Nanoscale Dynamics Research Articles

Nanoscale architecture and coordination of actin cores within the sealing zone of human osteoclasts.

Osteoclasts are unique in their capacity to degrade bone tissue. To achieve this process, osteoclasts form a specific structure called the sealing zone, which creates a close contact with bone and confines the release of protons and hydrolases for bone degradation. The sealing zone is composed of actin structures called podosomes nested in a dense actin network. The organization of these actin structures inside the sealing zone at the nano scale is still unknown. Here, we combine cutting-edge microscopy methods to reveal the nanoscale architecture and dynamics of the sealing zone formed by human osteoclasts on bone surface. Random illumination microscopy allowed the identification and live imaging of densely packed actin cores within the sealing zone. A cross-correlation analysis of the fluctuations of actin content at these cores indicates that they are locally synchronized. Further examination shows that the sealing zone is composed of groups of synchronized cores linked by α-actinin1 positive filaments, and encircled by adhesion complexes. Thus, we propose that the confinement of bone degradation mediators is achieved through the coordination of islets of actin cores and not by the global coordination of all podosomal subunits forming the sealing zone.

Mesoscopic approach for nanoscale liquid-vapor interfacial statics and dynamics

Nanoscale liquid-vapor interfacial transport phenomena are of great significance to a variety of applications including evaporation, condescension, boiling and micro/nano-fluidics. In this work, we propose a mesoscopic approach to describe the nanoscale liquid-vapor interfacial statics and dynamics by combining the pseudopotential multiphase lattice Boltzmann method, the theorem of corresponding states and the principle of dynamic similarity. We demonstrate that our mesoscopic predictions of density profile, interfacial thicknesses and surface tensions for planar liquid-vapor interfaces of various real fluids agree very well with the NIST recommended data and molecular theories of capillarity in a very wide temperature range. We quantify the size effects of nano-bubbles and nano-droplets on the surface tension of water under a saturation temperature of 100°C. We show that the surface tensions of nano-bubbles decrease while the surface tensions of nano-droplets increase with increasing size, and the predictions from static cases and dynamic cases are consistent. The mesoscopic approach proposed in this paper could well resolve nanoscale liquid-vapor interfacial phenomena for various real fluids with high resolution, high accuracy and affordable computation cost in a very wide temperature range, which paves the way for quantitative investigations of nanoscale liquid-vapor interfacial transport for real fluids in practical applications.

Subsynaptic mobility of presynaptic mGluR types is differentially regulated by intra- and extracellular interactions.

Presynaptic metabotropic glutamate receptors (mGluRs) are essential for the control of synaptic transmission. However, how the subsynaptic dynamics of these receptors is controlled and contributes to synaptic signaling remain poorly understood quantitatively. Particularly, since the affinity of individual mGluR subtypes for glutamate differs considerably, the activation of mGluR subtypes critically depends on their precise subsynaptic distribution. Here, using superresolution microscopy and single-molecule tracking, we unravel novel molecular mechanisms that control the nanoscale distribution and mobility of presynaptic mGluRs in hippocampal neurons. We demonstrate that the high-affinity group II receptor mGluR2 localizes diffusely along the axon, and is highly mobile, while the low-affinity group III receptor mGluR7 is stably anchored at the active zone. We demonstrate that intracellular interactions modulate surface diffusion of mGluR2, while immobilization of mGluR7 at the active zone relies on its extracellular domain. Receptor activation or increases in synaptic activity do not alter the surface mobility of presynaptic mGluRs. Finally, computational modeling of presynaptic mGluR activity revealed that this particular nanoscale arrangement directly impacts their ability to modulate neurotransmitter release. Altogether, this study demonstrates that distinct mechanisms control surface mobility of presynaptic mGluRs to contribute differentially to glutamatergic synaptic transmission.

