Chemical capacitance measurements reveal the impact of oxygen vacancies on the charge curve of LiNi0.5Mn1.5O4-δ thin films.

The level of oxygen deficiency δ in high-voltage spinels of the composition LiNi0.5Mn1.5O4-δ (LNMO) significantly influences the thermodynamic and kinetic properties of the material, ultimately affecting the cell performance of the corresponding lithium-ion batteries. This study presents a comprehensive defect chemical analysis of LNMO thin films with oxygen vacancy concentrations of 2.4% and 0.53%, focusing particularly on the oxygen vacancy regime around 4 V versus Li+/Li. A set of electrochemical properties is extracted from impedance measurements as a function of state-of-charge for the full tetrahedral-site regime (3.8 to 4.9 V versus Li+/Li). A defect chemical model (Brouwer diagram) is derived from the data, providing a coherent explanation for all important trends of the electrochemical properties and charge curve. Highly resolved chemical capacitance measurements allow a refining of the defect model for the oxygen vacancy regime, showing that a high level of oxygen deficiency not only impacts the amount of redox active Mn3+/4+, but also promotes the trapping of electrons in proximity to an oxygen vacancy. The resulting stabilisation of Mn3+ thereby mitigates the voltage reduction in the oxygen vacancy regime. These findings offer valuable insights into the complex influence of oxygen deficiency on the performance of lithium-ion batteries based on LNMO.

Adeno-Associated Virus-like Particles' Response to pH Changes as Revealed by nES-DMA.

Gas-phase electrophoresis on a nano-Electrospray Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) separates single-charged, native analytes according to the surface-dry particle size. A volatile electrolyte, often ammonium acetate, is a prerequisite for electrospraying. Over the years, nES GEMMA has demonstrated its unique capability to investigate (bio-)nanoparticle containing samples in respect to composition, analyte size, size distribution, and particle numbers. Virus-like particles (VLPs), being non-infectious vectors, are often employed for gene therapy applications. Focusing on adeno-associated virus 8 (AAV8) based VLPs, we investigated the response of these bionanoparticles to pH changes via nES GEMMA as ammonium acetate is known to exhibit these changes upon electrospraying. Indeed, slight yet significant differences in VLP diameters in relation to pH changes are found between empty and DNA-cargo-filled assemblies. Additionally, filled VLPs exhibit aggregation in dependence on the applied electrolyte's pH, as corroborated by atomic force microscopy. In contrast, cryogenic transmission electron microscopy did not relate to changes in the overall particle size but in the substantial particle's shape based on cargo conditions. Overall, we conclude that for VLP characterization, the pH of the applied electrolyte solution has to be closely monitored, as variations in pH might account for drastic changes in particles and VLP behavior. Likewise, extrapolation of VLP behavior from empty to filled particles has to be carried out with caution.

Performance modulation through selective, homogenous surface doping of lanthanum strontium ferrite electrodes revealed by in situ PLD impedance measurements.

Accelerating the oxygen reduction kinetics of solid oxide fuel cell (SOFC) cathodes is crucial to improve their efficiency and thus to provide the basis for an economically feasible application of intermediate temperature SOFCs. In this work, minor amounts of Pt were doped into lanthanum strontium ferrite (LSF) thin film electrodes to modulate the material's oxygen exchange performance. Surprisingly, Pt was found to be incorporated on the B-site of the perovskite electrode as non metallic Pt4+. The polarization resistance of LSF thin film electrodes with and without additional Pt surface doping was compared directly after film growth employing in situ electrochemical impedance spectroscopy inside a PLD chamber (i-PLD). This technique enables observation of the polarization resistance of pristine electrodes unaltered by degradation or any external contamination of the electrode surface. Moreover, growth of multi-layers of materials with different compositions on the very same single crystalline electrolyte substrate combined with in situ impedance measurements allow excellent comparability of different materials. Even a 5 nm layer of Pt doped LSF (1.5 at% Pt), i.e. a Pt loading of 80 ng cm-2, improved the polarization resistance by a factor of about 2.5. In addition, p(O2) and temperature dependent impedance measurements on both pure and Pt doped LSF were performed in situ and obtained similar activation energies and p(O2) dependence of the polarization resistance, which allow us to make far reaching mechanistic conclusions indicating that Pt4+ introduces additional active sites.

