Time-resolved Atomic Force Microscopy Research Articles

Nanoactuator for Neuronal Optoporation.

Light-driven modulation of neuronal activity at high spatial-temporal resolution is becoming of high interest in neuroscience. In addition to optogenetics, nongenetic membrane-targeted nanomachines that alter the electrical state of the neuronal membranes are in demand. Here, we engineered and characterized a photoswitchable conjugated compound (BV-1) that spontaneously partitions into the neuronal membrane and undergoes a charge transfer upon light stimulation. The activity of primary neurons is not affected in the dark, whereas millisecond light pulses of cyan light induce a progressive decrease in membrane resistance and an increase in inward current matched to a progressive depolarization and action potential firing. We found that illumination of BV-1 induces oxidation of membrane phospholipids, which is necessary for the electrophysiological effects and is associated with decreased membrane tension and increased membrane fluidity. Time-resolved atomic force microscopy and molecular dynamics simulations performed on planar lipid bilayers revealed that the underlying mechanism is a light-driven formation of pore-like structures across the plasma membrane. Such a phenomenon decreases membrane resistance and increases permeability to monovalent cations, namely, Na+, mimicking the effects of antifungal polyenes. The same effect on membrane resistance was also observed in nonexcitable cells. When sustained light stimulations are applied, neuronal swelling and death occur. The light-controlled pore-forming properties of BV-1 allow performing "on-demand" light-induced membrane poration to rapidly shift from cell-attached to perforated whole-cell patch-clamp configuration. Administration of BV-1 to ex vivo retinal explants or in vivo primary visual cortex elicited neuronal firing in response to short trains of light stimuli, followed by activity silencing upon prolonged light stimulations. BV-1 represents a versatile molecular nanomachine whose properties can be exploited to induce either photostimulation or space-specific cell death, depending on the pattern and duration of light stimulation.

Effect of methanol and photoinduced surface oxygen vacancies on the charge carrier dynamics in TiO2

The migration of holes in metal-oxide semiconductors such as TiO2 plays a vital role in (photo)catalytic applications. The dynamics of charge carriers under operation conditions can be influenced by both methanol addition and photoinduced surface oxygen vacancies (PI-SOVs). Nevertheless, the existing knowledge of the effect of methanol as a function of PI-SOVs solely concentrates on the chemical reduction process. For this reason, the fundamental understanding of the time-dependent charge carrier-vacancy interactions in the presence of methanol is impaired. Here, we conducted time-resolved atomic force microscopy measurements to quantitatively disclose the effect of methanol adsorption on the dynamics of hole migration in TiO2. Our results show that time constants associated with the migration of charge carriers significantly change due to methanol adsorption. Moreover, the energy landscape of the hole migration barrier was dominated and lowered by PI-SOVs. Our findings contribute to the physics of charge carrier dynamics by enabling the engineering of charge carrier-vacancy interactions.

Lipid bilayer fluidity and degree of order regulates small EVs adsorption on model cell membrane

Small extracellular vesicles (sEVs) are known to play an important role in the communication between distant cells and to deliver biological information throughout the body. To date, many studies have focused on the role of sEVs characteristics such as cell origin, surface composition, and molecular cargo on the resulting uptake by the recipient cell. Yet, a full understanding of the sEV fusion process with recipient cells and in particular the role of cell membrane physical properties on the uptake are still lacking. Here we explore this problem using sEVs from a cellular model of triple-negative breast cancer fusing to a range of synthetic planar lipid bilayers both with and without cholesterol, and designed to mimic the formation of ‘raft’-like nanodomains in cell membranes. Using time-resolved Atomic Force Microscopy we were able to track the sEVs interaction with the different model membranes, showing the process to be strongly dependent on the local membrane fluidity. The strongest interaction and fusion is observed over the less fluid regions, with sEVs even able to disrupt ordered domains at sufficiently high cholesterol concentration. Our findings suggest the biophysical characteristics of recipient cell membranes to be crucial for sEVs uptake regulation.

Customization of an atomic force microscope for multidimensional measurements under environmental conditions.

