Analysis Of Atomic Force Microscopy Images Research Articles

Rapid Electrochemical Monitoring of Bacterial Respiration for Gram-Positive and Gram-Negative Microbes: Potential Application in Antimicrobial Susceptibility Testing.

Antimicrobial resistance is a grave threat to human life. Currently used time-consuming antibiotic susceptibility test (AST) methods limit physicians in selecting proper antibiotics. Hence, we developed a rapid AST using electroanalysis with a 15 min assay time, called EAST, which is live-monitored by time-lapse microscopy video. The present work reports systematical electrochemical analysis and standardization of protocol for EAST measurement. The proposed EAST is successfully applied for Gram-positive Bacillus subtilis and Gram-negative Escherichia coli as model organisms to monitor bacterial concentration, decay kinetics in the presence of various antibiotics (ciprofloxacin, cefixime, and amoxycillin), drug efficacy, and IC50. Bacterial decay kinetics in the presence of antibiotics were validated by the colony counting method, field emission scanning electron microscopy, and atomic force microscopy image analysis. The EAST predicts the antibiotic susceptibility of bacteria within 15 min, which is a significant advantage over existing techniques that consume hours to days. The EAST was explored further by using bacteria-friendly l-lysine-functionalized cerium oxide nanoparticle coated indium tin oxide as a working electrode to observe the enhanced electron-transfer rate in the EAST. The results are very significant for future miniaturization and automation. The proposed EAST has huge potential in the development of a rapid AST device for applications in the clinical and pharmaceutical industries.

Investigation of the Influences of Post-Thermal Annealing on Physical Properties of TiO2 Thin Films Deposited by RF Sputtering

For this study, titanium dioxide (TiO2) thin films were deposited on glass substrates at room temperature by the RF magnetron sputtering technique. The preparation parameters that offer better control and reproducibility of film fabrication were first optimized. Then, the effects of post-deposition annealing temperature at 350, 450, and 550°C on the microstructure, surface morphology, and optical properties of the prepared films were investigated using X-ray diffraction (XRD), Raman spectroscopy (RS), scanning electron microscopy (SEM), atomic force microscopy (AFM), and UV–Visible (UV–Vis) spectrophotometry. Interestingly, XRD analysis shows that as-deposited and annealed TiO2 film possess anatase crystal structure only with a preferential orientation along the (101) plane. The intensity of the (101) diffraction peak and crystallite size are found to increase with increasing annealing temperature, which indicates an improvement in the crystallinity of the films. Raman spectra confirm that all samples possess anatase phase and the crystallinity is enhanced with increasing thermal annealing. From the analysis of SEM and AFM images, it is revealed that the heat treatment significantly affects the morphology, grain size, and surface roughness of the TiO2 films. The UV–Vis spectroscopy analysis shows that as-deposited TiO2 thin film is highly transparent in the visible region with an average transmittance about 84%, whereas the transmission decreases slightly with an increase in annealing temperature. Moreover, the optical band gap energy shows a red shift with increasing the annealing temperature.

The effects of anthracycline drugs on the conformational distribution of mouse P-glycoprotein explains their transport rate differences

P-glycoprotein (Pgp) is an ATP-dependent efflux transporter and plays a major role in anti-cancer drug resistance by pumping a chemically diverse range of cytotoxic drugs from cancerous tumors. Despite numerous studies with the transporter, the molecular features that drive anti-cancer drug efflux are not well understood. Even subtle differences in the anti-cancer drug molecular structure can lead to dramatic differences in their transport rates. To unmask these structural differences, this study focused on two closely-related anthracycline drugs, daunorubicin (DNR), and doxorubicin (DOX), with mouse Pgp. While only differing by a single hydroxyl functional group, DNR has a 4 to 5-fold higher transport rate than DOX. They both non-competitively inhibited Pgp-mediated ATP hydrolysis below basal levels. The Km of Pgp-mediated ATP hydrolysis extracted from the kinetics curves was lower for DOX than DNR. However, the dissociation constants (KDs) for these drugs determined by fluorescence quenching were virtually identical. Acrylamide quenching of Pgp tryptophan fluorescence to probe the tertiary structure of Pgp suggested that DNR shifts Pgp to a “closed” conformation, while DOX shifts Pgp to an “intermediate” conformation. The effects of these drugs on the Pgp conformational distributions in a lipid bilayer were also examined by atomic force microscopy (AFM). Analysis of AFM images revealed that DNR and DOX cause distinct and significant shifts in the conformational distribution of Pgp. The results were combined to build a conformational distribution model for anthracycline transport by Pgp.