AFM investigation of APAC (antiplatelet and anticoagulant heparin proteoglycan).

Antiplatelet and anticoagulant drugs are classified antithrombotic agents with the purpose to reduce blood clot formation. For a successful treatment of many known complex cardiovascular diseases driven by platelet and/or coagulation activity, the need of more than one antithrombotic agent is inevitable. However, combining drugs with different mechanisms of action enhances risk of bleeding. Dual anticoagulant and antiplatelet (APAC), a novel semisynthetic antithrombotic molecule, provides both anticoagulant and antiplatelet properties in preclinical studies. APAC is entering clinical studies with this new exciting approach to manage cardiovascular diseases. For a better understanding of the biological function of APAC, comprehensive knowledge of its structure is essential. In this study, atomic force microscopy (AFM) was used to characterize APAC according to its structure and to investigate the molecular interaction of APAC with von Willebrand factor (VWF), since specific binding of APAC to VWF could reduce platelet accumulation at vascular injury sites. By the optimization of drop-casting experiments, we were able to determine the volume of an individual APAC molecule at around 600 nm3, and confirm that APAC forms multimers, especially dimers and trimers under the experimental conditions. By studying the drop-casting behavior of APAC and VWF individually, we depictured their interaction by using an indirect approach. Moreover, in vitro and in vivo conducted experiments in pigs supported the AFM results further. Finally, the successful adsorption of APAC to a flat gold surface was confirmed by using photothermal-induced resonance, whereby attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) served as a reference method.

Adeno-associated Virus Virus-like Particle Characterization via Orthogonal Methods: Nanoelectrospray Differential Mobility Analysis, Asymmetric Flow Field-Flow Fractionation, and Atomic Force Microscopy.

Adeno-associated virus (AAV)-based virus-like particles (VLPs) are thriving vectors of choice in the biopharmaceutical field of gene therapy. Here, a method to investigate purified AAV serotype 8 (AAV8) batches via a nanoelectrospray gas-phase mobility molecular analyzer (nES GEMMA), also known as an nES differential mobility analyzer, is presented. Indeed, due to AAV's double-digit nanometer scale, nES GEMMA is an excellently suited technique to determine the surface-dry particle size termed electrophoretic mobility diameter of such VLPs in their native state at atmospheric pressure and with particle-number-based detection. Moreover, asymmetric flow field-flow fractionation (AF4, also known as AFFFF) and atomic force microscopy (AFM) techniques were employed as orthogonal techniques for VLP characterization. In addition, AF4 was implemented to size-separate as well as to enrich and collect fractions of AAV8 VLPs after inducing analyte aggregation in the liquid phase. Bionanoparticle aggregation was achieved by a combination of heat and shear stress. These fractions were later analyzed with nES GEMMA (in the gas phase) and AFM (on a solid surface). Both techniques confirm the presence of dimers, trimers, and putative VLP oligomers. Last, AFM reveals even larger AAV8 VLP aggregates, which were not detectable by nES GEMMA because their heterogeneity combined with low abundance was below the limit of detection of the instrument. Hence, the combination of the employed orthogonal sizing methods with the separation technique AF4 allow a comprehensive characterization of AAV8 VLPs applied as vectors.

Facile isolation and analysis of sporopollenin exine from bee pollen.

We present facile methods to obtain purified sporopollenin exine capsules, and provide mass balances for classical and novel purification procedures. An ionic liquid, tetrabutyl phosphonium hydroxide turned out to be the most effective in removing the intine wall. The sporopollenin capsules were investigated by fluorescent microscopy, AFM, solid-state NMR and infrared Raman spectroscopy. The latter two methods showed that sunflower and rape exines have different proportions of O-aliphatic and aromatic constituents. Purified exine capsules were coated with functionalized fluorophores. The procedures presented in this paper could contribute to further spread of the applications of this hollow, and chemically highly resistant material.

CuSO4/[Cu(NH3)4]SO4-Composite Thermochemical Energy Storage Materials.

