Real-time Atomic Force Microscopy Research Articles

In Situ Real-Time Atomic Force Microscopy Observations of Chain Mobility at Polymer/Water Interfaces of Poly(methyl methacrylate), Poly(2-hydroxyethyl methacrylate), and Poly(2-methoxyethyl methacrylate) Films in Water.

Polymer materials are widely used in water or in contact with an aqueous environment. However, evaluating the chain mobility, a crucial parameter, at a polymer-water interface is challenging. In this study, we, for the first time, observed poly(methyl methacrylate) (PMMA), poly(2-hydroxyethyl methacrylate) (PHEMA), and poly(2-methoxyethyl methacrylate) (PMEMA) film surfaces in water via in situ real-time atomic force microscopy (AFM) in tapping mode and quantified the chain mobility. The average displacement between adjacent images (nm/8.75 min) was evaluated using particle image velocimetry. The displacement of PMMA, which has a high bulk glass-transition temperature (Tg) (108 °C) and exhibits limited water absorption, was low both in air (0.54 nm/8.75 min) and water (0.86), while PHEMA, which has a high bulk Tg (99 °C) and exhibits high water absorption, exhibited low mobility in air (0.40) but two orders of magnitude higher mobility in water (60). PMEMA, which has a low bulk Tg (14 °C) and exhibits limited water absorption, already started to move in air (4.5), and its mobility moderately increased in water (20). These behaviors were reasonable, considering the bulk Tg and water absorption characteristics of the polymers. Further, the chain mobility in water was compared with that of dried samples at high temperatures in air. The mobility of PMMA, PHEMA, and PMEMA in water corresponded to that of the dried samples observed in air below the surface Tg (97 °C) for PMMA, at ∼125 °C for PHEMA, and at ∼35 °C for PMEMA. In situ real-time AFM analysis of polymer materials in water is an effective method for evaluating the chain mobility at the polymer/water interface.

N-Formylation modifies membrane damage associated with PSMα3 interfacial fibrillation.

The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably depends on the expression of the phenol soluble modulins α3 (PSMα3) peptides, able to self-assemble into amyloid-like cross-α fibrils. Despite remarkable advances evidencing the crucial, yet insufficient, role of fibrils in PSMα3 cytotoxic activities towards host cells, the relationship between its molecular structures, assembly propensities, and modes of action remains an open intriguing problem. In this study, combining atomic force microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the charge provided by the N-terminal capping of PSMα3 alters its interactions with model membranes of controlled lipid composition without compromising its fibrillation kinetics or morphology. N-formylation eventually dictates PSMα3-membrane binding via electrostatic interactions with the lipid head groups. Furthermore, PSMα3 insertion within the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains only in the fluid phase of membranes and not in the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, formed along PSMα3 self-assembly and promoted at the membrane interface, likely disrupt membrane integrity via peptide accumulation and subsequent membrane thinning in a peptide concentration and lipid-dependent manner. Overall, our multiscale and multimodal approach sheds new light on the key roles of N-formylation and intermediate self-assembling entities, rather than mature fibrils, in dictating deleterious interactions of PSMα3 with membrane lipids, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.

Real-time microscopy of the relaxation of a glass

The understanding of the dynamics of a glass above its devitrification temperature remains incomplete. Here, we build a spatio-temporal map of the relaxation dynamics of a highly stable glass into its supercooled liquid using real-time atomic force microscopy imaging. This methodology enables direct visualization of the progression of the liquid phase and clarifies and quantifies the presence of localized fast mobility regions separated by giant length scales. Our data establish a clear correlation between dynamic length and time scales in glasses. This approach may also be applicable to unveil the microscopic structure and dynamics of other glass-forming systems with much shorter length and time scales, including liquid-cooled glasses.

