Project description:A big step forward for composite application in the sector of structural materials is given by the use of Multi-Wall Carbon Nanotubes (MWCNTs) functionalized with hydrogen bonding moieties, such as barbiturate and thymine, to activate self-healing mechanisms and integrate additional functionalities. These materials with multiple healing properties at the same damaged site, imparted by hydrogen bonds, will also have the potential to improve material reliability, extend the service life, reduce replacement costs, and improve product safety. This revolutionary approach is obtained by integrating the non-covalent interactions coupled with the conventional covalent approach used to cross-link the polymer. The objective of this work is to characterize rubber-toughened supramolecular self-healing epoxy formulations based on unfunctionalized and functionalized MWCNTs using Tunneling Atomic Force Microscopy (TUNA). This advanced technique clearly shows the effect produced by the hydrogen bonding moieties acting as reversible healing elements by their simultaneous donor and acceptor character, and covalently linked to MWCNTs to originate self-healing nanocomposites. In particular, TUNA proved to be very effective for the morphology study of both the unfunctionalized and functionalized carbon nanotube-based conductive networks, thus providing useful insights aimed at understanding the influence of the intrinsic nature of the nanocharge on the final properties of the multifunctional composites.
Project description:The nuclear pore complex (NPC) is a proteinaceous assembly that regulates macromolecular transport into and out of the nucleus. Although the structure of its scaffold is being revealed in increasing detail, its transport functionality depends upon an assembly of intrinsically disordered proteins (called FG-Nups) anchored inside the pore's central channel, which have hitherto eluded structural characterization. Here, using high-resolution atomic force microscopy, we provide a structural and nanomechanical analysis of individual NPCs. Our data highlight the structural diversity and complexity at the nuclear envelope, showing the interplay between the lamina network, actin filaments, and the NPCs. It reveals the dynamic behaviour of NPC scaffolds and displays pores of varying sizes. Of functional importance, the NPC central channel shows large structural diversity, supporting the notion that FG-Nup cohesiveness is in a range that facilitates collective rearrangements at little energetic cost. Finally, different nuclear transport receptors are shown to interact in qualitatively different ways with the FG-Nups, with particularly strong binding of importin-β.
Project description:The CTCF protein has emerged as a key architectural protein involved in genome organization. Although hypothesized to initiate DNA looping, direct evidence of CTCF-induced DNA loop formation is still missing. Several studies have shown that the 11 zinc finger (11 ZF) domain of CTCF is actively involved in DNA binding. We here use atomic force microscopy to examine the effect of the 11 ZF domain comprising residues 266-579 (11 ZF CTCF) and the 3 ZF domain comprising residues 402-494 (6-8 ZF CTCF) of human CTCF on the DNA morphology. Our results show that both domains alter the DNA architecture from the relaxed morphology observed in control DNA samples to compact circular complexes, meshes, and networks, offering important insights into the multivalent character of the 11 ZF CTCF domain. Atomic force microscopy images reveal quasi-circular DNA/CTCF complexes, which are destabilized upon replacing the 11 ZF CTCF by the 6-8 ZF CTCF domain, highlighting the role of the 11 ZF motif in loop formation. Intriguingly, the formation of circular DNA/CTCF complexes is dominated by non-specific binding, whereby contour length and height profiles suggest a single DNA molecule twice wrapped around the protein.
Project description:Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM)1 has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules2. Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms3 to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset.
Project description:AFM has developed into a powerful tool in structural biology, providing topographs of proteins under close-to-native conditions and featuring an outstanding signal/noise ratio. However, the imaging mechanism exhibits particularities: fast and slow scan axis represent two independent image acquisition axes. Additionally, unknown tip geometry and tip-sample interaction render the contrast transfer function nondefinable. Hence, the interpretation of AFM topographs remained difficult. How can noise and distortions present in AFM images be quantified? How does the number of molecule topographs merged influence the structural information provided by averages? What is the resolution of topographs? Here, we find that in high-resolution AFM topographs, many molecule images are only slightly disturbed by noise, distortions, and tip-sample interactions. To identify these high-quality particles, we propose a selection criterion based on the internal symmetry of the imaged protein. We introduce a novel feature-based resolution analysis and show that AFM topographs of different proteins contain structural information beginning at different resolution thresholds: 10 A (AqpZ), 12 A (AQP0), 13 A (AQP2), and 20 A (light-harvesting-complex-2). Importantly, we highlight that the best single-molecule images are more accurate molecular representations than ensemble averages, because averaging downsizes the z-dimension and "blurs" structural details.
Project description:While offering high resolution atomic and electronic structure, scanning probe microscopy techniques have found greater challenges in providing reliable electrostatic characterization on the same scale. In this work, we offer electrostatic discovery atomic force microscopy, a machine learning based method which provides immediate maps of the electrostatic potential directly from atomic force microscopy images with functionalized tips. We apply this to characterize the electrostatic properties of a variety of molecular systems and compare directly to reference simulations, demonstrating good agreement. This approach offers reliable atomic scale electrostatic maps on any system with minimal computational overhead.
Project description:The Atomic Force Microscope (AFM) possesses several desirable imaging features including the ability to produce height profiles as well as two-dimensional images, in fluid or air, at high resolution. AFM has been used to study a vast selection of samples on the scale of angstroms to micrometers. However, current AFMs cannot access samples with vertical topography of the order of 100 μm or greater. Research efforts have produced AFM scanners capable of vertical motion greater than 100 μm, but commercially available probe tip lengths are still typically less than 10 μm high. Even the longest probe tips are below 100 μm and even at this range are problematic. In this paper, we present a method to hand-fabricate "Deep AFM" probes with tips of the order of 100 μm and longer so that AFM can be used to image samples with large scale vertical topography, such as fractured bone samples.
Project description:Anode free concepts are gaining traction in battery research. To improve cyclability, a better understanding of the deposition processes and morphologies is necessary. Correlative experiments enable a link between a variety of properties obtained, such as chemical, mechanical or electrochemical data. Here, electron paramagnetic resonance imaging (EPRI) is correlated with atomic force microscopy (AFM) to gain a deeper understanding of the microscopic topography and local stiffness at different intensities of the lithium selective EPRI map. Experiments were carried out on a sample of lithium deposited on copper foil from standard battery electrolyte. The correlation of both methods reveals that EPRI has a high sensitivity towards small lithium structures, while bulk lithium was not detected. The results demonstrate that EPRI can be used for prescreening to identify regions with different properties, which can then be analysed individually by AFM.
Project description:We present polynomial force reconstruction from experimental intermodulation atomic force microscopy (ImAFM) data. We study the tip-surface force during a slow surface approach and compare the results with amplitude-dependence force spectroscopy (ADFS). Based on polynomial force reconstruction we generate high-resolution surface-property maps of polymer blend samples. The polynomial method is described as a special example of a more general approximative force reconstruction, where the aim is to determine model parameters that best approximate the measured force spectrum. This approximative approach is not limited to spectral data, and we demonstrate how it can be adapted to a force quadrature picture.
Project description:Histones constitute the protein components of nucleosomes. Despite their small sizes, histones do not diffuse through the nuclear pore complex. Instead, they are transported to the nucleus by importins, either alone or in complex with histone chaperones. Determining the molecular size of the importin-histone complexes is key to understanding the mechanism of histone transport and also the potential roles of importins as histone chaperones and in the assembly of nucleosomes. Here we report a simple and reproducible sedimentation-velocity based method to determine the molecular sizes of importin-histone complexes using analytical ultracentrifugation. The method does not use any reporter tags or interaction with column resin thereby analyzing the interactions of the native proteins.