Nanoscale Structure and Dynamics of Model Membrane Lipid Raft Systems, Studied by Neutron Scattering Methods

Quasi-elastic neutron scattering (QENS) and small angle neutron scattering (SANS), in combination with isotopic contrast variation, have been used to determine the structure and dynamics of three-component lipid membranes, in the form of vesicles, comprising an unsaturated [palmitoyl-oleoyl-phosphatidylcholine (POPC) or dioleoyl-phosphatidylcholine (DOPC)], a saturated phospholipid (dipalmitoyl-phosphatidylcholine (DPPC)), and cholesterol, as a function temperature and composition. SANS studies showed vesicle membranes composed of a 1:1:1 molar ratio of DPPC:DOPC:cholesterol and a 2:2:1 molar ratio of DPPC:POPC:cholesterol phase separated, forming lipid rafts of ∼18 and ∼7 nm diameter respectively, when decreasing temperature from 308 to 297 K. Phase separation was reversible upon increasing temperature. The larger rafts observed in systems containing DOPC are attributed to the greater mis-match in lipid alkyl chains between DOPC and DPPC, than for POPC and DPPC. QENS studies, over the temperature range 283–323K, showed that the resulting data were best modelled by two Lorentzian functions: a narrow component, describing the “in-plane” lipid diffusion, and a broader component, describing the lipid alkyl chain segmental relaxation. The overall “in-plane” diffusion was found to show a significant reduction upon increasing temperature due to the vesicle membranes transitioning from one containing rafts to one where the component lipids are homogeneously mixed. The use of different isotopic combinations allowed the measured overall reduction of in-plane diffusion to be understood in terms of an increase in diffusion of the saturated DPPC lipid and a corresponding decrease in diffusion of the unsaturated DOPC/POPC lipid. As the rafts are considered to be composed principally of saturated lipid and cholesterol, the breakdown of rafts decreases the exposure of the DPPC to cholesterol whilst increasing the exposure of cholesterol to unsaturated lipid. These results show the sensitivity of lipid diffusion to local cholesterol concentration, and the importance of considering the local, rather that the global composition of a membrane when understanding the diffusion processes of lipids within the membrane. The novel combination of SANS and QENS allows a non-intrusive approach to characterize the structure and dynamics occurring in phase-separated model membranes which are designed to mimic the lateral heterogeneity of lipids seen in cellular membranes–a heterogeneity that can have pathological consequences.

Early stage growth of amorphous thin film: Average kinetics, nanoscale dynamics, and pressure dependence

We used the coherent grazing incidence small angle x-ray scattering technique to study the average kinetics and nanoscale dynamics during early-stage a-${\mathrm{WSi}}_{2}$ sputter deposition. The kinetic and dynamic properties are examined as a function of pressure, which is known to be a critical factor in determining final surface roughness. Surface growth kinetics and dynamics are characterized by time parameters extracted from the height-height structure factor and correlation functions. The roughness at a given length scale reaches a maximum before relaxing down to a steady state. The lateral length scale dependence and pressure dependence are then compared among the measured kinetics and dynamics time parameters. Surfaces grown at lower pressures are smoother, leading to longer correlation times. The time parameters to reach a dynamic steady state and a kinetic steady state show contrasting pressure dependence. A dynamic steady state is reached earlier than the kinetic steady state at high pressure. A more random deposition direction and lower kinetic energy at higher pressures can explain these phenomena, along with the hypothesis that larger nanoclusters form in vapor before arriving at the surface. A continuum model is applied to simulate the overall behavior with mixed success.

Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis

Clathrin polymerization and changes in plasma membrane architecture are necessary steps in forming vesicles to internalize cargo during clathrin-mediated endocytosis (CME). Simultaneous analysis of clathrin dynamics and membrane structure is challenging due to the limited axial resolution of fluorescence microscopes and the heterogeneity of CME. This has fueled conflicting models of vesicle assembly and obscured the roles of flat clathrin assemblies. Here, using Simultaneous Two-wavelength Axial Ratiometry (STAR) microscopy, we bridge this critical knowledge gap by quantifying the nanoscale dynamics of clathrin-coat shape change during vesicle assembly. We find that de novo clathrin accumulations generate both flat and curved structures. High-throughput analysis reveals that the initiation of vesicle curvature does not directly correlate with clathrin accumulation. We show clathrin accumulation is preferentially simultaneous with curvature formation at shorter-lived clathrin-coated vesicles (CCVs), but favors a flat-to-curved transition at longer-lived CCVs. The broad spectrum of curvature initiation dynamics revealed by STAR microscopy supports multiple productive mechanisms of vesicle formation and advocates for the flexible model of CME.

Nanotechnology Tools Enabling Biological Discovery.