Atomic force microscopy (AFM) is an analytical surface characterization tool that reveals the surface topography at a nanometer length scale while probing local chemical, mechanical, and even electronic sample properties. Both contact (performed with a constant deflection of the cantilever probe) and dynamic operation modes (enabled by demodulation of the oscillation signal under tip-sample interaction) can be employed to conduct AFM-based measurements. Although surface topography is accessible regardless of the operation mode, the resolution and the availability of the quantified surface properties depend on the mode of operation. However, advanced imaging techniques, such as frequency modulation, to achieve high resolution, quantitative surface properties are not implemented in many commercial systems. Here, we show the step-by-step customization of an atomic force microscope. The original system was capable of surface topography and basic force spectroscopy measurements while employing environmental control, such as temperature variation of the sample/tip, etc. We upgraded this original setup with additional hardware (e.g., a lock-in amplifier with phase-locked loop capacity, a high-voltage amplifier, and a new controller) and software integration while utilizing its environmental control features. We show the capabilities of the customized system with frequency modulation-based topography experiments and automated voltage and/or distance spectroscopy, time-resolved AFM, and two-dimensional force spectroscopy measurements under ambient conditions. We also illustrate the enhanced stability of the setup with active topography and frequency drift corrections. We believe that our methodology can be useful for the customization and automation of other scanning probe systems.

In Situ Observation of Dicalcium Phosphate Monohydrate Formation and Phase Transformation.

Calcium orthophosphates (CaPs), as important minerals in biomineralization and biomedicine, have attracted wide attention. Dicalcium phosphate monohydrate (DCPM, CaHPO4·H2O), the recently discovered crystalline CaP phase, has a higher metastability than dihydrate (DCPD, CaHPO4·2H2O) and anhydrate (DCPA, CaHPO4), which may lead to many potential applications in functional biomaterial development. However, the preparation of large-sized DCPM and the underlying mechanisms of its formation and phase evolution remain unclear. Herein, for the first time, we propose a method to prepare micrometer-sized DCPM under an acidic water-methanol mixture and using in situ time-resolved atomic force microscopy further explore its crystallization via dissolution of an acidic amorphous calcium phosphate. In support of the potential role of DCPM as the biomaterial, we demonstrate that DCPM can quickly evolve into more stable octacalcium phosphate in a near-physiological solution. This work provides a mechanistic understanding of the formation and phase transformation of DCPM, which may serve as a basis for subsequent synthesis and application of DCPM as functional biomaterials.

The Effect of Photoinduced Surface Oxygen Vacancies on the Charge Carrier Dynamics in TiO2 Films.

Metal-oxide semiconductors (MOS) are widely utilized for catalytic and photocatalytic applications in which the dynamics of charged carriers (e.g., electrons, holes) play important roles. Under operation conditions, photoinduced surface oxygen vacancies (PI-SOV) can greatly impact the dynamics of charge carriers. However, current knowledge regarding the effect of PI-SOV on the dynamics of hole migration in MOS films, such as titanium dioxide, is solely based upon volume-averaged measurements and/or vacuum conditions. This limits the basic understanding of hole-vacancy interactions, as they are not capable of revealing time-resolved variations during operation. Here, we measured the effect of PI-SOV on the dynamics of hole migration using time-resolved atomic force microscopy. Our findings demonstrate that the time constant associated with hole migration is strongly affected by PI-SOV, in a reversible manner. These results will nucleate an insightful understanding of the physics of hole dynamics and thus enable emerging technologies, facilitated by engineering hole-vacancy interactions.

Ergodic and Nonergodic Dynamics of Oxygen Vacancy Migration at the Nanoscale in Inorganic Perovskites.

Perovskites are widely utilized either as a primary component or as a substrate in which the dynamics of charged oxygen vacancy defects play an important role. Current knowledge regarding the dynamics of vacancy mobility in perovskites is solely based upon volume- and/or time-averaged measurements. This impedes our understanding of the basic physical principles governing defect migration in inorganic materials. Here, we measure the ergodic and nonergodic dynamics of vacancy migration at the relevant spatial and temporal scales using time-resolved atomic force microscopy techniques. Our findings demonstrate that the time constant associated with oxygen vacancy migration is a local property and can change drastically on short length and time scales, such that nonergodic states lead to a dramatic increase in the migration barrier. This correlated spatial and temporal variation in oxygen vacancy dynamics can extend hundreds of nanometers across the surface in inorganic perovskites.