The thermochemical energy-storage material couple CuSO4/[Cu(NH3)4]SO4 combines full reversibility, application in a medium temperature interval (<350 °C), and fast liberation of stored heat. During reaction with ammonia, a large change in the sulfate solid-state structure occurs, resulting in a 2.6-fold expansion of the bulk material due to NH3 uptake. In order to limit this volume work, as well as enhance the thermal conductivity of the solid material, several composites of anhydrous CuSO4 with inorganic inert support materials were prepared and characterized with regard to their energy storage density, reversibility of the storage reaction, thermal conductivity, and particle morphology. The best thermochemical energy storage properties were obtained for a 10:1 CuSO4-sepiolite composite, combining an attractive energy storage density with slightly improved thermal conductivity and decreased bulk volume work compared to the pure salt.

Nano electrospray differential mobility analysis based size-selection of liposomes and very-low density lipoprotein particles for offline hyphenation to MALDI mass spectrometry.

Gas-phase electrophoresis of single-charged analytes (nanoparticles) enables their separation according to the surface-dry particle size (Electrophoretic Mobility Diameter, EMD), which corresponds to the diameter of spherical shaped particles. Employing a nano Electrospray Differential Mobility Analyzer (nES DMA), also known as nES Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA), allows sizing/size-separation and determination of particle-number concentrations. Separations are based on a constant high laminar sheath flow and a tunable, orthogonal electric field enabling scanning of EMDs in the nanometer size range. Additionally, keeping the voltage constant, only nanoparticles of a given EMD pass the instrument and can be collected on corresponding supporting materials for subsequent nanoparticle analyses applying e.g. microscopic, immunologic or spectroscopic techniques. In our proof-of-concept study we now focus for the first time on mass spectrometric (MS) characterization of DMA size-selected material. We carried out size-selection of liposomes, vesicles consisting of a lipid bilayer and an aqueous lumen employed as carriers in e.g. pharmaceutic, cosmetic or nutritional applications. Particles of 85 nm EMD were collected on gold-coated silicon wafers. Subsequently, matrix was applied and Matrix-Assisted Laser Desorption / Ionization (MALDI) MS carried out. However, we not only focused on plain liposomes but also demonstrated the applicability of our approach for very heterogeneous low density lipoprotein (VLDL) particles, a transporter of lipid metabolism. Our novel offline hyphenation of gas-phase electrophoresis (termed nES DMA or nES GEMMA) and MALDI-MS opens the avenue to the molecular characterization of size-select nanoparticles of complex nature.

Cycle Stability and Hydration Behavior of Magnesium Oxide and Its Dependence on the Precursor-Related Particle Morphology.

Thermochemical energy storage is considered as an auspicious method for the recycling of medium-temperature waste heat. The reaction couple Mg(OH)2⁻MgO is intensely investigated for this purpose, suffering so far from limited cycle stability. To overcome this issue, Mg(OH)2, MgCO3, and MgC2O4·2H2O were compared as precursor materials for MgO production. Depending on the precursor, the particle morphology of the resulting MgO changes, resulting in different hydration behavior and cycle stability. Agglomeration of the material during cyclization was identified as main reason for the decreased reactivity. Immersion of the spent material in liquid H2O decomposes the agglomerates restoring the initial reactivity of the material, thus serving as a regeneration step.

Size and molecular weight determination of polysaccharides by means of nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA).

Size, size distribution and molecular weight (MW) determination of nanoparticles and that are for example large polymers, are of great interest and pose an analytical challenge. In this context, nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) is a valuable tool with growing impact. Separation of single-charged analytes according to their electrophoretic mobility diameter (EMD) starting from single-digit EMDs up to several hundred nm diameters is possible. In case of spherical analytes, the EMD corresponds to the dry nanoparticle size. Additionally, the instrument is capable of number-based, single-particle detection following the recommendation of the European Commission for nanoparticle characterization (2011/696/EU). In case an EMD/MW correlation for a particular compound class (based on availability of well-defined standards) exists, a nanoparticle's MW can be determined from its EMD. In the present study, we focused on nES GEMMA of linear and branched, water-soluble polysaccharides forming nanoparticles and were able to obtain spectra for both analyte classes regarding single-charged species. Based on EMDs for corresponding analytes, an excellent EMD/MW correlation could be obtained in case of the branched natural polymer (dextran). This enables the determination of dextran MWs from nES GEMMA spectra despite high analyte polydispersity and in a size/MW range, where classical mass spectrometry is limited. EMD/MW correlations based on linear (pullulans, oat-ß-glucans) polymers were significantly different, possibly indicating challenges in the exact MW determination of these compounds by, for example, chromatographic and light scattering means. Despite these observations, nES GEMMA of linear, monosaccharide-based polymers enabled the determination of size and size-distribution of such dry bionanoparticles.

