interactions for micelle-based targeting. Native chemical ligation was used as a chemoselective and efficient method to prepare relatively well-defined and stable micelles with a tunable average protein content between 0 and 20 copies of CNA35 per micelle. The thermodynamics and kinetics of CNA35 micelle binding to collagen was studied using solid-phase and surface plasmon resonance assays. Multivalent interactions between the micelles and collagen had a remarkable effect on micellar stability, since no dissociation of collagen-bound micelles was observed even after extensive washing. In addition, an impressive enhancement of collagen affinity was observed both in vitro and ex vivo resulting from multivalent display of a so-called "nonbinding" variant of CNA35. This "restoration" of collagen affinity was subsequently also observed for liposomes displaying the same low-affinity CNA35 variant at a sufficient density. These results demonstrate the importance of multivalent interactions for micelle-based targeting and illustrate the strength of multiple weak interactions when targeting intrinsically multivalent extracellular matrix (ECM) proteins such as collagen

A reduced SNARE model for membrane fusion

A minimal model system was developed to mimic the SNARE-protein-mediated fusion of biological membranes (see picture). Fusion between two populations of liposomes is controlled by a pair of complementary lipidated oligopeptides that form noncovalent coiled-coil complexes and thereby force the membranes into close proximity to promote fusion. The model system displays the key characteristics of in vivo fusion events