Tackling the Challenges of Dynamic Experiments Using Liquid-Cell Transmission Electron Microscopy.

Revolutions in science and engineering frequently result from the development, and wide adoption, of a new, powerful characterization or imaging technique. Beginning with the first glass lenses and telescopes in astronomy, to the development of visual-light microscopy, staining techniques, confocal microscopy, and fluorescence super-resolution microscopy in biology, and most recently aberration-corrected, cryogenic, and ultrafast (4D) electron microscopy, X-ray microscopy, and scanning probe microscopy in nanoscience. Through these developments, our perception and understanding of the physical nature of matter at length-scales beyond ordinary perception have been fundamentally transformed. Despite this progression in microscopy, techniques for observing nanoscale chemical processes and solvated/hydrated systems are limited, as the necessary spatial and temporal resolution presents significant technical challenges. However, the standard reliance on indirect or bulk phase characterization of nanoscale samples in liquids is undergoing a shift in recent times with the realization ( Williamson et al. Nat. Mater . 2003 , 2 , 532 - 536 ) of liquid-cell (scanning) transmission electron microscopy, LC(S)TEM, where picoliters of solution are hermetically sealed between electron-transparent "windows," which can be directly imaged or videoed at the nanoscale using conventional transmission electron microscopes. This Account seeks to open a discussion on the topic of standardizing strategies for conducting imaging experiments with a view to characterizing dynamics and motion of nanoscale materials. This is a challenge that could be described by critics and proponents alike, as analogous to doing chemistry in a lightning storm; where the nature of the solution, the nanomaterial, and the dynamic behaviors are all potentially subject to artifactual influence by the very act of our observation.

Impact of the green tea ingredient epigallocatechin gallate and a short pentapeptide (Ile-Ile-Ala-Glu-Lys) on the structural organization of mixed micelles and the related uptake of cholesterol.

BACKGROUND: High levels of blood cholesterol are conventionally linked to an increased risk of developing cardiovascular disease (Grundy, 1986). Here we examine the molecular mode of action of natural products with known cholesterol-lowering activity, such as for example the green tea ingredient epigallocatechin gallate and a short pentapeptide, Ile-Ile-Ala-Glu-Lys. METHODS: Molecular Dynamics simulations are used to gain insight into the formation process of mixed micelles and, correspondingly, how active agents epigallocatechin gallate and Ile-Ile-Ala-Glu-Lys could possibly interfere with it. RESULTS: Self-assembly of physiological micelles occurs on the order of 35-50 ns; most of the structural properties of mixed micelles are unaffected by epigallocatechin gallate or Ile-Ile-Ala-Glu-Lys which integrate into the micellar surface; the diffusive motion of constituting lipids palmitoyl-oleoyl-phosphatidylcholine and cholesterol is significantly down-regulated by both epigallocatechin gallate and Ile-Ile-Ala-Glu-Lys; CONCLUSIONS: The molecular mode of action of natural compounds epigallocatechin gallate and Ile-Ile-Ala-Glu-Lys is a significant down-regulation of the diffusive motion of micellar lipids. GENERAL SIGNIFICANCE: Natural compounds like the green tea ingredient epigallocatechin gallate and a short pentapeptide, Ile-Ile-Ala-Glu-Lys, lead to a significant down-regulation of the diffusive motion of micellar lipids thereby modulating cholesterol absorption into physiological micelles.

Directly Observing Micelle Fusion and Growth in Solution by Liquid-Cell Transmission Electron Microscopy.

