Force Microscopy In Liquid Research Articles

Biophysical Properties of the Fibril Structure of the Toxic Conformer of Amyloid-β42: Characterization by Atomic Force Microscopy in Liquid and Molecular Docking.

Alzheimer's disease is associated with the aggregation of the misfolded neuronal peptide, amyloid-β42 (Aβ42). Evidence has suggested that several reasons are responsible for the toxicity caused by the aggregation of Aβ42, including the conformational restriction of Aβ42. In this study, one of the toxic conformers of Aβ42, which contains a Glu-to-Pro substitution (E22P-Aβ42), was explored using atomic force microscopy and molecular docking to study the aggregation dynamics. We proposed a systematic model of fibril formation to better understand the molecular basis of conformational transitions in the Aβ42 species. Our results demonstrated the formation of amorphous aggregates in E22P-Aβ42 that are stem-based, network-like structures, while the formation of mature fibrils occurred in the less toxic conformer of Aβ42, E22-Aβ42, that are sphere-like flexible structures. A comparison was made between the biophysical properties of E22P-Aβ42 and E22-Aβ42 that revealed that E22P-Aβ42 had greater stiffness, dihedral angle, number of β sheets involved, and elasticity, compared with E22-Aβ42. These findings will have considerable implications toward our understanding of the structural basis of the toxicity caused by conformational diversity in Aβ42 species.

Membrane destabilization and pore formation induced by the Synechocystis IM30 protein

The inner membrane-associated protein of 30 kDa (IM30) is essential in chloroplasts and cyanobacteria. The spatio-temporal cellular localization of the protein appears to be highly dynamic and triggered by internal as well as external stimuli, mainly light intensity. The soluble fraction of the protein is localized in the cyanobacterial cytoplasm or the chloroplast stroma, respectively. Additionally, the protein attaches to the thylakoid membrane as well as to the chloroplast inner envelope or the cyanobacterial cytoplasmic membrane, respectively, especially under conditions of membrane stress. IM30 is involved in thylakoid membrane biogenesis and/or maintenance, where it either stabilizes membranes and/or triggers membrane-fusion processes. These apparently contradicting functions have to be tightly controlled and separated spatiotemporally in chloroplasts and cyanobacteria. IM30’s fusogenic activity depends on Mg2+ binding to IM30; yet, it still is unclear how Mg2+-loaded IM30 interacts with membranes and promotes membrane fusion. Here, we show that the interaction of Mg2+ with IM30 results in increased binding of IM30 to native, as well as model, membranes. Via atomic force microscopy in liquid, IM30-induced bilayer defects were observed in solid-supported bilayers in the presence of Mg2+. These structures differ dramatically from the membrane-stabilizing carpet structures that were previously observed in the absence of Mg2+. Thus, Mg2+-induced alterations of the IM30 structure switch the IM30 activity from a membrane-stabilizing to a membrane-destabilizing function, a crucial step in membrane fusion.

Visualization of DNA Damage and Protection by Atomic Force Microscopy in Liquid.

DNA damage is closely related to cancer and many aging-related diseases. Peroxynitrite is a strong oxidant, thus a typical DNA damage agent, and is a major mediator of the inflammation-associated pathogenesis. For the first time, we directly visualized the process of DNA damage by peroxynitrite and DNA protection by ectoine via atomic force microscopy in liquid. We found that the persistence length of DNA decreases significantly by adding a small amount of peroxynitrite, but the observed DNA chains are still intact. Specifically, the persistence length of linear DNA in a low concentration of peroxynitrite (0 µM to 200 µM) solution decreases from about 47 nm to 4 nm. For circular plasmid DNA, we observed the enhanced superhelices of plasmid DNA due to the chain soften. When the concentration of peroxynitrite was above 300 µM, we observed the fragments of DNA. Interestingly, we also identified single-stranded DNAs during the damage process, which is also confirmed by ultraviolet spectroscopy. However, if we added 500 mM ectoine to the high concentration PN solution, almost no DNA fragments due to double strand breaks were observed because of the protection of ectoine. This protection is consistent with the similar effect for DNA damage caused by ionizing radiation and oxygenation. We ascribe DNA protection to the preferential hydration of ectoine.