Nanoscale force sensing of an ultrafast nonlinear optical response

The nonlinear optical response of a material is a sensitive probe of electronic and structural dynamics under strong light fields. The induced microscopic polarizations are usually detected via their far-field light emission, thus limiting spatial resolution. Several powerful near-field techniques circumvent this limitation by employing local nanoscale scatterers; however, their signal strength scales unfavorably as the probe volume decreases. Here, we demonstrate that time-resolved atomic force microscopy is capable of temporally and spatially resolving the microscopic, electrostatic forces arising from a nonlinear optical polarization in an insulating dielectric driven by femtosecond optical fields. The measured forces can be qualitatively explained by a second-order nonlinear interaction in the sample. The force resulting from this nonlinear interaction has frequency components below the mechanical resonance frequency of the cantilever and is thus detectable by regular atomic force microscopy methods. The capability to measure a nonlinear polarization through its electrostatic force is a powerful means to revisit nonlinear optical effects at the nanoscale, without the need for emitted photons or electrons from the surface.

Phosphorylated/Nonphosphorylated Motifs in Amelotin Turn Off/On the Acidic Amorphous Calcium Phosphate-to-Apatite Phase Transformation.

Amelotin (AMTN) as a matrix protein exerts a direct effect on biomineralization by modulating apatite (HAP) formation during the dental enamel maturation stage through the specific interaction of a potentially phosphorylated Ser-Ser-Glu-Glu-Leu (SSEEL) peptide fragment with calcium phosphate (Ca-P) surfaces. However, the roles of (non)phosphorylation of this evolutionarily conserved subdomain within AMTN remain poorly understood. Here, we show, by time-resolved atomic force microscopy (AFM) imaging of in situ HAP crystallization via the HPO42--rich amorphous calcium phosphate (acidic ACP), the on/off switching of the phase transformation process through a nonphosphorylation-to-phosphorylation transition of the SSEEL motif. Using high-resolution transmission electron microscopy (HRTEM), we observed that the acidic ACP phase is stabilized by the phosphorylated SSEEL motif, delaying its transformation to HAP, whereas the nonphosphorylated counterpart promotes HAP formation by accelerating the dissolution-recrystallization of the acidic ACP substrate. Dynamic force spectroscopy measurements demonstrate greater binding energies of nonphosphorylated SSEEL to the acidic ACP substrate by the formation of molecular peptide-ACP bonding, explaining the enhanced dissolution of the acidic ACP substrate by stronger complexion with surface Ca2+ ions. Our findings demonstrate direct evidence for the switching role of (non)phosphorylation of an evolutionarily conserved subdomain within AMTN in controlling the phase transition of growing enamel and designing tissue regeneration biomaterials.

Quantification of Biomolecular Dynamics Inside Real and Synthetic Nuclear Pore Complexes Using Time-Resolved Atomic Force Microscopy.

Over the past decades, atomic force microscopy (AFM) has emerged as an increasingly powerful tool to study the dynamics of biomolecules at nanometer length scales. However, the more stochastic the nature of such biomolecular dynamics, the harder it becomes to distinguish them from AFM measurement noise. Rapid, stochastic dynamics are inherent to biological systems comprising intrinsically disordered proteins. One role of such proteins is in the formation of the transport barrier of the nuclear pore complex (NPC): the selective gateway for macromolecular traffic entering or exiting the nucleus. Here, we use AFM to observe the dynamics of intrinsically disordered proteins from two systems: the transport barrier of native NPCs and the transport barrier of a mimetic NPC made using a DNA origami scaffold. Analyzing data recorded with 50–200 ms temporal resolution, we highlight the importance of drift correction and appropriate baseline measurements in such experiments. In addition, we describe an autocorrelation analysis to quantify time scales of observed dynamics and to assess their veracity—an analysis protocol that lends itself to the quantification of stochastic fluctuations in other biomolecular systems. The results reveal the surprisingly slow rate of stochastic, collective transitions inside mimetic NPCs, highlighting the importance of FG-nup cohesive interactions.

Time-Resolved Imaging of Bacterial Surfaces Using Atomic Force Microscopy.

Time-resolved atomic force microscopy (AFM) offers countless new modes by which to study bacterial cell physiology on relevant time scales, from mere milliseconds to hours and days on end. In addition, time-lapse AFM acts as a complementary tool to optical fluorescence microscopy (OFM), for which the combination offers a correlative link between the physical manifestation of bacterial phenotypes and molecular mechanisms obeying those principles. Herein we describe the essential materials and methods necessary for conducting time-resolved AFM and dual AFM/OFM experiments on bacteria.

Time-Resolved Study of Nanomorphology and Nanomechanic Change of Early-Stage Mineralized Electrospun Poly(lactic acid) Fiber by Scanning Electron Microscopy, Raman Spectroscopy and Atomic Force Microscopy.