CARACTERIZACIÓN DE MEMBRANAS DE ÓSMOSIS INVERSA PARA TRATAMIENTO DE AGUA POR TÉCNICAS DE MICROSCOPIA DE FUERZA ATÓMICA

Membrane technology, applied to separation processes on residual or contaminated water has evolved as chemical and physical properties improve. Our main goal was to develop a method for atomic force microscopy image analysis, to eliminate subjective criteria and obtain pore size data. Tools from WxSM 5.0 Develop 8.3 software were used to invert images, and statistical analysis was performed from median ranges to obtain pore size distribution. Results were compared to data obtained from dynamic mechanical analysis (DMA), stiffness, storage and loss module, and glass transition temperature (Tg). Most uniformed pore size distribution was obtained by membrane prepared at 180 oC with FeCl 3 , μ p was 35 nm and MWCO of 268 KDa, the same membrane presented the best mechanical properties, 0.86 GPa Young modulus, and 26 MPa tensile rupture strain. These results will relate preparation conditions with structure and later functionality on filtration processes.

DNA accessibility of chromatosomes quantified by automated image analysis of AFM data

DNA compaction and accessibility in eukaryotes are governed by nucleosomes and orchestrated through interactions between DNA and DNA-binding proteins. Using QuantAFM, a method for automated image analysis of atomic force microscopy (AFM) data, we performed a detailed statistical analysis of structural properties of mono-nucleosomes. QuantAFM allows fast analysis of AFM images, including image preprocessing, object segmentation, and quantification of different structural parameters to assess DNA accessibility of nucleosomes. A comparison of nucleosomes reconstituted with and without linker histone H1 quantified H1’s already described ability of compacting the nucleosome. We further employed nucleosomes bearing two charge-modifying mutations at position R81 and R88 in histone H2A (H2A R81E/R88E) to characterize DNA accessibility under destabilizing conditions. Upon H2A mutation, even in presence of H1, the DNA opening angle at the entry/exit site was increased and the DNA wrapping length around the histone core was reduced. Interestingly, a distinct opening of the less bendable DNA side was observed upon H2A mutation, indicating an enhancement of the intrinsic asymmetry of the Widom-601 nucleosomes. This study validates AFM as a technique to investigate structural parameters of nucleosomes and highlights how the DNA sequence, together with nucleosome modifications, can influence the DNA accessibility.

Assembly and Optical Properties of Metal Nanoparticles

In this paper, the deposition and optical properties of charge-stabilized gold nanoparticles on silicon oxide substrates is studied, which have been derivatised with (aminopropyl) triemethoxysilane. Monodispersed charged-stabilized colloidal gold nanoparticles with diameters between 20-150 nm were prepared and their self-assembly and optical properties on silica substrates is studied. Atomic force microscopy (AFM) is employed to investigate the nanoparticle monolayers ex situ. Analysis of AFM images provide evidence that the formation of the colloidal nanoparticle monolayers is governed by random sequential adsorption. The results indicate that the ionic strength of the suspension influences the spatial distribution of the nanoparticles. For all sizes of the Au nanoparticles tested, optical simulations of extinction coefficients made by finite-difference time domain (FDTD) indicate a resonance peak in the range of 510-600 nm wavelength of the visible range of the electromagnetic spectrum. The results indicate a simple and inexpensive approach of assembly of plasmonic nanostructures that can find applications in metamaterials and light waveguides.