Amphiphilic small molecules and polymers form commonplace nanoscale macromolecular compartments and bilayers, and as such are truly essential components in all cells and in many cellular processes. The nature of these architectures, including their formation, phase changes, and stimuli-response behaviors, is necessary for the most basic functions of life, and over the past half-century, these natural micellar structures have inspired a vast diversity of industrial products, from biomedicines to detergents, lubricants, and coatings. The importance of these materials and their ubiquity have made them the subject of intense investigation regarding their nanoscale dynamics with increasing interest in obtaining sufficient temporal and spatial resolution to directly observe nanoscale processes. However, the vast majority of experimental methods involve either bulk-averaging techniques including light, neutron, and X-ray scattering, or are static in nature including even the most advanced cryogenic transmission electron microscopy techniques. Here, we employ in situ liquid-cell transmission electron microscopy (LCTEM) to directly observe the evolution of individual amphiphilic block copolymer micellar nanoparticles in solution, in real time with nanometer spatial resolution. These observations, made on a proof-of-concept bioconjugate polymer amphiphile, revealed growth and evolution occurring by unimer addition processes and by particle-particle collision-and-fusion events. The experimental approach, combining direct LCTEM observation, quantitative analysis of LCTEM data, and correlated in silico simulations, provides a unique view of solvated soft matter nanoassemblies as they morph and evolve in time and space, enabling us to capture these phenomena in solution.

Complex Media and Enzymatic Kinetics.

Enzymatic reactions in complex environments often take place with concentrations of enzyme comparable to that of substrate molecules. Two such cases occur when an enzyme is used to detect low concentrations of substrate/analyte or inside a living cell. Such concentrations do not agree with standard in vitro conditions, aimed at satisfying one of the founding hypotheses of the Michaelis-Menten reaction scheme, MM. It would be desirable to generalize the classical approach and show its applicability to complex systems. A permeable micrometrically structured hydrogel matrix was fabricated by protein cross-linking. Glucose oxidase enzyme (GOx) was embedded in the matrix and used as a prototypical system. The concentration of H2O2 was monitored in time and fitted by an accurate solution of the enzymatic kinetic scheme, which is expressed in terms of simple functions. The approach can also find applications in digital microfluidics and in systems biology where the kinetics response in the linear regimes often employed must be replaced.

Are two-station biased random walkers always potential molecular motors?

The short answer to the title question is no. Despite their tremendous complexity, many nanomachines are simply one-dimensional systems undergoing a biased, that is, unidirectional, walk on a two-minima potential energy curve. The initially prepared state, or station, is higher in energy than the final equilibrium state that is reached after overcoming an energy barrier. All chemical reactions comply with this scheme, which does not necessarily imply that a generic chemical reaction is a potential molecular motor. If the barrier is low, the system may walk back and the motion will have a large purely Brownian component. Alternatively, a large distance from the barrier of either of the two stations may introduce a Brownian component. Starting from a general inequality that leverages on the idea that the amount of heat dissipated along the potential energy curve is a good indication of the effectiveness of the biased walk, we provide guidelines for the selection of the features of artificial molecular motors.

Crossover of two power laws in the anomalous diffusion of a two lipid membrane.

Molecular dynamics simulations of a bi-layer membrane made by the same number of 1-palmitoyl-2-oleoyl-glycero-3-phospho-ethanolamine and palmitoyl-oleoyl phosphatidylserine lipids reveal sub-diffusional motion, which presents a crossover between two different power laws. Fractional Brownian motion is the stochastic mechanism that governs the motion in both regimes. The location of the crossover point is justified with simple geometrical arguments and is due to the activation of the mechanism of circumrotation of lipids about each other.

Operations and thermodynamics of an artificial rotary molecular motor in solution.

A general framework is provided that makes possible the estimation of time-dependent properties of a stochastic system moving far from equilibrium. The process is investigated and discussed in general terms of nonequilibrium thermodynamics. The approach is simple and can be exploited to gain insight into the dynamics of any molecular-level machine. As a case study, the dynamics of an artificial molecular rotary motor, similar to the inversion of a helix, which drives the motor from a metastable state to equilibrium, are examined. The energy path that the motor walks was obtained from the results of atomistic calculations. The motor undergoes unidirectional rotation and its entropy, internal energy, free energy, and net exerted force are given as a function of time, starting from the solution of Smoluchowski's equation. The rather low value of the organization index, that is, the ratio of the work done by the particle against friction during the unidirectional motion per available free energy, reveals that the motion is mainly subject to randomness, and the amount of energy converted to heat due to the directional motion is very small.

Perturbation theory in the catalytic rate constant of the Henri-Michaelis-Menten enzymatic reaction.

