Analysis Of Atomic Force Microscopy Images Research Articles

Assembly of Centromere Chromatin for Characterization by High-Speed Time-Lapse Atomic Force Microscopy.

Atomic force microscopy (AFM) is an imaging technique that enables single molecule characterization of biological systems at nanometer resolution. Imaging in ambient conditions can provide details of the conformational states and interactions of a population of molecules which is well complemented by single-molecule imaging of the systems dynamics using time-lapse AFM imaging, in which images are capture at rates of 10-15 frames per second in an aqueous buffer. Here we describe the assembly and preparation of nucleosomes containing centromere protein A (CENP-A) for AFM imaging in both static and time-lapse modes. The AFM imaging and data analysis techniques described enable characterization of the extent of DNA wrapping around the histone core and time-resolved visualization of the systems intrinsic dynamic behaviors.

Laboratory exercise for studying the morphology of heat-denatured and amyloid aggregates of lysozyme by atomic force microscopy.

To facilitate learning advanced instrumental techniques, essential tools for visualizing biomaterials, a simple and versatile laboratory exercise demonstrating the use of Atomic Force Microscopy (AFM) in biomedical applications was developed. In this experiment, the morphology of heat-denatured and amyloid-type aggregates formed from a low-cost and well-characterized model protein, hen egg white lysozyme (HEWL), are compared. Structural differences between the amorphous and ordered particles are quantified using ImageJ for the analysis of AFM images as a postlaboratory assignment. The laboratory exercise allows the direct observation of changes in the protein structure and helps students to understand the operation of AFM, as well as protein folding and misfolding related to many physiological and pathological processes. The described protocol stands alone, but also fits well into a larger module on protein structure and function or microscopic techniques as it can be linked easily to existing laboratory exercises on these topics. It can be easily adapted to the upper level undergraduate laboratory courses with limited lab hours as well as graduate level courses to improve students' research skills. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):162-171, 2018.

Fractal analysis of nanostructured silver film surface

The electrodeposition method is used to fabricate silver thin films at different thicknesses. The samples then were characterized using X-ray diffraction (XRD) and atomic force microscopy (AFM) techniques. We found that the crystalline structure of the Ag thin films depends on film thickness. The surface morphology and fractal dimension were studied by the AFM image analysis approach. Calculation of standard deviation surface roughness, indicated that it grows as a power-law in time or thickness with the growth exponent of β = 0.67. The average value of Hurst exponent is found to be 0.68 using rescaled range analyses. The results indicate that the morphology and grain size of samples can easily be controlled using different thicknesses. Fractal analysis shows that the fractal dimension of the Ag films increases as the film thickness increases. We proved that the fractal analysis present a useful explanation of the irregularities in the surface film.

Combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces.

Over the past five years, atomic force microscopy (AFM)-based approaches have evolved into a powerful multiparametric tool set capable of imaging the surfaces of biological samples ranging from single receptors to membranes and tissues. One of these approaches, force-distance curve-based AFM (FD-based AFM), uses a probing tip functionalized with a ligand to image living cells at high-resolution and simultaneously localize and characterize specific ligand-receptor binding events. Analyzing data from FD-based AFM experiments using appropriate probabilistic models allows quantification of the kinetic and thermodynamic parameters that describe the free-energy landscape of the ligand-receptor bond. We have recently developed an FD-based AFM approach to quantify the binding events of single enveloped viruses to surface receptors of living animal cells while simultaneously observing them by fluorescence microscopy. This approach has provided insights into the early stages of the interaction between a virus and a cell. Applied to a model virus, we probed the specific interaction with cells expressing viral cognate receptors and measured the affinity of the interaction. Furthermore, we observed that the virus rapidly established specific multivalent interactions and found that each bond formed in sequence strengthened the attachment of the virus to the cell. Here we describe detailed procedures for probing the specific interactions of viruses with living cells; these procedures cover tip preparation, cell sample preparation, step-by-step FD-based AFM imaging and data analysis. Experienced microscopists should be able to master the entire set of protocols in 1 month.