Minkowski functional characterization and fractal analysis of surfaces of titanium nitride films

The aim of this study is to gain a deeper understanding of the micromorphology characteristics of thin titanium nitride (TiN) films sputtered on glass substrates by using ion beam sputtering (IBS). For that purpose, TiN samples were deposited onto glass substrates from gas mixtures with different contents of molecular nitrogen and argon atoms. Atomic force microscopy (AFM) was used to characterize the surface microtexture of obtained thin films at high magnification. The detailed analysis of AFM images using Minkowski functionals and fractal analysis reveals a significant effect of the preparation conditions on the surface features with non-monotonic dependences. Presented results suggest that non-classical spatial characteristics of the variability of the surface topography, including fractal dimension, corner frequency, roughness, and feature shape and size, can be tuned having control over the relative flow rates of the gas mixture during film deposition.

Development of a New Electrochemical Atomic Force Microscopy Tool for Obtaining Surface Morphology Through Electrochemical Reaction.

Electrochemical atomic force microscopy is a complex electrochemical analysis method that has been applied in many fields. Electrochemical atomic force microscopy consists of an electrochemical cell, electrochemical analysis system, and atomic force microscopy. To simultaneously analyze the electrochemical system and the atomic force microscopy, an electrochemical cell with a suitable structure is needed. We developed the electrochemical atomic force microscopy analysis system using a self-developed electrochemical cell. To confirm the in-situ analytical ability of the developed electrochemical atomic force microscopy analysis system, we observed the electrochemical corrosion process of copper with respect to changes in pH of a sulfuric acid solution. The operation of the electrochemical atomic force microscopy tool was verified by experiments on the electrochemical corrosion of copper, and the factors affecting the corrosion process were examined. It was confirmed that electrochemical atomic force microscopy can perform electrochemical analysis and atomic force microscopy image analysis at the same time.

Dense electronic excitation induced modifications in nanocrystalline zirconium oxide thin films: Detailed analysis of optical and surface topographical

Ion irradiation is known to create structural disorder and crystallization improvement in the material. In the present study, focus is on the effect of high energy ion beam irradiation on the optical, surface morphological and structural properties of ZrO2 thin films of 200 nm grown on Si and glass substrate by RF sputtering technique. The prepared thin films were annealed at 600 °C and irradiated with swift heavy ions (SHI) of 120 MeV Ti9+ ion beam with varied fluences ranging from 5E11 to 2E13 ions/cm2. SRIM simulation reveals that the electronic energy losses (Se) dominate over the nuclear energy losses (Sn) and the projected range of the Ti ions was found to be 13.64 μm. Optical properties were studied by using UV-Vis and Photoluminescence (PL) spectroscopy. The transmittance of the ZrO2 samples lies in the range of (46–79%) and the Tauc's plot was used to calculate the optical band gap. PL spectroscopy demonstrates the emission spectrum using the excitation wavelength of 310 nm. The influence of SHI irradiation on the surface morphology of ZrO2 thin films was examined by Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FESEM). The sectional analysis of AFM images reveals that the grain size decreases with increasing the ion fluences and lies in the range of 51-29 nm. The structural analysis of pristine and irradiated samples was done by using X-ray diffraction (XRD) technique. The crystallite size increases for the lower fluence up to 1E12 ions/cm2 and decreases at higher fluence due to the development of fragmentation by the effect of SHI irradiation. XRD patterns were used to calculate strain, stress, dislocation density and texture coefficient of the ZrO2 samples. Rutherford Backscattering Spectrometry (RBS) ensures the presence of Zr and O elements with the atomic fraction of 1: 2 and thickness (200 nm) of the samples was confirmed by simulating the RBS spectrum. The modifications induced in the properties of the ZrO2 thin films were mainly due to the electronic energy losses through the inelastic collision in the target material.