Identifying a Mesomorphic Boundary Zone within Polybutene-1 Single Crystals Emerging During a Crystal–Crystal Transformation

We present experimental evidence that the transformation from one solid phase to another solid phase within a faceted polymer single crystal passes through a transient mesomorphic phase. This phase exists in the boundary zone separating the two crystalline phases of isotactic polybutene-1 (PB-1), i.e., Form II and Form I of PB-1. Employing real-time atomic force microscopy in tapping mode (TM-AFM), we followed the crystal–crystal transformation in time. TM-AFM measurements performed at about 20 °C under a nitrogen atmosphere with high humidity have allowed through differences in viscoelastic properties and associated changes in thickness to distinguish the two crystalline phases as well as the boundary zone. Measurements at an elevated temperature of 90 °C (which was far below the melting temperatures of both Form II and Form I crystals) further revealed that this boundary zone shows a transient and diffuse character and a distinctly increased thickness. Using high-resolution phase imaging of TM-AFM, we observed that within this rather extended boundary zone of up to some hundred nanometers at elevated temperatures, Form I crystals with a diameter of around 30 nm were growing in a softer and featureless matrix phase. The boundary zone and so the growth front of the Form I crystal advanced at an almost constant rate, suggesting that chains in the mesomorphic phase were considerably mobile. Our observation of an extended boundary zone probably indicates that compared to a direct crystal–crystal transformation, the corresponding free energy barrier for this transformation is lower within such a zone. Hence, our here presented model study sheds light on the poorly understood and complex pathways involved in a solid–solid transformation within a polymer single crystal.

P009 IL-3 receptor signalling suppresses chronic intestinal inflammation by controlling mechanobiology and tissue egress of regulatory T cells

Abstract Background The pathogenesis of inflammatory bowel diseases (IBD) such as Crohn’s disease (CD) and ulcerative colitis (UC) is still incompletely understood. However, despite having been described back in the 1980s and reported to be involved in chronic inflammatory arthritis, the role of interleukin 3 (IL-3) signaling via the IL-3 receptor (IL-3R) in the development and perpetuation of chronic intestinal inflammation remains largely unexplored. In this study, we therefore aimed to explore the role of IL-3 signaling via IL-3R in experimental colitis as well as human IBD. Methods We analysed IL-3 expression in lamina propria mononuclear cells (LPMCs) from patients with IBD and from mice with experimental colitis. We compared the development of T cell transfer colitis in Rag1-/- mice after transfer of Il3r-/- and Il3r+/+ naïve T cells and investigated the underlying mechanisms by immunofluorescence and functional cell trafficking assays. The mechanical properties of Il3r-/- and Il3r+/+ T cells were determined by real-time deformability cytometry (RT-DC) and atomic force microscopy (AFM) and cytoskeleton structure was analysed by scanning electron microscopy (SEM) and fluorescence recovery after photobleaching (FRAP). Results The expression of IL-3 and IL3+ T cells were increased in the gut of patients with IBD and IL-3 expression was associated to shorter flare-free survival. In vivo, experimental chronic colitis upon T cell transfer was exacerbated in the absence of IL-3 or IL-3R signalling. This was attributable to IL-3-induced changes in kinase signalling and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of regulatory T (Treg) cells from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Treg cells. (A) IL3 mRNA expression in colon tissue from patients.(B) Flare-free survival of IBD patients.(C) Colitis in Rag1–/– mice after transfer of naïve CD4+ T cells from Il3r+/+ and Il3r-/- mice. Mini-endoscopy (left) and IVIS (right).(D) RT-DC plot (left) and quantification (right) of thymic lymphocytes from Il3r-/- and Il3r+/+ mice.(E) Lightsheet microscopy (left) and quantification of TReg index (right, TReg fraction in Ozanimod/DATK32 per fraction in placebo) in mLNs from Rag1-/- mice with transfer colitis induced by Il3r-/- or Il3r+/+ T cells. Conclusion We uncover a crucial role of IL-3R signalling in regulating TReg mechanobiology and tissue egress. Together, our data demonstrate a central – probably counter-regulatory – role for IL-3 receptor signalling in Treg cells in the pathogenesis of murine and human chronic intestinal inflammation and therefore suggest that IL-3 might be a novel target for future therapeutic approaches in IBD.