Nanoporous Polyether Sulfone Membrane, Preparation and Characterization: Effect of Porosity and Mean Pore Size on Performance

Flat sheet membranes were prepared by phase inversion technique using polyether sulfone (PES) dissolved in dimethylacetamide (DMAc) with and without adding polyvinylpyrrolidone (PVP) or polyethyleneglycol (PEG). The characteristics of the prepared membranes were evaluated using Scanning Electron Microscope (SEM) images, Atomic Force Microscopy (AFM), and Optical Contact Angle (OCA) measurements, and porosity tests. The porosity test and SEM images show that increasing additives to a certain value increases the porosity of the membrane. Also, as the coagulation bath temperature is increased, the porosity of the membrane is increased. The roughness of the membrane is increased by increasing the additive concentration. The analysis of AFM images confirms the nanoporous structure of the prepared membranes, and that the membranes with appropriate pore size distribution can be prepared by the applied method. Permeability tests using single-layer membranes show that the direct relationship between porosity and the flux of pure water or salt solution is dominated by the effect of applied additive while the salt rejection shows an inverse relationship with the mean pore size regardless of the applied additive. The salt permeation flux is a function of total porosity while the salt rejection is a function of surface porosity. Pervaporation tests show that both permeation flux and enrichment factor depend on the total porosity of the support membrane.

Dynamic remodeling of the dynamin helix during membrane constriction

Dynamin is a dimeric GTPase that assembles into a helix around the neck of endocytic buds. Upon GTP hydrolysis, dynamin breaks these necks, a reaction called membrane fission. Fission requires dynamin to first constrict the membrane. It is unclear, however, how dynamin helix constriction works. Here we undertake a direct high-speed atomic force microscopy imaging analysis to visualize the constriction of single dynamin-coated membrane tubules. We show GTP-induced dynamic rearrangements of the dynamin helix turns: the average distances between turns reduce with GTP hydrolysis. These distances vary, however, over time because helical turns were observed to transiently pair and dissociate. At fission sites, these cycles of association and dissociation were correlated with relative lateral displacement of the turns and constriction. Our findings show relative longitudinal and lateral displacements of helical turns related to constriction. Our work highlights the potential of high-speed atomic force microscopy for the observation of mechanochemical proteins onto membranes during action at almost molecular resolution.

Heteroprotein Complex Formation of Bovine Lactoferrin and Pea Protein Isolate: A Multiscale Structural Analysis.

Associative electrostatic interactions between two oppositely charged globular proteins, lactoferrin (LF) and pea protein isolate (PPI), the latter being a mixture of vicilin, legumin, and convicilin, was studied with a specific PPI/LF molar ratio at room temperature. Structural aspects of the electrostatic complexes probed at different length scales were investigated as a function of pH by means of different complementary techniques, namely, with dynamic light scattering, small-angle X-ray scattering (SAXS), turbidity measurements, and atomic force microscopy (AFM). Irrespective of the applied techniques, the results consistently displayed that complexation between LF and PPI did occur. In an optimum narrow range of pH 5.0-5.8, a viscous liquid phase of complex coacervate was obtained upon mild centrifugation of the turbid LF-PPI mixture with a maximum Rh, turbidity and the ζ-potential being close to zero observed at pH 5.4. In particular, the SAXS data demonstrated that the coacervates were densely assembled with a roughly spherical size distribution exhibiting a maximum extension of ∼80 nm at pH 5.4. Equally, AFM image analysis showed size distributions containing most frequent cluster sizes around 40-80 nm with spherical to elliptical shapes (axis aspect ratio ≤ 2) as well as less frequent elongated to chainlike structures. The most frequently observed compact complexes, we identify as mainly leading to LF-PPI coacervation, whereas for the less frequent chain-like aggregates, we hypothesize that additionally PPI-PPI facilitated complexes exist.

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein-DNA Complexes That Carry Out DNA Repair.

Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of single biomolecules and complexes deposited on a surface with nanometer resolution. AFM is a powerful tool for characterizing protein-protein and protein-DNA interactions. It can be used to capture snapshots of protein-DNA solution dynamics, which in turn, enables the characterization of the conformational properties of transient protein-protein and protein-DNA interactions. With AFM, it is possible to determine the stoichiometries and binding affinities of protein-protein and protein-DNA associations, the specificity of proteins binding to specific sites on DNA, and the conformations of the complexes. We describe methods to prepare and deposit samples, including surface treatments for optimal depositions, and how to quantitatively analyze images. We also discuss a new electrostatic force imaging technique called DREEM, which allows the visualization of the path of DNA within proteins in protein-DNA complexes. Collectively, these methods facilitate the development of comprehensive models of DNA repair and provide a broader understanding of all protein-protein and protein-nucleic acid interactions. The structural details gleaned from analysis of AFM images coupled with biochemistry provide vital information toward establishing the structure-function relationships that govern DNA repair processes.

Synthesis and Properties of Novel Silver-Containing DNA Molecules.

Migration of silver atoms from silver nano-particles selectively to a double-stranded poly(dG)-poly(dC) polymer leads to metallization of the DNA. As a result the DNA molecules become shorter and thicker (higher), as evident from the atomic force microscopy imaging analysis. The metalized molecules can be detected by transmission and scanning electron microscopy in contrast to the initial non-metalized ones.

Acceleration effect of copper(II) ions on the rate of citrate synthesis of gold nanoparticles

The gold nanoparticles (GNPs) were obtained by modification of the classical Turkevich citrate method by the addition of copper(II) ions in the initial reaction mixture. We studied the kinetics of the nanoparticles growth with or without copper(II) ions, synthesized at two different temperatures. The kinetic curves obtained by means of UV–vis spectroscopy from the plasmon maxima demonstrated the acceleration effects of the copper(II) ions on nucleation and growth of the GNPs. These effects were also confirmed from the Atomic Force Microscope (AFM) imaging data where the AFM was applied as a tool for studying the kinetics of the GNPs’ growth. From the analysis of AFM images in the course of the synthesis process were obtained the sizes (i.e., diameters) of the GNPs and the GNPs’ growth profiles were fitted with the Finke–Watzky (F–W) kinetic model. From the experimental data and the theoretical fits we found that the citrate reduction of the tetrachloroauric acid was accelerated by the addition of copper(II) ions as well as the GNPs’ growth was accelerated following the F–W kinetics model. Using Transmission Electron Microscopy (TEM) we compared the morphology and the size distribution of the nanoparticles synthesized by the citrate method with or without copper(II) ions. The results showed that the addition of copper(II) ions in the initial reaction mixture of the Turkevich citrate synthesis let to acceleration of both the nucleation and the growth processes following the F–W kinetic model.

The influence of incident angle on physical properties of a novel back contact prepared by oblique angle deposition

In this paper, oblique vacuum thermal evaporation and direct current (DC) magnetron sputtering technique are used to produce a novel back contact electrode (BCE) of CuInS2 solar cell. These novel back contact electrodes (BCEs) are based on a layered structure of Mo/Ag/Mo (MAM). The influence of vapor source incidence angle θ on optical-electrical properties of novel BCE is investigated by X-ray Diffraction (XRD), Surface Profiler, Atomic Force Microscope (AFM), UV-vis-IR Spectrometer, and Four-point Probe Method. According to the analysis of AFM images of BCEs, the variation tendencies of surface roughness and uniformity are closely related to the incidence angle θ. The surface roughness increases with the increase of incidence angle θ, but the uniformity becomes poor at same time. This phenomenon can be attributed to the variation of interlayer Ag films (the density and inclined angle of Ag nanorods). The results of four-point probe test show that the novel BCE deposited by vapor source incidence angle θ equal to 45° owns the lowest resistance value of 3.71×10−8Ωm, which is probably due to a loose and multi-point contact interface between Ag layer and top layer (Mo2). The reflectance of novel BCEs deposited by incident angle less than 45° is higher than that of normal bi-layer Mo (Mo12) BCE. As a result, the efficiency of corresponding solar cell may be upgraded.