In this study, scanning electron microscopy (SEM), Raman spectroscopy and high-resolution atomic force microscopy (AFM) were used to reveal the early-stage change of nanomorphology and nanomechanical properties of poly(lactic acid) (PLA) fibers in a time-resolved manner during the mineralization process. Electrospun PLA nanofibers were soaked in simulated body fluid (SBF) for different periods of time (0, 1, 3, 5, 7 and 21 days) at 10 °C, much lower than the conventional 37 °C, to simulate the slow biomineralization process. Time-resolved Raman spectroscopy analysis can confirm that apatites were deposited on PLA nanofibers after 21 days of mineralization. However, there is no significant signal change among several Raman spectra before 21 days. SEM images can reveal the mineral deposit on PLA nanofibers during the process of mineralization. In this work, for the first time, time-resolved AFM was used to monitor early-stage nanomorphology and nanomechanical changes of PLA nanofibers. The Surface Roughness and Young’s Modulus of the PLA nanofiber quantitatively increased with the time of mineralization. The electrospun PLA nanofibers with delicate porous structure could mimic the extracellular matrix (ECM) and serve as a model to study the early-stage mineralization. Tested by the mode of PLA nanofibers, we demonstrated that AFM technique could be developed as a potential diagnostic tool to monitor the early onset of pathologic mineralization of soft tissues.

Assembly Mechanism and Nanomechanics of Aβ in Alzheimer's Disease

Alzheimer's disease (AD) is characterized by progressive dementia and eventual loss of bodily functions that, historically, is associated with the formation of plaques composed of amyloid beta (Aβ), a 36-43 amino acid peptide. A much more complex picture is now emerging that involves many different species of Aβ oliogomers along the assembly pathway. We have used atomic force microscopy (AFM) to investigate Aβ assembly dynamics in order to observe fibril morphology and to characterize fibril elongation and nanomechanics in physiologically relevant buffers on mica as well as on supported lipid bilayers. We have especially focused on resolving small oligomers that is currently thought to initiate deterioration of brain function in AD and its drug intervention and on defining early stage oligomer to fibril growth by integrations between optical spectroscopy determination of protein conformation and AFM observations. We aim to apply quantitative kinetic schemes to identify the number of steps and kinetic constants using time-resolved AFM, thioflavin T fluorescence and circular dichroism data to define the transition from lag to log phase and the broader assembly mechanism.

In situ imaging of silicalite-1 surface growth reveals the mechanism of crystallization.

The growth mechanism of silicalite-1 (MFI zeolite) is juxtaposed between classical models that postulate silica molecules as primary growth units and nonclassical pathways based on the aggregation of metastable silica nanoparticle precursors. Although experimental evidence gathered over the past two decades suggests that precursor attachment is the dominant pathway, direct validation of this hypothesis and the relative roles of molecular and precursor species has remained elusive. We present an in situ study of silicalite-1 crystallization at characteristic synthesis conditions. Using time-resolved atomic force microscopy images, we observed silica precursor attachment to crystal surfaces, followed by concomitant structural rearrangement and three-dimensional growth by accretion of silica molecules. We confirm that silicalite-1 growth occurs via the addition of both silica molecules and precursors, bridging classical and nonclassical mechanisms.

Direct measurement of activation time and nucleation rate in capillary-condensed water nanomeniscus

We demonstrate real-time observation of nucleation of the single water nanomeniscus formed via capillary condensation. We directly measure (i) activation time by time-resolved atomic force microscopy and (ii) nucleation rate by statistical analysis of its exponential distribution, which is the experimental evidence that the activation process is stochastic and follows the Poisson statistics. It implies that formation of the water nanomeniscus is triggered by nucleation, which requires activation for producing a nucleus. We also find the dependence of the nucleation rate on the tip-sample distance and temperature.

Direct observation of dynamic shape transformation and coalescence in platinum nanosheets on graphite surface at room temperature by time-resolved AFM

The shape transformation of platinum (Pt) nanosheets with a uniform thickness of as thin as 3.5 ± 1 nm supported on graphite was investigated by in situ atomic force microscopy (AFM). The AFM observations revealed the shape transformation and the coalescence in preferred directions for the Pt nanosheets at room temperature (25 °C), which is much lower than the melting point of bulk metallic platinum (1769 °C). The behavior may be attributed to the high surface energy for the edge parts of Pt nanosheets with the small curvature of the nanometer scale.