Enzyme Synergy in Transient Clusters of Endo- and Exocellulase Enables a Multilayer Mode of Processive Depolymerization of Cellulose.

Biological degradation of cellulosic materials relies on the molecular-mechanistic principle that internally chain-cleaving endocellulases work synergistically with chain end-cleaving exocellulases in polysaccharide chain depolymerization. How endo–exo synergy becomes effective in the deconstruction of a solid substrate that presents cellulose chains assembled into crystalline material is an open question of the mechanism, with immediate implications on the bioconversion efficiency of cellulases. Here, based on single-molecule evidence from real-time atomic force microscopy, we discover that endo- and exocellulases engage in the formation of transient clusters of typically three to four enzymes at the cellulose surface. The clusters form specifically at regular domains of crystalline cellulose microfibrils that feature molecular defects in the polysaccharide chain organization. The dynamics of cluster formation correlates with substrate degradation through a multilayer-processive mode of chain depolymerization, overall leading to the directed ablation of single microfibrils from the cellulose surface. Each multilayer-processive step involves the spatiotemporally coordinated and mechanistically concerted activity of the endo- and exocellulases in close proximity. Mechanistically, the cooperativity with the endocellulase enables the exocellulase to pass through its processive cycles ∼100-fold faster than when acting alone. Our results suggest an advanced paradigm of efficient multienzymatic degradation of structurally organized polymer materials by endo–exo synergetic chain depolymerization.

Processive Enzymes Kept on a Leash: How Cellulase Activity in Multienzyme Complexes Directs Nanoscale Deconstruction of Cellulose.

Biological deconstruction of polymer materials gains efficiency from the spatiotemporally coordinated action of enzymes with synergetic function in polymer chain depolymerization. To perpetuate enzyme synergy on a solid substrate undergoing deconstruction, the overall attack must alternate between focusing the individual enzymes locally and dissipating them again to other surface sites. Natural cellulases working as multienzyme complexes assembled on a scaffold protein (the cellulosome) maximize the effect of local concentration yet restrain the dispersion of individual enzymes. Here, with evidence from real-time atomic force microscopy to track nanoscale deconstruction of single cellulose fibers, we show that the cellulosome forces the fiber degradation into the transversal direction, to produce smaller fragments from multiple local attacks (“cuts”). Noncomplexed enzymes, as in fungal cellulases or obtained by dissociating the cellulosome, release the confining force so that fiber degradation proceeds laterally, observed as directed ablation of surface fibrils and leading to whole fiber “thinning”. Processive cellulases that are enabled to freely disperse evoke the lateral degradation and determine its efficiency. Our results suggest that among natural cellulases, the dispersed enzymes are more generally and globally effective in depolymerization, while the cellulosome represents a specialized, fiber-fragmenting machinery.

Statistical Characterization of Heterogeneous Dissolution Rates of Calcite from In situ and Real-Time AFM Imaging

The evolution of the surface topography of a calcite crystal subject to dissolution is documented through in situ real-time imaging obtained via atomic force microscopy (AFM). The dissolution process takes place by exposing the crystal surface to deionized water. AFM data allow detection of nucleation and expansion of mono- and multilayer rhombic etch pits and are employed to estimate the spreading rate of these structures. Spatially heterogeneous distributions of local dissolution rate are evaluated from the difference between topographic measurements taken at prescribed time intervals. We rest on a stochastic framework of analysis viewing the dissolution rate as a generalized sub-Gaussian (GSG) spatially correlated random process. Our analysis yields: (i) a quantitative assessment of the temporal evolution of the statistics of the dissolution rates as well as their spatial increments; (ii) a characterization of the degree of spatial correlation of dissolution rates and of the way this is linked to the various mechanisms involved in the dissolution process and highlighted through the experimental evidences. Our results indicate that the parameters driving the statistics of the GSG distribution and the spreading rate of the multilayer pits display a similar trend in time, thus suggesting that the evolution of these structures imprints the statistical features of local dissolution rates.Article HighlightsWe investigate dynamics of dissolution patterns on a calcite crystal in contact with deionized water via AFM imagingTemporal behavior of parameters of our statistical model is consistent with surface pattern evolutionA nested model for the spatial correlation of rates embeds multiple mechanisms driving dissolution rate.