Advancing fluorescence microscopy and spectroscopy for the benefit of cell science and drug delivery

Here we report on two recent advances we made applying advanced fluorescence microscopy and spectroscopy to further the study of cell biology and drug delivery. At first, we detail a new instrumental set-up combining atomic force microscopy in liquid and super-resolution fluorescence microscopy in novel configuration such that long-term simultaneous and co-localized observation with both techniques becomes feasible. We believe this will contribute to the study of fast membrane activated cell-signalling processes in the years to come. Furthermore, we report on a novel application of fluorescence cross-correlation spectroscopy (FCCS) for the characterization of lipid-nucleic acid complexes. We could determine the number of nucleic acid particles incorporated in each liposome, a parameter not readily accessible on ensemble basis with other methods. This parameter is crucial for co-delivery applications, and we believe FCCS can play an important role in the future development of new drug delivery systems.

Ultra-microstructures and mechanics of plant mucilage functional interfaces investigated via atomic force microscopy in liquid

<p indent="0mm">Mucus plays an important role in regulating biological activities. Accordingly, investigating the structures and properties of mucus can be beneficial for providing a better understanding of physiological and pathological changes. Due to a lack of adequate observation tools, the ultra-microstructures of native mucus functional interfaces are not yet fully understood. Atomic force microscopy (AFM) provides a novel and powerful tool for investigating the structures and properties of biomaterials in their native states. Nevertheless, to date, studies of biomaterials using AFM are commonly performed in air, which cannot faithfully reflect the structures of biomaterials in liquids. In this paper, by adopting the mucilage secreted by the carnivorous sundew plant as an example, high-resolution AFM imaging and analysis of mucous nanostructures in aqueous conditions were achieved. Sundew mucilage was coated onto the surface of a glass slide or mica, and the subsequent AFM imaging of the mucilage-coated substrates in pure water revealed that a large number of nanoparticles were contained in the sundew mucilage. In contrast, AFM imaging of sundew mucilage-coated substrates in air showed that dried mucilage contained a large number of nanofibers. This underscored the structural differences in sundew mucilage in the air and in a solution. Furthermore, AFM multiparametric imaging was employed to visually characterize the mechanical properties of sundew mucilage nanostructures in pure water and showed significant differences in the mechanics between nanoparticles and nanofibers. This study provides a novel approach for investigating the ultra-microstructures and mechanics of mucus’ functional interfaces in their native states <italic>in situ</italic>, which has fundamental impacts on research focused on biomaterials.

In Situ AFM Imaging of Adsorption Kinetics of DPPG Liposomes: A Quantitative Analysis of Surface Roughness.

The adsorption of intact liposomes on surfaces is of great importance for the development of sensors and drug delivery systems and, also, strongly dependent on the surface roughness where the liposomes are adsorbed. In this paper, we analyzed, by using atomic force microscopy in liquid, the evolution of the morphology of gold surfaces and of poly(allylamine hydrochloride) (PAH) surfaces with different roughness during the adsorption of liposomes prepared with the synthetic phospholipid 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]. Our results reveal the following. On smooth surfaces of Au only and Au with PAH, the liposomes open and deploy on the substrate, creating a supported-lipid bilayer, with the opening process being faster on the Au/PAH surface. On rough substrates of Au coated with polyelectrolyte multilayers, the liposomes were adsorbed intact on the surface. This was corroborated by power spectral density analysis that demonstrates the presence of superstructures with an average lateral size of 43 and 87 nm, in accordance with two and four times the mean liposome hydrodynamic diameter of about 21 nm. In addition, this work presents an adequate and effective methodology for analysis of adsorption phenomena of liposomes on rough surfaces.

Power transfer in bimodal amplitude modulation atomic force microscopy in liquids: A numerical investigation

Bimodal amplitude modulation atomic force microscopy (AM-AFM) is an emerging technique for compositional imaging in liquids. In this work, we investigate the power transfer in bimodal AM-AFM in liquids by a numerical analysis. Power items are calculated by direct numerical integral and the corresponding amplitude and phase response is presented. Results show power balance is satisfied for each mode. The power transfer in each mode is significantly small compared to the external input power and most of the power is dissipated into the surrounding medium, especially for a large setpoint or cantilever-sample separation. The power transfer among different modes is complex and strongly depends on the cantilever and imaging parameters. Power transfer between different modes goes up with increasing free amplitude of the second mode. In addition, a stiffer sample will produce a more complex force spectra, which perturbs the cantilever oscillation more heavily compared to a compliant sample. Besides, the non-driven higher mode of a softer cantilever is more likely to be momentarily excited. The power items and cantilever response during imaging are also provided, revealing the phases in bimodal AFM in liquids may not be utilized to characterize the sample elasticity due to the non-monotonic trends. Instead, the amplitude of the second mode could be used to characterize the elasticity of the sample with moderate to high moduli.