The Henry-Michaelis-Menten (HMM) mechanism of enzymatic reaction is studied by means of perturbation theory in the reaction rate constant k (2) of product formation. We present analytical solutions that provide the concentrations of the enzyme (E), the substrate (S), as well as those of the enzyme-substrate complex (C), and the product (P) as functions of time. For k (2) small compared to k (-1), we properly describe the entire enzymatic activity from the beginning of the reaction up to longer times without imposing extra conditions on the initial concentrations E ( o ) and S ( o ), which can be comparable or much different.

Photothermal motion: effect of low-intensity irradiation on the thermal motion of organic nanoparticles.

The effect of local photo-triggered heat release on the motion of organic nanopartcles (NP), a process that is itself thermal, is largely unexplored under low-intensity irradiation. Here, we develop organic NP specifically tailored for this study and demonstrate, comparing three different irradiation intensity regimes, that indeed the NP undergo "acceleration" upon light absorption (Photothermal Motion). These NP have a well-defined chemical composition and extremely high molar absorbance coefficient, and upon excitation, they deactivate mostly non radiatively with localized heat dissipation. The residual fluorescence efficiency is high enough to allow the detection of their trajectory in a simple wide field fluorescence microscope under low-intensity irradiation, a typical condition for NP bio-applications. The NP were characterized in detail from the photophysical point of view using UV-VIS absorption, steady-state and time-resolved fluorescence spectroscopy and ultra-fast transient absorption (UF-TA). A detailed analysis of the trajectories of the NP reveals a strong dependency of the diffusion coefficient on the irradiation intensity even in a low power regime. This behavior demonstrates the inhomogeneity of the environment surrounding the NP as a result of local heat generation. Upon irradiation, the effective temperature increase, that emerges from the analysis, is much larger than that expected for plasmonic NP. Anomalous diffusion object-motion analysis (ADOMA) revealed that, in the more intense irradiation regime, the motion of the NP is a fractional Brownian motion, which is a simple generalization of Brownian motion where the steps are not independent of each other.

Universal Markers Unveil Metastatic Cancerous Cross-Sections at Nanoscale.

The characterization of cancer histological sections as metastatic, M, or not-metastatic, NM, at the cellular size level is important for early diagnosis and treatment. We present timely warning markers of metastasis, not identified by existing protocols and used methods. Digitized atomic force microscopy images of human histological cross-sections of M and NM colorectal cancer cells were analyzed by multifractal detrended fluctuation analysis and the generalized moments method analysis. Findings emphasize the multifractal character of all samples and accentuate room for the differentiation of M from NM cross-sections. Two universal markers emphatically achieve this goal performing very well: (a) the ratio of the singularity parameters (left/right), which are defined relative to weak/strong fluctuations in the multifractal spectrum, is always greater than 0.8 for NM tissues; and (b) the index of multifractality, used to classify universal multifractals, points to log-normal distribution for NM and to log-Cauchy for M tissues. An immediate large-scale screening of cancerous sections is doable based on these findings.

Viscoelasticity and Noise Properties Reveal the Formation of Biomemory in Cells.

Living cells are neither perfectly elastic nor liquid and return a viscoelastic response to external stimuli. Nanoindentation provides force-distance curves, allowing the investigation of cell mechanical properties, and yet, these curves can differ from point to point on the cell surface, revealing its inhomogeneous character. In the present work, we propose a mathematical method to estimate both viscoelastic and noise properties of cells as these are depicted on the values of the scaling exponents of relaxation function and power spectral density, respectively. The method uses as input the time derivative of the response force in a nanoindentation experiment. Generalized moments method and/or rescaled range analysis is used to study the resulting time series depending on their nonstationary or stationary nature. We conducted experiments in living Ulocladium chartarum spores. We found that spores in the approaching phase present a viscoelastic behavior with the corresponding scaling exponent in the range 0.25-0.52 and in the retracting phase present a liquid-like behavior with exponents in the range 0.67-0.85. This substantial difference of the scaling exponents in the two phases suggests the formation of biomemory as a response of the spores to the indenting AFM mechanical stimulus. The retracting phase may be described as a process driven by bluish noises, while the approaching one is driven by persistent noise.

Complex Nanoparticle Diffusional Motion in Liquid-Cell Transmission Electron Microscopy.