The mechanism driving a solid-solid phase transition in a biomacromolecular crystal.

Solid-solid phase transitions (SSPTs) occur between distinguishable crystalline forms. Because of their importance in application and theory in materials science and condensed-matter physics, SSPTs have been studied most extensively in metallic alloys, inorganic salts and small organic molecular crystals, but much less so in biomacromolecular crystals. In general, the mechanisms of SSPTs at the atomic and molecular levels are not well understood. Here, the ordered molecular rearrangements in biomacromolecular crystals of the adenine riboswitch aptamer are described using real-time serial crystallography and solution atomic force microscopy. Large, ligand-induced conformational changes drive the initial phase transition from the apo unit cell (AUC) to the trans unit cell 1 (TUC1). During this transition, coaxial stacking of P1 duplexes becomes the dominant packing interface, whereas P2-P2 interactions are almost completely disrupted, resulting in 'floating' layers of molecules. The coupling points in TUC1 and their local conformational flexibility allow the molecules to reorganize to achieve the more densely packed and energetically favorable bound unit cell (BUC). This study thus reveals the interplay between the conformational changes and the crystal phases - the underlying mechanism that drives the phase transition. Using polarized video microscopy to monitor SSPTs in small crystals at high ligand concentration, the time window during which the major conformational changes take place was identified, and the in crystallo kinetics have been simulated. Together, these results provide the spatiotemporal information necessary for informing time-resolved crystallography experiments. Moreover, this study illustrates a practical approach to characterization of SSPTs in transparent crystals.

Preparation of DNA Origami Belt and Effect of pH on Its Stability

Abstract The long single-stranded DNA containing multiple copy units prepared by rolling circle amplification technique was used as a scaffold strand. The scaffold strands were hybridized to three short staple strands by molecular self-assembly and produced DNA origami belt. Using the principle of SYBR Green real-time fluorescence quantitative PCR method and atomic force microscopy (AFM) imaging technology, the formation of double-stranded structure was examined by the change of fluorescence signal intensity in the reaction solution while the morphology of DNA belts was observed by AFM intuitively. The effect of pH value on the stability of DNA belt was studied, and its performance of pH-resistant in the wide pH range of 3–11 was obtained. DNA belts exhibited the best stability in neutral pH enviroment (pH = 7), which showed a significantly higher fluorescence signal than that of other groups within 24 h. DNA belts had poor resistance to acid and alkali environment, especially to extreme pH conditions (pH = 3, pH = 11). At extreme pH, even instantaneous treatments (

Observation of Stress Corrosion Cracking of Stainless Steel Using Real-Time In-Situ High-Speed Atomic Force Microscopy and Correlative Techniques

Observation of Stress Corrosion Cracking of Stainless Steel Using Real-Time In-Situ High-Speed Atomic Force Microscopy and Correlative Techniques

Advance application of Raman spectroscopy for quantitative analysis of noncrystalline components in thin films of poly(ε‐caprolactone)/poly(butadiene) blends

AbstractA quantitative analysis method for the distribution of noncrystalline poly(butadiene) component in poly(ε‐caprolactone)/poly(butadiene) (PCL/PB) binary blends have been analyzed by advance application of Raman spectroscopy, optical microscopy, and differential scanning calorimetry (DSC) techniques. Thin films of different compositions of PCL/PB binary blends were prepared from solution and isothermally crystallized at a certain temperature. After calibration with real data, quantitative analyses by Raman spectroscopy revealed the amorphous PB are trapped inside the PCL crystals. Polarized optical microscopy and real time atomic force microscopy were used to collect data for the crystal morphology and crystal growth rate. For pure PCL crystals, a morphology of truncated lozenge shape was observed, independent of crystallization temperature and regardless of the blends compositions. For the pure PCL and their blends, almost unique crystal growth rate was found. The miscibility behaviors using DSC were drawn through melting point depression method. The Hoffman‐Weeks extrapolations of the blends were found to be linear and identical with those of the neat PCL. The interaction parameter for the blends indicating that the PCL and PB blends have no intermolecular interaction, confirming the blends are immiscible. Despite the immiscibility of the blend, the PCL crystals do not bend during the growth process and do not reduce the growth rate as they do for miscible blend systems.