Over the years, the engineering aspect of nanotechnology has been significantly exploited. Medical intervention strategies have been developed by leveraging existing molecular biology knowledge and combining it with nanotechnology tools to improve outcomes. However, little attention has been paid to harnessing the strengths of nanotechnology as a biological discovery tool. Fundamental understanding of controlling dynamic biological processes at the subcellular level is key to developing personalized therapeutic and diagnostic interventions. Single-cell analyses using intravital microscopy, expansion microscopy, and microfluidic-based platforms have been helping to better understand cell heterogeneity in healthy and diseased cells, a major challenge in oncology. Also, single-cell analysis has revealed critical signaling pathways and biological intracellular components with key biological functions. The physical manipulation enabled by nanotools can allow real-time monitoring of biological changes at a single-cell level by sampling intracellular fluid from the same cell. The formation of intercellular highways by nanotube-like structures has important clinical implications such as metastasis development. The integration of nanomaterials into optical and molecular imaging techniques has rendered valuable morphological, structural, and biological information. Nanoscale imaging unravels mechanisms of temporality by enabling the visualization of nanoscale dynamics never observed or measured between individual cells with standard biological techniques. The exceptional sensitivity of nanozymes, artificial enzymes, make them perfect components of the next-generation mobile diagnostics devices. Here, we highlight these impactful cancer-related biological discoveries enabled by nanotechnology and producing a paradigm shift in cancer research and oncology.

Multi-Dimensional Spectral Single Molecule Localization Microscopy.

Single molecule localization (SML) and tracking (SPT) techniques, such as (spt)PALM, (u/DNA)PAINT and quantum dot tracking, have given unprecedented insight into the nanoscale molecular organization and dynamics in living cells. They allow monitoring individual proteins with millisecond temporal resolution and high spatial resolution (<30 nm) by precisely localizing the point spread function (PSF) of individual emitters and tracking their position over time. While SPT methods have been extended to study the temporal dynamics and co-organization of multiple proteins, conventional experimental setups are restricted in the number of proteins they can probe simultaneously and usually have to tradeoff between the number of colors, the spatio-temporal resolution, and the field of view. Yet, localizing and tracking several proteins simultaneously at high spatial and temporal resolution within large field of views can provide important biological insights. By employing a dual-objective spectral imaging configuration compatible with live cell imaging combined with dedicated computation tools, we demonstrate simultaneous 3D single particle localization and tracking of multiple distinct species over large field of views to be feasible without compromising spatio-temporal resolution. The dispersive element introduced into the second optical path induces a spectrally dependent displacement, which we used to analytically separate up to five different fluorescent species of single emitters based on their emission spectra. We used commercially available microscope bodies aligned one on top of the other, offering biologists with a very ergonomic and flexible instrument covering a broad range of SMLM applications. Finally, we developed a powerful freely available software, called PALMTracer, which allows to quantitatively assess 3D + t + λ SMLM data. We illustrate the capacity of our approach by performing multi-color 3D DNA-PAINT of fixed samples, and demonstrate simultaneous tracking of multiple receptors in live fibroblast and neuron cultures.

Ratiometric imaging of viral lipid order utilizing a solvatochromic fluorescent probe

The complex interplay between lipid phase structure of the host cells and nanoscale dynamics at the viral lipid envelope is considered critical for the pathogen’s ability to interact and fuse with the host-cell membrane. Despite decades of research, details about the exact role played by viral lipids in regulating its’ infectivity remain unknown. In particular, a clear mechanistic picture of viral lipid phase structure, and its impact on viral fusion is still lacking. This is partially owing to a dearth of techniques that reveal viral lipid organization in a holistic fashion.

Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A

Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective strategies for bioethanol production. Cel7A from the fungus Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. Here, we used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A molecules binding to and moving processively along immobilized cellulose.

MPP1 Determines the Mobility of Flotillins and Controls the Confinement of Raft-Associated Molecules.

MPP1 (membrane palmitoylated protein 1) belongs to the MAGUK (membrane-associated guanylate kinase homologs) scaffolding protein family. These proteins organize molecules into complexes, thereby maintaining the structural heterogeneity of the plasma membrane (PM). Our previous results indicated that direct, high-affinity interactions between MPP1 and flotillins (raft marker proteins) display dominant PM-modulating capacity in erythroid cells. In this study, with high-resolution structured illuminated imaging, we investigated how these complexes are organized within erythroid cells on the nanometer scale. Furthermore, using other spectroscopic techniques, namely fluorescence recovery after photobleaching (FRAP) and spot-variation fluorescence correlation spectroscopy (svFCS), we revealed that MPP1 acts as a key raft-capturing molecule, regulating temporal immobilization of flotillin-based nanoclusters, and controls local concentration and confinement of sphingomyelin and Thy-1 in raft nanodomains. Our data enabled us to uncover molecular principles governing the key involvement of MPP1-flotillin complexes in the dynamic nanoscale organization of PM of erythroid cells.