Effects of substrate temperature and precursor amount on optical properties and microstructure of CVD deposited amorphous TiO2 thin films

In this research, TiO2 thin films were deposited on glass substrate by chemical vapor deposition using tetra isopropylorthotitanate as a precursor at low temperature. The TiO2thin films were formed without using an oxygen source or annealing. Effects of substrate temperature and amount of the precursor on surface microstructure and optical properties of deposited TiO2 thin films were investigated. X-ray diffraction (XRD), UV-visible spectrophotometry and atomic force microscopy (AFM) were used to analyze the fabricated layers. The XRD analysis revealed that structure of all prepared layers was in amorphous phase. Analysis of AFM images showed that by increasing substrate temperature, the layer surfaces became rougher and simultaneously nanoparticle size was more uniform. Texture analysis revealed that increasing substrate temperature and decreasing precursor amount both increased surface isotropy. Thickness of layers was calculated by applying the Swanepoel method to the transmittance spectra. Variations in optical parameters were studied, including of optical band gap, refractive index, extinction coefficient, complex dielectric function, Urbach energy, single oscillator energy and dispersion energy. The results indicated that optical properties were functions of substrate temperature and precursor amount.

General Resolution Enhancement Method in Atomic Force Microscopy Using Deep Learning

AbstractHere, a resolution enhancement method is developed for post‐processing images from atomic force microscopy (AFM). This method is based on deep learning neural networks in the AFM topography measurements. In this study, a very deep convolution neural network is developed to derive a high‐resolution topography image from a low‐resolution topography image. The AFM measured images from various materials are tested in this study. The derived high‐resolution AFM images are comparable with the experimental measured high‐resolution images measured at the same locations. The results suggest that this method can be developed as a general post‐processing method for AFM image analysis.

Investigating the Nanodomain Organization of Rhodopsin in Native Membranes by Atomic Force Microscopy.

Membrane proteins play an integral role in cellular communication. They are often organized within the crowded cell membrane into nanoscale domains (i.e., nanodomains), which facilitates their function in cell signaling processes. The visualization of membrane proteins and nanodomains within biological membranes under physiological conditions presents a challenge and is not possible using conventional microscopy methods. Atomic force microscopy (AFM) provides an opportunity to study the organization of membrane proteins within biological membranes with sub-nanometer resolution. An example of a membrane protein organized into nanodomains is rhodopsin. Rhodopsin is expressed in photoreceptor cells of the retina and upon photoactivation initiates a series of biochemical reactions called phototransduction, which represents the first steps of vision. AFM has provided an opportunity to directly visualize the packing of rhodopsin in native retinal membranes and the quantitative analysis of AFM images is beginning to reveal insights about the nanodomain organization of rhodopsin in the membrane. In this report, we outline procedures for imaging rhodopsin nanodomains by AFM and the quantitative analysis of those AFM images.

The conformation and dynamics of P-glycoprotein in a lipid bilayer investigated by atomic force microscopy

The membrane-bound P-glycoprotein (Pgp) transporter plays a major role in human disease and drug disposition because of its ability to efflux a chemically diverse range of drugs through ATP hydrolysis and ligand-induced conformational changes. Deciphering these structural changes is key to understanding the molecular basis of transport and to developing molecules that can modulate efflux. Here, atomic force microscopy (AFM) is used to directly image individual Pgp transporter molecules in a lipid bilayer under physiological pH and ambient temperature. Analysis of the Pgp AFM images revealed “small” and “large” protrusions from the lipid bilayer with significant differences in protrusion height and volume. The geometry of these “small” and “large” protrusions correlated to the predicted extracellular (EC) and cytosolic (C) domains of the Pgp X-ray crystal structure, respectively. To assign these protrusions, simulated AFM images were produced from the Pgp X-ray crystal structures with membrane planes defined by three computational approaches, and a simulated 80 Å AFM cantilever tip. The theoretical AFM images of the EC and C domains had similar heights and volumes to the “small” and “large” protrusions in the experimental AFM images, respectively. The assignment of the protrusions in the AFM images to the EC and C domains was confirmed by changes in protrusion volume by Pgp-specific antibodies. The Pgp domains showed a considerable degree of conformational dynamics in time resolved AFM images. With this information, a model of Pgp conformational dynamics in a lipid bilayer is proposed within the context of the known Pgp X-ray crystal structures.