Dislocations Accelerate Oxygen Ion Diffusion in La0.8Sr0.2MnO3 Epitaxial Thin Films.

Revealing whether dislocations accelerate oxygen ion transport is important for providing abilities in tuning the ionic conductivity of ceramic materials. In this study, we report how dislocations affect oxygen ion diffusion in Sr-doped LaMnO3 (LSM), a model perovskite oxide that serves in energy conversion technologies. LSM epitaxial thin films with thicknesses ranging from 10 nm to more than 100 nm were prepared by pulsed laser deposition on single-crystal LaAlO3 and SrTiO3 substrates. The lattice mismatch between the film and substrates induces compressive or tensile in-plane strain in the LSM layers. This lattice strain is partially reduced by dislocations, especially in the LSM films on LaAlO3. Oxygen isotope exchange measured by secondary ion mass spectrometry revealed the existence of at least two very different diffusion coefficients in the LSM films on LaAlO3. The diffusion profiles can be quantitatively explained by the existence of fast oxygen ion diffusion along threading dislocations that is faster by up to 3 orders of magnitude compared to that in LSM bulk.

A bio-inspired method for direct measurement of local wall shear rates with micrometer localization using the multimeric protein von Willebrand factor as sensor molecule.

Wall shear rates are critical for a broad variety of fluidic phenomena and are taken into account in nearly every experimental or simulation study. Generally, shear rates are not observable directly but rather derived from other parameters such as pressure and flow, often assuming somehow idealized systems. However, there is a biological system which is able to constantly measure the wall shear as a part of a regulatory circuit: The blood circulation system takes advantage of shear rate sensor (protein)molecules (multimeric forms of von Willebrand Factor, VWF), which are dissolved in the blood plasma and dramatically change their conformation under shear conditions. The conformational changes are accompanied by several functional variations and therefore interplay with the regulation of the coagulation system. In this study, we use a recombinantly produced and therefore well-defined multimeric form of VWF as a sensor which directly responds to shear rates. Shear rates, up to 32.000 s-1, were obtained using a kind of micro-plate-to-plate rheometer capable of adsorbing shear-stretched VWF oligomeric molecules on a surface to conserve their differently stretched conformation and so allow detection of their elongation by atomic force microscopy. The laminar flow in this geometrically simple device has been characterized by adopting classical fluid dynamical models, in order to ensure well-known, stable shear rates which could be correlated quantitatively with an observed stretching of sensor molecules.

Image-Based Chemical Structure Determination.

Chemical imaging is a powerful tool for understanding the chemical composition and nature of heterogeneous samples. Recent developments in elemental, vibrational, and mass-spectrometric chemical imaging with high spatial resolution (50-200 nm) and reasonable timescale (a few hours) are capable of providing complementary chemical information about various samples. However, a single technique is insufficient to provide a comprehensive understanding of chemically complex materials. For bulk samples, the combination of different analytical methods and the application of statistical methods for extracting correlated information across different techniques is a well-established and powerful concept. However, combined multivariate analytics of chemical images obtained via different imaging techniques is still in its infancy, hampered by a lack of analytical methodologies for data fusion and analysis. This study demonstrates the application of multivariate statistics to chemical images taken from the same sample via various methods to assist in chemical structure determination.

Advanced portrayal of SMIL coating by allying CZE performance with in-capillary topographic and charge-related surface characterization.