Real-Time AFM and Impedance Corrosion Monitoring of Environmentally Friendly Ceria Films on AA7075

Cerium-based conversion coatings have emerged as promising green alternatives to the harmful chromium-based ones, but the mechanism of corrosive protection still remains a subject of academic and industrial research. This study focuses at small scale phenomena of corrosion inhibition imparted by ceria (CeO2) to AA7075. Ceria nanoparticles were deposited from diluted and concentrated CeO2 sols by immersion. A multi-analytical approach, combining Atomic Force microscopy (AFM), Scanning Kelvin Probe Force Microscopy, Glow Discharge Optical Emission Spectroscopy, open circuit potential and electrochemical impedance spectroscopy was employed. Deposition of ceria films led to deactivation of cathodic sites, i.e. decreased Volta potential difference, resulting in increased corrosion inhibition. In situ AFM real-time monitoring revealed that during exposure to NaCl electrolyte, the changes in size of deposited ceria aggregates occurred: nanoparticles disintegrated/desorbed and re-deposited at the coating surface. The process was found to be dynamic in nature. Small particles size and inherent reactivity are believed to accelerate this phenomenon. Due to the greater CeO2 reservoir, this phenomenon was more pronounced with a thicker film, imparting longer term protection.

Erratum: Spheroid Culture of Mesenchymal Stromal Cells Results in Morphorheological Properties Appropriate for Improved Microcirculation.

[This corrects the article DOI: 10.1002/advs.201802104.].

Real Time and Quantitative Imaging of Lignocellulosic Films Hydrolysis by Atomic Force Microscopy Reveals Lignin Recalcitrance at Nanoscale.

Lignocellulosic biomass is considered as a sustainable source of energy and chemicals, but its recalcitrance to bioconversion still limits its use. In this paper, a strategy based on two aspects was developed to improve our knowledge on the lignin recalcitrance to enzymatic hydrolysis. First, lignocellulosic films of cellulose nanofibrils (CNFs) with increasing content of lignin (up to 40%) were prepared. Thanks to in situ real time Atomic Force Microscopy (AFM) measurements during the hydrolysis and by comparison with biochemical assays, the use of such films allows to fully assess the importance of the lignin content and of the arrangement between CNFs and lignin on the hydrolysis efficiency. In a second time, contrary to other studies by AFM which only followed a specific structure during enzymatic processes mostly on simple systems (CNFs or cellulose nanocrystals), a quantitative analysis of in-situ time-lapse measurements was developed.Itenablesto accurately address lignocellulosic biomass recalcitrance mechanisms mediated by lignin at nanoscale. Such analysis could pave the way for the use of a quantitative criteria to visualize in situ deconstruction of complex lignocellulosic substrates. Coupling the use of lignocellulosic films and dynamical AFM quantitative analysis to follow the evolution of the structure at nanoscale might lead to an effective targeting of new promising bioconversion strategies.

Kinetics of photo-dissolution within Ag/As2S3 heterostructure

Chalcogenide glass – silver heterostructures are candidates for photoresist and diffractive optical applications. To optimize their processing, we report the kinetics of Ag photo-dissolution in As2S3 matrix using in-situ optical transmission/reflection measurements and real time atomic force microscopy (AFM) imaging under optical illumination. The results indicate that photo-dissolution occurs in three stages with the extent and kinetics of each stage depending strongly on Ag film thickness. By contrast, the photo-dissolution is found to be independent of As2S3 matrix thickness. The extent of three stages also depends strongly on the laser dose and can be reduced to two stages at higher laser fluence. A comparative study of two oppositely stacked sample configurations: As2S3/Ag/glass and Ag/As2S3/glass show that the heterostructures respond differently to light illumination. For the former, Ag dissolves completely into As2S3 matrix at a faster rate than for the latter case. The origin of this difference is established by energy dispersive X-ray spectroscopy and AFM measurements.