Ultrastructural changes in red blood cells in aortic dissection patients under extracorporeal circulation: Atomic force microscopy study.

During extracorporeal circulation in heart surgery, blood is exposed to non-physiological conditions, such as high shear stress, foreign surfaces, turbulence, and hypothermia. These factors cause damage to the red blood cells, which is manifested by immediate and delayed hemolysis or some changes in the mechanical properties of red blood cells, defined as sublethal trauma. Unfortunately, sublethal trauma is hard to detect, and there is not enough morphological evidence regarding red blood cell sublethal trauma. In this study, red blood cell sublethal trauma was observed after extracorporeal circulation by describing ultrastructural changes in red blood cell membranes using atomic force microscopy and scanning electron microscopy. Venous blood (2 mL) was collected into heparin tubes from preoperative, intraoperative and postoperative aortic dissection patients for comparison with blood from healthy patients. The red blood cell morphological study (malformations percentage, diameter, height, concavity, and roughness) was performed with scanning electron microscopy and atomic force microscopy. Scanning electron microscopy and atomic force microscopy imaging analysis revealed that the red blood cell shape changed during extracorporeal circulation and that the red blood cell malformation percentage in the postoperative group was higher than those in the preoperative and intraoperative groups. Most morphological parameters had no obvious changes, except roughness (Ra and Rq) in aortic dissection patients. Atomic force microscopy quantitative analysis indicated that the roughness of red blood cell membranes increased during extracorporeal circulation. This study demonstrates that ultrastructural morphological damage occurs to red blood cells membranes due to extracorporeal circulation in aortic dissection patients. In addition, we provided a new parameter (Ra and Rq) to evaluate red blood cell sublethal trauma.

Self-organized nanopatterning of polycarbonate surfaces by argon ion sputtering

Polycarbonate surfaces were sputtered using 40 keV Ar+ ions at off normal incidence of 30° with ion fluences ranging from 1 × 1016 Ar+ cm−2 to 5 × 1016 Ar+ cm−2. Surface topography along with structural modification has been studied using atomic force microscope (AFM) and Raman spectroscopy. Substantial smoothing of polymeric surfaces along with evolution of some random dot like nano structures after ion irradiation has been revealed. Average size of dots varied from 34 to 95 nm while density of dots varied from 0.17 to 14.7 × 107 dots cm−2 for various ion fluences. Power spectral density spectrum has been obtained from fast Fourier analysis of respective AFM images to get the information about underlying mechanism of sputtering induced nano structuring and smoothing of polymeric surfaces. AFM results have been well correlated with the disordering parameters calculated from the deconvoluted Raman spectra. These nanopatterned substrates are useful in a wide range of important applications, for example, in bioengineering, binary optics, making organic thin-film transistors and light-emitting diodes.

High-resolution AFM structure of DNA G-wires in aqueous solution

We investigate the self-assembly of short pieces of the Tetrahymena telomeric DNA sequence d[G4T2G4] in physiologically relevant aqueous solution using atomic force microscopy (AFM). Wire-like structures (G-wires) of 3.0 nm height with well-defined surface periodic features were observed. Analysis of high-resolution AFM images allowed their classification based on the periodicity of these features. A major species is identified with periodic features of 4.3 nm displaying left-handed ridges or zigzag features on the molecular surface. A minor species shows primarily left-handed periodic features of 2.2 nm. In addition to 4.3 and 2.2 nm ridges, background features with periodicity of 0.9 nm are also observed. Using molecular modeling and simulation, we identify a molecular structure that can explain both the periodicity and handedness of the major G-wire species. Our results demonstrate the potential structural diversity of G-wire formation and provide valuable insight into the structure of higher-order intermolecular G-quadruplexes. Our results also demonstrate how AFM can be combined with simulation to gain insight into biomolecular structure.