Nanoscale dynamics of actin filaments in the red blood cell membrane skeleton.

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15–18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200–300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability.

Effect of Water on a Hydrophobic Deep Eutectic Solvent.

Deep eutectic solvents (DESs) formed by hydrogen bond donors and acceptors are a promising new class of solvents. Both hydrophilic and hydrophobic binary DESs readily absorb water, making them ternary mixtures, and a small water content is always inevitable under ambient conditions. We present a thorough study of a typical hydrophobic DES formed by a 1:2 mole ratio of tetrabutyl ammonium chloride and decanoic acid, focusing on the effects of a low water content caused by absorbed water vapor, using multinuclear NMR techniques, molecular modeling, and several other physicochemical techniques. Already very low water contents cause dynamic nanoscale phase segregation, reduce solvent viscosity and fragility, increase self-diffusion coefficients and conductivity, and enhance local dynamics. Water interferes with the hydrogen-bonding network between the chloride ions and carboxylic acid groups by solvating them, which enhances carboxylic acid self-correlation and ion pair formation between tetrabutyl ammonium and chloride. Simulations show that the component molar ratio can be varied, with an effect on the internal structure. The water-induced changes in the physical properties are beneficial for most prospective applications but water creates an acidic aqueous nanophase with a high halide ion concentration, which may have chemically adverse effects.

A Multiphysics Modeling of Electromagnetic Signaling Phenomena at kHz-GHz Frequencies in Bacterial Biofilms

This paper presents a model that describes a possible mechanism for electromagnetic (EM) signal transmission and reception by bacterial cells within their biofilm communities. Bacterial cells in biofilms are embedded into a complex extracellular matrix containing, among other components, charged helical nanofibrils from amyloid-forming peptides. Based on the current knowledge about the nanoscale structure and dynamics of the amyloids, we explore a hypothetical model that the mechanical vibration of these nanofibrils allows the cells to transmit EM signals to their neighboring cells and the surrounding environment. For the reception, the induced electric field can either exert force on the charges of adjacent nanofibrils associated with the neighboring cells or affect the placement/conformation of a certain charged messenger protein within the cell. The proposed model is based on a coupled system of electrical and mechanical nanoscale structures, which predicts signal transmission and reception within kHz-GHz frequency ranges. Different mechanisms for generating EM signals at various frequency bands related to the structure of the cell and their biofilm constituents are discussed.

Development of fluorescence labeling technique for multi-modal live-cell super-resolution microscopy

Live-cell super-resolution microscopy is a powerful tool that reveals spatiotemporal dynamics of biomolecules with nano-scale resolution in live specimens. However, the photobleaching of fluorescent molecules labeled to the target molecule is a significant obstacle to continuously obtaining super-resolution images for a long period. In this study, we developed a fluorescent labeling technique called de-quenching of organic dye emission (DeQODE) that enables long-term observation with a super-resolution microscope without being affected by photobleaching. In the DeQODE, a non-fluorescent probe is designed to become fluorescent by sequential binding to tag protein, an antibody against the probe, enabling maintaining the fluorescence signal for a long period. After optimizing the binding-dissociating property between tag protein and the probe, several intracellular organelles and the distribution of proteins labeled with DeQODE were imaged with a nano-scale resolution for tens of minutes by multiple super-resolution microscopies including stimulated emission depletion microscopy (STED), single-molecule localization microscopy (SMLM), and structured illumination microscopy (SIM). Our results show that DeQODE can contribute to research clarifying the nano-scale dynamics of functional molecules by live-cell super-resolution imaging.

Thermal Stability and Mechanical Properties of Al-Zn and Al-Bi-Zn Alloys Deformed by ECAP

It is well known that ultrafine grained and nanocrystalline materials show enhanced strength, while they are susceptible to thermally induced grain coarsening. The present work aims to enhance the thermal stability of ultrafine Al grains produced by equal channel angular pressing (ECAP) via dynamically precipitation. Detailed characterization by electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM) has been carried out to reveal the microstructural evolution during both ECAP and post-ECAP annealing. After five passes of ECAP, both Al-8Zn and Al-6Bi-8Zn alloys show an ultrafine grain structure together with dynamic precipitated nanoscale Zn particles along grain boundaries. Upon annealing at 200 °C, ultrafine grains in the Al-8Zn and Al-6Bi-8Zn alloys show a remarkable thermal stability compared to the Al-8Bi alloy, which is mainly due to the presence of nanoscale Zn precipitates along grain boundaries. The present work reveals that nanoscale Zn particles have a positive effect on preserving the ultrafine grains during annealing, which is useful for the design of UFG Al alloys with improved thermal stability.