Direct Imaging of Atomic-Scale Surface Structures of Brookite TiO2 Nanoparticles by Frequency Modulation Atomic Force Microscopy in Liquid

Direct imaging of atomic-scale surface structures of TiO2 nanoparticles is a promising method for understanding the detailed mechanism of their photocatalytic activities. Atomic force microscopy (AFM) is one of the analytical tools used for direct imaging of surfaces, but atomic-scale AFM studies of TiO2 nanoparticles in liquid have not been reported. Here, we report two methods for fixing of TiO2 nanocrystals: an electrostatic method and a cross-linking method. Both methods enabled visualization of subnanoscale surface structures of brookite TiO2 nanoparticles in liquid by frequency modulation AFM (FM-AFM). The FM-AFM imaging results and density functional theory molecular dynamics calculations suggest that the subnanoscale structures in the FM-AFM images can be explained by the arrangement of the surface topmost atoms or their hydration structures on the brookite TiO2(210) surface. Our results open up various possibilities for studying atomic-scale surface structures of nanoparticles in liquids by AFM.

Fibrous matrix component of cell wall in the giant-celled green alga Valonia utricularis observed by atomic force microscopy in liquid.

The cell walls of the giant-celled green alga Valonia exhibit the "crossed-fibril" arrangement of cellulose microfibrils (CMFs). The existence of fibrous matrix components coiling around the CMFs was recently reported using atomic force microscopy (AFM). To understand the biological role of this fibrous wall component, we attempted to reveal the detailed morphology of the fibrous structure of the cell walls isolated from V. utricularis specimens in artificial seawater (ASW), by using an improved method of AFM. We also investigated the effect of incubation in an acidic environment that had been known to make the cell walls liable to separate into layers, on the morphology of the fibrous structures. Thin, fibrous structures were found to entangle around a single CMF or multiple adjacent CMFs, the thickness of which was 0.85nm on average. Incubation in acidic ASW (pH4), greatly modified the morphology of the fibrous structures in quality and quantity, compared with those incubated in the original ASW (pH8). Thickness of the fibrous structures was increased to 4.63nm on average, remarkable deformations were observed, and the density of the structures was reduced to less than half by incubation in the acidic ASW. In addition, same fibrous structures extended over CMFs and forming aggregates were observed on the surface of the cell wall layers separated artificially. Fluorescence microscopy of cell walls treated with Vicia villosa lectin conjugated with fluorescein isothiocyanate showed specific labelling of cell wall regions where the fibrous structures were present abundantly as observed by AFM. From these observations, it can be assumed that the fibrous structures of V. utricularis are the matrix component of the cell wall containing N-acetylgalactosamine, involved in the maintenance of the cell wall integrity through bonding the neighboring cell wall layers, and their morphology and function of V. utricularis are sensitive to acidic pH.

Measurement of Adsorbed Gas Molecules on Graphite Surface by Using Frequency Modulation Atomic Force Microscopy in Liquid

Measurement of Adsorbed Gas Molecules on Graphite Surface by Using Frequency Modulation Atomic Force Microscopy in Liquid

Self-assembled monolayers of sulfonate-terminated alkanethiols investigated by frequency modulation atomic force microscopy in liquid

A molecular-scale understanding of self-assembled monolayers (SAMs) of sulfonate-terminated alkanethiols is crucial for interfacial studies of functionalized SAMs and their various applications. However, such an understanding has been difficult to achieve because of the lack of direct information on these molecular-scale structures in real space. In this study, we investigated the structures of sulfonate SAMs of sodium 11-mercapto-1-undecanesulfonate (MUS) by frequency modulation atomic force microscopy (FM-AFM) in liquid. The subnanometer-resolution FM-AFM images showed that the single-component MUS SAM prepared in pure water had random surface structures. In contrast, the MUS SAM prepared in a water–ethanol mixed solvent showed periodic striped structures with a flat-lying conformation. The results suggest a significant solvent effect on molecular-scale structures of long-chain sulfonate SAMs. In addition, we investigated the molecular-scale structures of mixed SAMs of MUS and 11-mercapto-1-undecanol (MUO) with alkane chains of the same length. The FM-AFM images of the mixed SAMs showed clear phase separation between MUS SAM and MUO SAM domains. In the MUO SAM domains, the incorporated MUS molecules appeared as protrusions. The results obtained in this study provide direct structural information on long-chain sulfonate and mixed SAMs.

Interface structure between tetraglyme and graphite.