Morphology and properties of globular polymeric materials in the solid state: A composite material of DNA with a cationic surfactant

The aim of the present study was to control entanglements in order to regulate the properties of polymeric solids. Initially, fabrication of polymeric solids with few entanglements was attempted. Films of the DNA–cationic surfactant, cetyltrimethylammonium bromide (CTAB) (DNA–CTA), were cast from ethanol solution at room temperature. Morphological examination of DNA–CTA complex films using atomic force microscopy (AFM) revealed that these films were constructed by particle-like substances. Geometrical analysis of AFM images showed that the particle-like substances were the aggregates of several DNA–CTA globules. Mechanical characterization suggested that there were fewer entanglements than with normal plastic films. Small angle X-ray scattering experiments during annealing indicated that molecular motions were highly excited in the surface region of each particle. In conclusion, a globular polymeric film with fewer entanglements was fabricated. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016, 54, 730–738

Detection of CD28/CD86 co‐stimulatory molecules and surface properties of T and dendritic cells: An AFM study

Although the importance of B7/CD28 co-stimulation has been widely studied, little is known about their nano-spatial localization and their corresponding cells' biophysical and biomechanical properties. Here, we investigated the morphological, biophysical, and biomechanical properties of T cells and dendritic cells (DCs) by atomic force microscopy (AFM) and force curves. The nano-spatial distribution of CD28 and CD86 antigen on T cells and DCs was detected by CD86 or CD28 antibody-functionalized AFM tip. Single-molecule force spectroscopy (SMFS)-based force volumes and quantum dots (QDs)-based fluorescence imaging demonstrated that the co-stimulatory molecules were not randomly distributed over the cells' surface, but more than 80% of CD28 and CD86 molecules appeared to be expressed as 100-200 nm nanoclusters and polarize dominantly in the peak of the cell membrane fluctuations. AFM imaging and quantitative analysis showed that the roughness of mature DCs (mDCs) was higher than that of immature DCs (iDCs). The adhesion force distribution of iDCs and mDCs was heterogeneous while the elasticity distribution was homogeneous locally. In addition, mDCs had a fourfold increase of Young's modulus of iDCs, indicating the contribution of the actin cytoskeleton to the elastic properties of the cells. Taken together, the nano-cluster distribution of CD28 and CD86, the rough mDCs surface, the higher adhesion force and elasticity of mDCs may facilitate to the occurrence of B7/CD28 co-stimulation signals and the formation of immune synapse. These nanoscale findings provide new insights into the antigen-presenting function of DCs, the T cell activation and ultimate immune response. SCANNING 38:365-375, 2016. © 2015 Wiley Periodicals, Inc.

Cell Membrane Disruption and DNA Binding of Staphylococcus Aureus Induced by Antibacterial Peptide F1 from Tibetan Kefir

The purpose of this work was to investigate antimicrobial mechanisms of a unique peptide F1 (found in Tibetan Kefir) against Staphylococcus aureus. The cell membrane permeability assay and potassium ion release assay were employed to examine the integrity of the bacterial membrane after the treatment with antimicrobial peptide F1. The transmission electron microscopy was used to analyze the effect of antimicrobial peptide F1 on the cell morphology of Staphylococcus aureus. The interaction between antimicrobial peptide F1 and genomic DNA of Staphylococcus aureus was investigated by DNA gel retardation assay and atomic force microscopy imaging analysis. The results showed that treatment with the antimicrobial peptide F1 led to the permeabilization of the cell membrane of Staphylococcus aureus. Moreover, the antimicrobial peptide F1 can bind to the DNA and inhibit related cellular functions. Having multiple targets means that antimicrobial peptide F1 may be more effective in killing bacterial cells, and bacteria have additional hurdles to overcome to build resistance. These results indicate that antimicrobial peptide F1 is a potential candidate as an effective biological preservative in the agricultural and food industry.