A successive multiple ionic polymer layer (SMIL) coating composed of four layers improved the capillary electrophoretic separation of a recombinant major birch pollen allergen and closely related variants when poly(acrylamide-co-2-acrylamido-2-methyl-1-propansulfonate) (55% PAMAMPS) replaced dextran sulfate as terminal SMIL layer. 55% PAMAMPS decelerated the electroosmotic flow (EOF) due to its lower charge density. Atomic force microscopy (AFM) was used to investigate SMIL properties directly on the inner capillary surface and to relate them to EOF measurements and results of associated CZE separations of a mixture of model proteins and peptides that were performed in the same capillary. For the first time, AFM-based biosensing topography and recognition imaging mode (TREC) under liquid conditions was applied for a sequential characterization of the inner surface of a SMIL coated capillary after selected treatments including pristine SMIL, SMIL after contact with the model mixture, after alkaline rinsing, and the replenishment of the terminal polyelectrolyte layer. A cantilever with tip-tethered avidin was used to determine the charge homogeneity of the SMIL surface in the TREC mode. SMIL coated rectangular capillaries with 100 µm internal diameter assured accessibility of the inner surface for this cantilever type. Observed changes in CZE performance and EOF mobility during capillary treatment were also reflected by alterations in surface roughness and charge distribution of the SMIL coating. A renewal of the terminal SMIL layer restored the original surface properties of SMIL and the separation performance. The alliance of the novel TREC approach and CZE results allows for an improved understanding and a comprehensive insight in effects occurring on capillary coatings.

Nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) of liposomes: applicability of the technique for nano vesicle batch control.

Liposomes are biodegradable nanoparticle vesicles consisting of a lipid bilayer encapsulating an aqueous core. Entrapped cargo material is shielded from the extra-vesicular medium and sustained release of encapsulated material can be achieved. However, application of liposomes as nano-carriers demands their characterization concerning size and size distribution, particle-number concentration, occurrence of vesicle building blocks in solution and determination of the resulting vesicle encapsulation capacity. These questions can be targeted via gas-phase electrophoretic mobility molecular analysis (GEMMA) based on a nano electrospray (nES) charge-reduction source. This instrument separates single-charged nanoparticles in the gas-phase according to size in a high-laminar sheath-flow by means of an orthogonal, tunable electric field. nES GEMMA analysis enables to confirm liposome integrity after passage through the instrument (in combination with atomic force microscopy) as well as to exclude vesicle aggregation. Additionally, nanoparticle diameters at peak apexes and size distribution data are obtained. Differences of hydrodynamic and dry particle diameter values, as well as the effect of number- and mass-based concentration data analysis on obtained liposome diameters are shown. Furthermore, the repeatability of liposome preparation is studied, especially upon incorporation of PEGylated lipids in the bilayer. Finally, the instruments applicability to monitor mechanical stress applied to vesicles is demonstrated.

Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging.

Capillary zone electrophoresis (CZE) is a powerful analytical technique for fast and efficient separation of different analytes ranging from small inorganic ions to large proteins. However electrophoretic resolution significantly depends on the coating of the inner capillary surface. High technical efforts like Successive Multiple Ionic Polymer Layer (SMIL) generation have been taken to develop stable coatings with switchable surface charges fulfilling the requirements needed for optimal separation. Although the performance can be easily proven in normalized test runs, characterization of the coating itself remains challenging. Atomic force microscopy (AFM) allows for topographical investigation of biological and analytical relevant surfaces with nanometer resolution and yields information about the surface roughness and homogeneity. Upgrading the scanning tip to a molecular biosensor by adhesive molecules (like partly inverted charged molecules) allows for performing topography and recognition imaging (TREC). As a result, simultaneously acquired sample topography and adhesion maps can be recorded. We optimized this technique for electrophoresis capillaries and investigated the charge distribution of differently composed and treated SMIL coatings. By using the positively charged protein avidin as a single molecule sensor, we compared these SMIL coatings with respect to negative charges, resulting in adhesion maps with nanometer resolution. The capability of TREC as a functional investigation technique at the nanoscale was successfully demonstrated.

Shear-Dependent Interactions of von Willebrand Factor with Factor VIII and Protease ADAMTS 13 Demonstrated at a Single Molecule Level by Atomic Force Microscopy.

Vital functions of mammals are only possible due to the behavior of blood to coagulate most efficiently in vessels with particularly high wall shear rates. This is caused by the functional changes of the von Willebrand Factor (VWF), which mediates coagulation of blood platelets (primary hemostasis) especially when it is stretched under shear stress. Our data show that shear stretching also affects other functions of VWF: Using a customized device to simulate shear conditions and to conserve the VWF molecules in their unstable, elongated conformation, we visualize at single molecule level by AFM that VWF is preferentially cleaved by the protease ADAMTS13 at higher shear rates. In contrast to this high shear-rate-selective behavior, VWF binds FVIII more effectively only below a critical shear rate of ∼30.000 s(-1), indicating that under harsh shear conditions FVIII is released from its carrier protein. This may be required to facilitate delivery of FVIII locally to promote secondary hemostasis.