Formation of Si-based nano-island array on porous anodic alumina

Si-based nano-island arrays were fabricated on porous anodic alumina by two methods. In the first method, a thick silicon film was first deposited onto the surface with highly ordered bowl array prepared by anodizing an Al foil, followed by the formation of a polycrystalline silicon nano-island array on the surface close to the bowl array after dissolving aluminum. In the second method, porous anodization was performed on an Al thin film on Si and a SiO 2 nano-island array was subsequently formed electrochemically. Time-resolved atomic force microscopy and photoluminescence were used to investigate the growth process as well as the mechanism of the growth process. Our proposed mechanism as well as assumptions made to formulate the model were found to be in agreement with the experimental results.

Evolution of a Rippled Membrane during Phospholipase A 2 Hydrolysis Studied by Time-Resolved AFM

The sensitivity of phospholipase A 2 (PLA 2) for lipid membrane curvature is explored by monitoring, through time-resolved atomic force microscopy, the hydrolysis of supported double bilayers in the ripple phase. The ripple phase presents a corrugated morphology. PLA 2 is shown to have higher activity toward the ripple phase compared to the gel phase in 1,2-dimyristoyl -sn-glycero-3-phosphocholine (DMPC) membranes, indicating its preference for this highly curved membrane morphology. Hydrolysis of the stable and metastable ripple structures is monitored for equimolar DMPC/1,2-distearoyl -sn-glycero-3-phosphocholine (DSPC)-supported double bilayers. As shown by high-performance liquid chromatography results, DSPC is resistant to hydrolysis at this temperature, resulting in a more gradual hydrolysis of the surface that leads to a change in membrane morphology without loss of membrane integrity. This is reflected in an increase in ripple spacing, followed by a sudden flattening of the lipid membrane during hydrolysis. Hydrolysis of the ripple phase results in anisotropic holes running parallel to the ripples, suggesting that the ripple phase has strip regions of higher sensitivity to enzymatic attack. Bulk high-performance liquid chromatography measurements indicate that PLA 2 preferentially hydrolyzes DMPC in the DMPC/DSPC ripples. We suggest that this leads to the formation of a flat gel-phase lipid membrane due to enrichment in DSPC. The results point to the ability of PLA 2 for inducing a compositional phase transition in multicomponent membranes through preferential hydrolysis while preserving membrane integrity.

Time-resolved atomic force microscopy imaging studies of asymmetric PS-b-PMMA ultrathin films: Dislocation and disclination transformations, defect mobility, and evolution of nanoscale morphology

Time-sequenced atomic force microscopy (AFM) studies of ultrathin films of cylinder-forming polystyrene-block-polymethylmethacrylate (PS-b-PMMA) copolymer are presented which delineate thin film mobility kinetics and the morphological changes which occur in microphase-separated films as a function of annealing temperature. Of particular interest are defect mobilities in the single layer (L thick) region, as well as the interfacial morphological changes which occur between L thick and adjacent 3L/2 thick layers, i.e., structural changes which occur during multilayer evolution. These measurements have revealed the dominant pathways by which disclinations and dislocations transform, annihilate, and topologically evolve during thermal annealing of such films. Mathematical combining equations are given to better explain such defect transformations and show the topological outcomes which result from defect–defect encounters. We also report a collective, Arrhenius-type flow of defects in localized L thick regions of the film; these are characterized by an activation energy of 377 kJ/mol. These measurements represent the first direct investigation of time-lapse interfacial morphological changes including associated defect evolution pathways for polymeric ultrathin films. Such observations will facilitate a more thorough and predictive understanding of diblock copolymer thin film dynamics, which in turn will further enable the utilization of these nanoscale phase-separated materials in a range of physical and chemical applications.

Reversible Liquid−Liquid Transitions in the Early Stages of Monolayer Self-Assembly

Time-resolved atomic force microscopy, contact angle analysis, and X-ray photoelectron spectroscopy were employed to study the effect of cyclic heating and cooling of a partial octadecyltrichlorosilane (OTS) monolayer on the oxidized Si(100) surface. A reversible structural change of the partial monolayer between high density and low density two-dimensional liquid phases was observed as a function of temperature at constant coverage. These studies show that the monolayer exists in a highly mobile hydrogen-bonded state akin to the equilibrium state of Langmuir films at the air−water interface. The lifetime of the mobile state was measured to be on the order of several minutes, after which grafting and cross-linking immobilize the monolayer. The results show that the mobile state permits large scale rearrangements of the molecules within the monolayers.