Fourier analysis of scratches generated on m‐GaN substrates during polishing

Generation of scratches on surface of m‐plane GaN substrates due to polishing was studied by atomic force microscopy (AFM). For epi‐ready substrates AFM images confirm a flat surface with the atomic step roughness while a lot of scratches are visible in AFM images for partially polished GaN substrates. The Fourier analysis of AFM images show that scratches propagate easier along {c‐plane} and {a‐plane} directions on m‐plane GaN surface. This observation is an evidence of anisotropy of mechanical properties of GaN crystals in the micro‐scale. This anisotropy is directly correlated with the symmetry and atomic arrangement of m‐plane GaN.

Quantitative analysis of drug-induced complement-mediated cytotoxic effect on single tumor cells using atomic force microscopy and fluorescence microscopy.

In the antibody-based targeted therapies of B-cell lymphomas, complement-mediated cytotoxicity (CMC) is an important mechanism. CMC is activated after the binding of drugs (monoclonal antibodies) to tumor cells. The activation of CMC ultimately leads to the lysis of tumor cells. However, it remains poorly understood how CMC alters the morphology and mechanics of single tumor cells at the nanoscale. In recent years, nanoscopic observations of cellular behaviors with the use of atomic force microscopy (AFM) have contributed much to the field of cell biology. In this work, by combining AFM with fluorescence microscopy, the detailed changes in cellular ultra-microstructures and mechanical properties during the process of CMC were quantitatively investigated on single tumor cells. AFM imaging distinctly showed that the CMC effect could lead to the formation of nano holes on the tumor cells. Quantitative analysis of AFM images indicated that cell surface became lower and rougher after the CMC process. The cellular mechanics measurements showed that during the process of CMC cells firstly softened and finally stiffened, which was validated by dynamically monitoring the mechanical changes of single living cells during CMC. The experimental results provide novel insights into the antibody-dependent CMC.

Fractal analysis of AFM images of the surface of Bowman's membrane of the human cornea.

The objective of this study is to further investigate the ultrastructural details of the surface of Bowman's membrane of the human cornea, using atomic force microscopy (AFM) images. One representative image acquired of Bowman's membrane of a human cornea was investigated. The three-dimensional (3-D) surface of the sample was imaged using AFM in contact mode, while the sample was completely submerged in optisol solution. Height and deflection images were acquired at multiple scan lengths using the MFP-3D AFM system software (Asylum Research, Santa Barbara, CA), based in IGOR Pro (WaveMetrics, Lake Oswego, OR). A novel approach, based on computational algorithms for fractal analysis of surfaces applied for AFM data, was utilized to analyze the surface structure. The surfaces revealed a fractal structure at the nanometer scale. The fractal dimension, D, provided quantitative values that characterize the scale properties of surface geometry. Detailed characterization of the surface topography was obtained using statistical parameters, in accordance with ISO 25178-2: 2012. Results obtained by fractal analysis confirm the relationship between the value of the fractal dimension and the statistical surface roughness parameters. The surface structure of Bowman's membrane of the human cornea is complex. The analyzed AFM images confirm a fractal nature of the surface, which is not taken into account by classical surface statistical parameters. Surface fractal dimension could be useful in ophthalmology to quantify corneal architectural changes associated with different disease states to further our understanding of disease evolution.

Surface Roughness and Critical Exponent Analyses of Boron-Doped Diamond Films Using Atomic Force Microscopy Imaging: Application of Autocorrelation and Power Spectral Density Functions