Clarification of the details of the interface structure between liquids and solids is crucial for understanding the fundamental processes of physical functions. Herein, we investigate the structure of the interface between tetraglyme and graphite and propose a model for the interface structure based on the observation of frequency-modulation atomic force microscopy in liquids. The ordering and distorted adsorption of tetraglyme on graphite were observed. It is found that tetraglyme stably adsorbs on graphite. Density functional theory calculations supported the adsorption structure. In the liquid phase, there is a layered structure of the molecular distribution with an average distance of 0.60 nm between layers.

Accurate nanoscale flexibility measurement of DNA and DNA-protein complexes by atomic force microscopy in liquid.

The elasticity of double-stranded DNA (dsDNA), as described by its persistence length, is critical for many biological processes, including genomic regulation. A persistence length value can be obtained using atomic force microscopy (AFM) imaging. However, most AFM studies have been done by depositing the sample on a surface using adhesive ligands and fitting the contour to a two-dimensional (2D) wormlike chain (WLC) model. This often results in a persistence length measurement that is different from the value determined using bulk and single molecule methods. We describe a method for obtaining accurate three-dimensional (3D) persistence length measurements for DNA and DNA-protein complexes by using a previously developed liquid AFM imaging method and then applying the 3D WLC model. To demonstrate the method, we image in both air and liquid several different dsDNA constructs and DNA-protein complexes that both increase (HIV-1 Vpr) and decrease (yeast HMO1) dsDNA persistence length. Fitting the liquid AFM-imaging contour to the 3D WLC model results in a value in agreement with measurements obtained in optical tweezers experiments. Because AFM also allows characterization of local DNA properties, the ability to correctly measure global flexibility will strongly increase the impact of measurements that use AFM imaging.

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown that when operated in amplitude-modulation (tapping) mode, atomic or molecular-level resolution images can be achieved over a wide range of soft and hard samples in liquid. In these situations, small oscillation amplitudes (SAM-AFM) enhance the resolution by exploiting the solvated liquid at the surface of the sample. Although the technique has been successfully applied across fields as diverse as materials science, biology and biophysics and surface chemistry, obtaining high-resolution images in liquid can still remain challenging for novice users. This is partly due to the large number of variables to control and optimize such as the choice of cantilever, the sample preparation, and the correct manipulation of the imaging parameters. Here, we present a protocol for achieving high-resolution images of hard and soft samples in fluid using SAM-AFM on a commercial instrument. Our goal is to provide a step-by-step practical guide to achieving high-resolution images, including the cleaning and preparation of the apparatus and the sample, the choice of cantilever and optimization of the imaging parameters. For each step, we explain the scientific rationale behind our choices to facilitate the adaptation of the methodology to every user's specific system.

Sub-nanometer Resolution Imaging with Amplitude-modulation Atomic Force Microscopy in Liquid.

Atomic force microscopy (AFM) has become a well-established technique for nanoscale imaging of samples in air and in liquid. Recent studies have shown that when operated in amplitude-modulation (tapping) mode, atomic or molecular-level resolution images can be achieved over a wide range of soft and hard samples in liquid. In these situations, small oscillation amplitudes (SAM-AFM) enhance the resolution by exploiting the solvated liquid at the surface of the sample. Although the technique has been successfully applied across fields as diverse as materials science, biology and biophysics and surface chemistry, obtaining high-resolution images in liquid can still remain challenging for novice users. This is partly due to the large number of variables to control and optimize such as the choice of cantilever, the sample preparation, and the correct manipulation of the imaging parameters. Here, we present a protocol for achieving high-resolution images of hard and soft samples in fluid using SAM-AFM on a commercial instrument. Our goal is to provide a step-by-step practical guide to achieving high-resolution images, including the cleaning and preparation of the apparatus and the sample, the choice of cantilever and optimization of the imaging parameters. For each step, we explain the scientific rationale behind our choices to facilitate the adaptation of the methodology to every user's specific system.

Imaging of surface nanobubbles by atomic force microscopy in liquids: Influence of drive frequency on the characterization of ultrasoft matter.

Imaging of soft matter with atomic force microscopy (AFM) is challenging due to tip-induced deformation, which convolutes with the measurement. The challenges are generally more serious in liquid environments due to a severe loss of sensitivity of the vibrating microcantilever to external forces, as well as due to the presence of undesirable mechanical resonances when piezoelectric excitation systems are used. Furthermore, the choice of imaging parameters can have a significant impact on the quality of the results, such that the customary practices used for tuning the cantilever are not always appropriate. Here we explore the influence of the chosen drive frequency on the imaging of ultrasoft matter, using surface gas nanobubbles on gold-coated glass and highly oriented pyrolytic graphite (HOPG) as a test platform. We carry out single- and multifrequency AFM experiments using both the traditional amplitude-peak method and the recently proposed phase-slope-peak method for tuning the cantilever, as well as piezoelectric and photothermal excitation, providing an extensive discussion on the factors governing the level of tip-induced sample deformation and the quality of the phase contrast obtained for each of the methods. The general conclusion is that there is no "one-size-fits-all" approach for tuning the cantilever for low-impact tapping-mode AFM, although rational optimization of the imaging process is generally possible, whereby the choice of the drive frequency plays a prominent role. The physical insight and guidelines provided here can be extremely useful for the gentle imaging of a wide range of biological and other soft materials in liquid environments. Microsc. Res. Tech. 80:41-49, 2017. © 2016 Wiley Periodicals, Inc.