Comprehensive size-determination of whole virus vaccine particles using gas-phase electrophoretic mobility macromolecular analyzer, atomic force microscopy, and transmission electron microscopy.

Biophysical properties including particle size distribution, integrity, and shape of whole virus vaccine particles at different stages in tick-borne encephalitis (TBE) vaccines formulation were analyzed by a new set of methods. Size-exclusion chromatography (SEC) was used as a conservative sample preparation for vaccine particle fractionation and gas-phase electrophoretic mobility macromolecular analyzer (GEMMA) for analyzing electrophoretic mobility diameters of isolated TBE virions. The derived particle diameter was then correlated with molecular weight. The diameter of the TBE virions determined after SEC by GEMMA instrumentation was 46.8 ± 1.1 nm. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) were implemented for comparison purposes and to gain morphological information on the virion particle. Western blotting (Dot Blot) as an immunological method confirmed biological activity of the particles at various stages of the developed analytical strategy. AFM and TEM measurements revealed higher diameters with much higher SD for a limited number of virions, 60.4 ± 8.5 and 53.5 ± 5.3 nm, respectively. GEMMA instrumentation was also used for fractionation of virions with specifically selected diameters in the gas-phase, which were finally collected by means of an electrostatic sampler. At that point (i.e., after particle collection), AFM and TEM showed that the sampled virions were still intact, exhibiting a narrow size distribution (i.e., 59.8 ± 7.8 nm for AFM and 47.5 ± 5.2 nm for TEM images), and most importantly, dot blotting confirmed immunological activity of the collected samples. Furthermore dimers and virion artifacts were detected, too.

Chemometric analysis of multisensor hyperspectral images of precipitated atmospheric particulate matter.

The chemometric analysis of multisensor hyperspectral data allows a comprehensive image-based analysis of precipitated atmospheric particles. Atmospheric particulate matter was precipitated on aluminum foils and analyzed by Raman microspectroscopy and subsequently by electron microscopy and energy dispersive X-ray spectroscopy. All obtained images were of the same spot of an area of 100 × 100 µm(2). The two hyperspectral data sets and the high-resolution scanning electron microscope images were fused into a combined multisensor hyperspectral data set. This multisensor data cube was analyzed using principal component analysis, hierarchical cluster analysis, k-means clustering, and vertex component analysis. The detailed chemometric analysis of the multisensor data allowed an extensive chemical interpretation of the precipitated particles, and their structure and composition led to a comprehensive understanding of atmospheric particulate matter.

Ca(2+) concentration-dependent conformational change of FVIII B-domain observed by atomic force microscopy.

FVIII is a multi-domain protein organized in a heavy and a light chain, and a B-domain whose biological function is still a matter of debate. The 3D structure of a B-domain-deleted FVIII variant has been determined by X-ray crystallography, leaving unexplained the functional nature of the flexible B-domain which could play an important role in the structure-function relationship since it is removed during the activation process. To obtain clues on the function of the B-domain, the morphology of full-length FVIII and its isolated domains was determined in the absence or presence of Ca(2+). Recombinant full-length FVIII, the purified heavy chain, light chain and B-domain as well as B-domain-deleted rFVIII were analysed in buffers of different Ca(2+) concentrations by atomic force microscopy. In the absence of Ca(2+), FVIII appeared as a globular molecule, whereas at high amounts of Ca(2+) up to 50-nm long tail structures emerged. These tails could be identified as unravelled B-domains, as images of isolated B-domains showed the same morphology and heavy chains which include the B-domain were also rich of tails, whereas the isolated light chains and B-domain-deleted FVIII lacked any deviation from a globular shape. The images further suggested that the B-domain interacts with the light chain particularly at low Ca(2+) concentrations. Our results show a Ca(2+)-regulated conformational change of the B-domain in the context of full-length rFVIII. As the B-domain tightly associated with the core of the FVIII molecule under low Ca(2+)-concentrations, a stabilizing function on FVIII under non-activating conditions may be proposed.