The evolution of the surface roughness of growing metal or semiconductor thin films provides much needed information about their growth kinetics and corresponding mechanism. While some systems show stages of nucleation, coalescence, and growth, others exhibit varying microstructures for different process conditions. In view of these classifications, we report herein detailed analyses based on atomic force microscopy (AFM) characterization to extract the surface roughness and growth kinetics exponents of relatively low boron-doped diamond (BDD) films by utilizing the analytical power spectral density (PSD) and autocorrelation function (ACF) as mathematical tools. The machining industry has applied PSD for a number of years for tool design and analysis of wear and machined surface quality. Herein, we present similar analyses at the mesoscale to study the surface morphology as well as quality of BDD films grown using the microwave plasma-assisted chemical vapor deposition technique. PSD spectra as a function of boron concentration (in gaseous phase) are compared with those for samples grown without boron. We find that relatively higher boron concentration yields higher amplitudes of the longer-wavelength power spectral lines, with amplitudes decreasing in an exponential or power-law fashion towards shorter wavelengths, determining the roughness exponent (α ≈ 0.16 ± 0.03) and growth exponent (β ≈ 0.54), albeit indirectly. A unique application of the ACF, which is widely used in signal processing, was also applied to one-dimensional or line analyses (i.e., along the x- and y-axes) of AFM images, revealing surface topology datasets with varying boron concentration. Here, the ACF was used to cancel random surface “noise” and identify any spatial periodicity via repetitive ACF peaks or spatially correlated noise. Periodicity at shorter spatial wavelengths was observed for no doping and low doping levels, while smaller correlations were observed for relatively higher boron concentration. These semiquantitative spatial analyses may prove useful in comparing synthesis techniques and varying compositional makeups of diamond films and other technologically important electronic materials. These findings in terms of critical exponents are also correlated with traditional Raman spectroscopy and x-ray diffraction structural properties, thus helping to provide insight into the growth kinetics, albeit in reverse manner.

AFM imaging and fractal analysis of surface roughness of AlN epilayers on sapphire substrates

The paper deals with AFM imaging and characterization of 3D surface morphology of aluminum nitride (AlN) epilayers on sapphire substrates prepared by magnetron sputtering. Due to the effect of temperature changes on epilayer's surface during the fabrication, a surface morphology is studied by combination of atomic force microscopy (AFM) and fractal analysis methods. Both methods are useful tools that may assist manufacturers in developing and fabricating AlN thin films with optimal surface characteristics. Furthermore, they provide different yet complementary information to that offered by traditional surface statistical parameters. This combination is used for the first time for measurement on AlN epilayers on sapphire substrates, and provides the overall 3D morphology of the sample surfaces (by AFM imaging), and reveals fractal characteristics in the surface morphology (fractal analysis).

Resveratrol Protects Chondrocytes from Apoptosis via Altering the Ultrastructural and Biomechanical Properties: An AFM Study

Osteoarthritis (OA), a degenerative joint disease with high prevalence among older people, occurs from molecular or nanometer level and extends gradually to higher degrees of the ultrastructure of cartilage, finally resulting in irreversible structural and functional damages. This report aims to use atomic force microscopy (AFM) to investigate the protective effects of resveratrol (RV), a drug with good anti-inflammatory properties, on cellular morphology, membrane architecture, cytoskeleton, cell surface adhesion and stiffness at nanometer level in sodium nitroprusside (SNP)-induced apoptotic chondrocytes, a typical cellular OA model. CCK-8 assay showed that 100 μM RV significantly prevented SNP-induced cytotoxicity. AFM imaging and quantitative analysis showed that SNP potently induced chondrocytes changes including shrunk, round, lamellipodia contraction and decrease in adherent junctions among cells, as well as the destruction of biomechanics: 90% decrease in elasticity and 30% decrease in adhesion. In addition, confocal imaging analysis showed that SNP induced aggregation of the cytoskeleton and decrease in the expression of cytoskeletal proteins. More importantly, these SNP-induced damages to chondrocytes could be potently prevented by RV pretreatment. Interestingly, the biomechanical changes occurred before morphological changes could be clearly observed during SNP-induced apoptosis, indicating that the biomechanics of cellular membrane may be a more robust indicator of cell function. Collectively, our data demonstrate that RV prevents SNP-induced apoptosis of chondrocytes by regulating actin organization, and that AFM-based technology can be developed into a powerful and sensitive method to study the interaction mechanisms between chondrocytes and drugs.