Quaternary Amine-Terminated Quantum Dots Induce Structural Changes to Supported Lipid Bilayers.

The cytoplasmic membrane represents an essential barrier between the cytoplasm and the environment external to cells. Interaction with nanomaterials can alter the integrity of the cytoplasmic membrane through the formation of holes and membrane thinning, which can ultimately lead to adverse biological impacts. Here we use supported lipid bilayers as experimental models for the cytoplasmic membrane to investigate the impact of quantum dots functionalized with the cationic polymer poly(diallyldimethylammonium chloride) (PDDA) on membrane structure. Using a quartz crystal microbalance with dissipation monitoring we show that the positively charged quantum dots attach to and induce structural rearrangement to zwitterionic bilayers in solely the liquid-disordered phase and in those containing phase-segregated liquid-ordered domains. Real-time atomic force microscopy imaging revealed that PDDA-coated quantum dots and, to a lesser extent, PDDA itself induced the disappearance of liquid-ordered domains. We hypothesize this effect is due to an increase in energy per unit area caused by collisions between PDDA-coated quantum dots at the membrane surface. This increase in free energy per area exceeds the approximate free-energy change associated with membrane mixing between the liquid-ordered and liquid-disordered phases and results in the destabilization of membrane domains.

Interfacial Mechanism in Lithium-Sulfur Batteries: How Salts Mediate the Structure Evolution and Dynamics.

Lithium-sulfur batteries possess favorable potential for energy-storage applications because of their high specific capacity and the low cost of sulfur. Intensive understanding of the interfacial mechanism, especially the polysulfide formation and transformation under complex electrochemical environment, is crucial for the buildup of advanced batteries. Here, we report the direct visualization of interfacial evolution and dynamic transformation of the sulfides mediated by the lithium salts via real-time atomic force microscopy monitoring inside a working battery. The observations indicate that the lithium salts influence the structures and processes of sulfide deposition/decomposition during discharge/charge. Moreover, the distinct ion interaction and the diffusion in electrolytes manipulate the interfacial reactions determining the kinetics of the sulfide transformation. Our findings provide deep insights into surface dynamics of lithium-sulfur reactions revealing the salt-mediated mechanisms at nanoscale, which contribute to the profound understanding of the interfacial processes for the optimized design of lithium-sulfur batteries.

Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response. Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm2 for 12hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness. Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness. Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease.

Spontaneous oscillation in cell adhesion and stiffness measured using atomic force microscopy

Atomic force microscopy (AFM) is an attractive technique for studying biomechanical and morphological changes in live cells. Using real-time AFM monitoring of cellular mechanical properties, spontaneous oscillations in cell stiffness and cell adhesion to the extracellular matrix (ECM) have been found. However, the lack of automated analytical approaches to systematically extract oscillatory signals, and noise filtering from a large set of AFM data, is a significant obstacle when quantifying and interpreting the dynamic characteristics of live cells. Here we demonstrate a method that extends the usage of AFM to quantitatively investigate live cell dynamics. Approaches such as singular spectrum analysis (SSA), and fast Fourier transform (FFT) were introduced to analyze a real-time recording of cell stiffness and the unbinding force between the ECM protein-decorated AFM probe and vascular smooth muscle cells (VSMCs). The time series cell adhesion and stiffness data were first filtered with SSA and the principal oscillatory components were isolated from the noise floor with the computed eigenvalue from the lagged-covariance matrix. Following the SSA, the oscillatory parameters were detected by FFT from the noise-reduced time series data sets and the sinusoidal oscillatory components were constructed with the parameters obtained by FFT.