Liquid-cell transmission electron microscopy (LCTEM) is a powerful in situ videography technique that has the potential to allow us to observe solution-phase dynamic processes at the nanoscale, including imaging the diffusion and interaction of nanoparticles. Artefactual effects imposed by the irradiated and confined liquid-cell vessel alter the system from normal "bulk-like" behavior in multiple ways. These artefactual LCTEM effects will leave their fingerprints in the motion behavior of the diffusing objects, which can be revealed through careful analysis of the object-motion trajectories. Improper treatment of the motion data can lead to erroneous descriptions of the LCTEM system's conditions. Here, we advance our anomalous diffusion object-motion analysis (ADOMA) method to extract a detailed description of the liquid-cell system conditions during any LCTEM experiment by applying a multistep analysis of the data and treating the x/y vectors of motion independently and in correlation with each other and with the object's orientation/angle.

Entropy and Random Walk Trails Water Confinement and Non-Thermal Equilibrium in Photon-Induced Nanocavities.

Molecules near surfaces are regularly trapped in small cavitations. Molecular confinement, especially water confinement, shows intriguing and unexpected behavior including surface entropy adjustment; nevertheless, observations of entropic variation during molecular confinement are scarce. An experimental assessment of the correlation between surface strain and entropy during molecular confinement in tiny crevices is difficult because strain variances fall in the nanometer scale. In this work, entropic variations during water confinement in 2D nano/micro cavitations were observed. Experimental results and random walk simulations of water molecules inside different size nanocavitations show that the mean escaping time of molecular water from nanocavities largely deviates from the mean collision time of water molecules near surfaces, crafted by 157 nm vacuum ultraviolet laser light on polyacrylamide matrixes. The mean escape time distribution of a few molecules indicates a non-thermal equilibrium state inside the cavity. The time differentiation inside and outside nanocavities reveals an additional state of ordered arrangements between nanocavities and molecular water ensembles of fixed molecular length near the surface. The configured number of microstates correctly counts for the experimental surface entropy deviation during molecular water confinement. The methodology has the potential to identify confined water molecules in nanocavities with life science importance.

Breathing modes of Kolumbo submarine volcano (Santorini, Greece).

Submarine volcanoes, such as Kolumbo (Santorini, Greece) are natural laboratories for fostering multidisciplinary studies. Their investigation requires the most innovative marine technology together with advanced data analysis. Conductivity and temperature of seawater were recorded directly above Kolumbo's hydrothermal vent system. The respective time series have been analyzed in terms of non-equilibrium techniques. The energy dissipation of the volcanic activity is monitored by the temperature variations of seawater. The venting dynamics of chemical products is monitored by water conductivity. The analysis of the time series in terms of stochastic processes delivers scaling exponents with turning points between consecutive regimes for both conductivity and temperature. Changes of conductivity are shown to behave as a universal multifractal and their variance is subdiffusive as the scaling exponents indicate. Temperature is constant over volcanic rest periods and a universal multifractal behavior describes its changes in line with a subdiffusive character otherwise. The universal multifractal description illustrates the presence of non-conservative conductivity and temperature fields showing that the system never retains a real equilibrium state. The existence of a repeated pattern of the combined effect of both seawater and volcanic activity is predicted. The findings can shed light on the dynamics of chemical products emitted from the vents and point to the presence of underlying mechanisms that govern potentially hazardous, underwater volcanic environments.

Interactions in concentric carbon nanotubes: the radius vs the chirality angle contributions.

In multiwall carbon nanotubes in general, and in double wall carbon nanotubes, DWCNTs, in particular, the guest-host interactions depend primarily on the difference of the nanotubes radii, Deltar. The chirality angle mismatch of the two tubes, Deltatheta, also matters since it determines the pattern of pi-stacking interactions that ultimately is responsible for the shift of graphite layers into the so-called A-B structure. Here we calculate the minimum energy structures of 198 DWCNTs and construct two functions of Deltar and Deltatheta that fit the calculated data. Cross terms exists between Deltar and Deltatheta. The shape of the functions is rationalized in simple physical terms and can be used to construct minimum energy multiwall nanotubes.