Observation of Liquid-Liquid-Phase Separation and Vesicle Spreading during Supported Bilayer Formation via Liquid-Phase Transmission Electron Microscopy.

Liquid-phase transmission electron microscopy (LP-TEM) enables the real-time visualization of nanoscale dynamics in solution. This technique has been used to study the formation and transformation mechanisms of organic and inorganic nanomaterials. Here, we study the formation of block-copolymer-supported bilayers using LP-TEM. We observe two formation pathways that involve either liquid droplets or vesicles as intermediates toward supported bilayers. Quantitative image analysis methods are used to characterize vesicle spread rates and show the origin of defect formation in supported bilayers. Our results suggest that bilayer assembly methods that proceed via liquid droplet intermediates should be beneficial for forming pristine supported bilayers. Furthermore, supported bilayers inside the liquid cells may be used to image membrane interactions with proteins and nanoparticles in the future.

Charge-Transfer Landscape Manifesting the Structure-Rate Relationship in the Condensed Phase Via Machine Learning.

In this work, we develop a machine learning (ML) strategy to map the molecular structure to condensed phase charge-transfer (CT) properties including CT rate constants, energy levels, electronic couplings, energy gaps, reorganization energies, and reaction free energies which are called CT fingerprints. The CT fingerprints of selected landmark structures covering the conformation space of an organic photovoltaic molecule dissolved in an explicit solvent are computed and used to train ML models using kernel ridge regression. The ML models show high predictive power with R2 > 0.97 and both mean absolute error and root-mean-square error within chemical accuracy. The CT landscape for millions of molecular dynamics sampled structures is thus constructed, which allows for instant prediction of CT rate properties, given any conformation of the molecule. We demonstrate some immediate utilities of the CT landscape such as calculating the ensemble-averaged CT rate constant and interpreting the effects of molecular structural features on the CT rate. The unprecedented CT landscape will be useful for investigating real-time CT dynamics in nanoscale- and mesoscale-condensed phase systems and for the optimal fabrication design for homogeneous and heterogeneous optoelectronic devices.

Multi-phase field simulation of Al[formula omitted]Ni[formula omitted] intermetallic growth at liquid Al/solid Ni interface using MD computed interfacial energies

Considering its application in developing Raney-type Ni catalysts and in metal surface coatings, the study on the growth behavior of Al3Ni2 intermetallic compound (IMC) at the Al/Ni material interface is of utmost importance. The present work integrates nanoscale molecular dynamics (MD) calculation with mesoscale phase field model for studying the interfacial phenomena associated with Al3Ni2 growth in Al/Ni interface at 1173.15 K. The interfacial energies computed from MD are in the range 0.9–1.2 J/m2 with FCC/IMC featuring as the interface with the largest value and IMC/LIQUID as the one with the lowest value. Phase field model parameters characterizing a varying interface energy formulation are established to simulate the 2D growth of interfacial IMC grains. With the help of an atomistically informed phase field model, it has been revealed that the phase areas and morphology are obviously sensitive to the interfacial properties. The methodologies and results of these multiscale simulations for IMC interfaced between Al and Ni microstructures offer the complementary and accelerated design route of in-silico studies for materials systems experimented at high temperature.

Nanoscale Charge Density and Dynamics in Graphene Oxide.

Graphene oxide (GO) is widely used as a component in thin film optoelectronic device structures for practical reasons because its electronic and optical properties can be controlled. Progress critically depends on elucidating the nanoscale electronic structure of GO. However, direct experimental access is challenging because of its disordered and nonconductive character. Here, we quantitatively mapped the nanoscopic charge distribution and charge dynamics of an individual GO sheet by using Kelvin probe force microscopy (KPFM). Charge domains are identified, presenting important charge interactions below distances of 20 nm. Charge dynamics with very long relaxation times of at least several hours and a logarithmic decay of the time correlation function are in excellent agreement with Monte Carlo simulations, revealing an universal hopping transport mechanism best described by Efros–Shklovskii’s law.