High resolution atomic force microscopy of double-stranded RNA.

Double-stranded (ds) RNA mediates the suppression of specific gene expression, it is the genetic material of a number of viruses, and a key activator of the innate immune response against viral infections. The ever increasing list of roles played by dsRNA in the cell and its potential biotechnological applications over the last decade has raised an interest for the characterization of its mechanical properties and structure, and that includes approaches using Atomic Force Microscopy (AFM) and other single-molecule techniques. Recent reports have resolved the structure of dsDNA with AFM at unprecedented resolution. However, an equivalent study with dsRNA is still lacking. Here, we have visualized the double helix of dsRNA under near-physiological conditions and at sufficient resolution to resolve the A-form sub-helical pitch periodicity. We have employed different high-sensitive force-detection methods and obtained images with similar spatial resolution. Therefore, we show here that the limiting factors for high-resolution AFM imaging of soft materials in liquid medium are, rather than the imaging mode, the force between the tip and the sample and the sharpness of the tip apex.

Magnetic Force Microscopy in Liquids.

In this work, the use of magnetic force microscopy (MFM) to acquire images of magnetic nanostructures in liquid environments is presented. Optimization of the MFM signal acquisition in liquid media is performed and it is applied to characterize the magnetic signal of magnetite nanoparticles. The ability for detecting magnetic nanostructures along with the well-known capabilities of atomic force microscopy in liquids suggests potential applications in fields such as nanomedicine, nanobiotechnology, or nanocatalysis.

Growth and Dissolution of Calcite in the Presence of Adsorbed Stearic Acid.

The interaction of organic molecules with the surface of calcite plays a central role in many geochemical, petrochemical, and industrial processes and in biomineralization. Adsorbed organics, typically fatty acids, can interfere with the evolution of calcite when immersed in aqueous solutions. Here we use atomic force microscopy in liquid to explore in real-time the evolution of the (1014) surface of calcite covered with various densities of stearic acid and exposed to different saline solutions. Our results show that the stearic acid molecules tend to act as "pinning points" on the calcite's surface and slow down the crystal's restructuring kinetics. Depending on the amount of material adsorbed, the organic molecules can form monolayers or bilayer islands that become embedded into the growing crystal. The growth process can also displaces the organic molecules and actively concentrate them into stacked multilayers. Our results provide molecular-level insights into the interplay between the adsorbed fatty acid molecules and the evolving calcite crystal, highlighting mechanisms that could have important implications for several biochemical and geochemical processes and for the oil industry.

Fabrication of electron beam deposited tip for atomic-scale atomic force microscopy in liquid

Recently, possibilities of improving operation speed and force sensitivity in atomic-scale atomic force microscopy (AFM) in liquid using a small cantilever with an electron beam deposited (EBD) tip have been intensively explored. However, the structure and properties of an EBD tip suitable for such an application have not been well-understood and hence its fabrication process has not been established. In this study, we perform atomic-scale AFM measurements with a small cantilever and clarify two major problems: contaminations from a cantilever and tip surface, and insufficient mechanical strength of an EBD tip having a high aspect ratio. To solve these problems, here we propose a fabrication process of an EBD tip, where we attach a 2 μm silica bead at the cantilever end and fabricate a 500–700 nm EBD tip on the bead. The bead height ensures sufficient cantilever-sample distance and enables to suppress long-range interaction between them even with a short EBD tip having high mechanical strength. After the tip fabrication, we coat the whole cantilever and tip surface with Si (30 nm) to prevent the generation of contamination. We perform atomic-scale AFM imaging and hydration force measurements at a mica–water interface using the fabricated tip and demonstrate its applicability to such an atomic-scale application. With a repeated use of the proposed process, we can reuse a small cantilever for atomic-scale measurements for several times. Therefore, the proposed method solves the two major problems and enables the practical use of a small cantilever in atomic-scale studies on various solid–liquid interfacial phenomena.