Effect of Ascorbic Acid Additions on the Mechanism Underlying the Growth of Nanostructured PbSe Films via Hydrochemical Deposition

The effect of ascorbic acid additions on the growth mechanism of thin PbSe films produced by hydrochemical deposition has been studied by atomic force microscopy (AFM). A fractal analysis of AFM images of the lead selenide films has shown that their formation follows the “cluster–particle” aggregation mechanism. The fractal dimension assessed by the box-counting method is 2.26 at an average grain size of ~150 nm. From the transmission spectra of the films grown at deposition times from 20 to 120 min, we have evaluated their optical band gap, which has been shown to decrease from 0.595 to 0.53 eV with increasing deposition time. These values are attributable to the manifestation of quantum size effects in the grown PbSe films.

Surfactant Copolymer Annealing of Chemically Permeabilized Cell Membranes.

Structural breakdown of the cell membrane is a primary mediator in trauma induced tissue necrosis. When membrane disruption exceeds intrinsic membrane sealing processes, biocompatible multi-block amphiphilic copolymer surfactants such as Poloxamer 188 (P188) have been found to be effective in catalyze or augment sealing. Although in living cells copolymer induced sealing of membrane defects has been detected by changes in membrane transport properties, it has not been directly imaged. In this project we used Atomic force microscopy (AFM) to directly image saponin permeabilized and poloxamer sealed plasma membranes of monolayer cultured MDCK and 3T3 fibroblasts. AFM image analysis resulted in the density and diameter ranges for membrane indentations per 5×5 μm area. For control, saponin lysed, and P188 treatment of saponin lysed membranes, the supra-threshold indentation density was 3.6 ± 2.8, 13.8 ± 6.7, and 4.9 ± 3.3/cell, respectively. These results indicated that P188 catalyzed reduction in size of AFM indentations which correlated with increase cell survival. This evidence confirm that biocompatible surfactant P188 augment natural cell membrane sealing capability when intrinsic processes are incapable alone.

Impact of formulation pH on physicochemical protein characteristics at the liquid-air interface

Both, formulation parameters and the presence of liquid-air interfaces are known to affect the aggregation of protein drugs. In this study, the impact of pH on the liquid-air interfacial behavior of three proteins, a polyclonal and two monoclonal antibodies (IgG, mAB1 and mAB2) was investigated using different surface sensitive methods. Equilibrium surface pressure values revealed only a minor impact of pH. Infrared Reflectance Absorbance Spectroscopy (IRRAS) proved not only the presence of the proteins at the interface but also showed that the secondary structure was not considerably affected by the adsorption to the interface independent of pH between pH 3 and 9. Additionally, the physical resistance of the film as determined by the interfacial compressibility in a Langmuir trough was not affected by pH. Compression of the interfacial film caused the formation of telescoped areas which were no longer present after decompression at all pH values as investigated by underwater Atomic Force Microscopy (AFM). Brewster Angle Microscopy (BAM) showed some slight changes in the film reflectivity depending on pH, indicating changes in the interfacial film thickness. IRRAS experiments at different angles of incidence as well as section analysis of AFM images proved not only that the film thickness increased upon compression, but also that the interfacial film is thinner at pH 4 than at pH 9. Continuous compression and decompression of the protein film resulted in particle formation with increasing numbers of particles at higher pH value as detected by Light Obscuration (LO) and Micro-Flow Imaging (MFI). The use of different surface sensitive methods provides expedient information on how liquid-air interfacial events are affected by formulation pH. These findings enable a better understanding of not only the events and processes happening at the interface but can also be directly linked to the interface-related formation of particles.

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.