Analysis Of Atomic Force Microscopy Images Research Articles

Quantitative atomic force microscopy image analysis of unusual filaments formed by the Acanthamoeba castellanii myosin II rod domain

We describe a quantitative analysis of Acanthamoeba castellanii myosin II rod domain images collected from atomic force microscope experiments. These images reveal that the rod domain forms a novel filament structure, most likely requiring unusual head-to-tail interactions. Similar filaments are seen also in negatively stained electron microscopy images. Truncated myosins from Acanthamoeba and other model organisms have been visualized before, revealing laterally associated bipolar minifilaments. In contrast, the filament structures that we observe are dominated by axial rather than lateral polymerization. The unusually small features in this structure (1–5 nm) required the development of quantitative and statistical techniques for filament image analysis. These techniques enhance the extraction of features that hitherto have been difficult to ascertain from more qualitative imaging approaches. The heights of the filaments are observed to have a bimodal distribution consistent with the diameters of a single rod domain and a pair of close-packed rod domains. Further quantitative analysis indicates that in-plane association is limited to at most a pair of rod domains. Taken together, this implies that the filaments contain no more than four rod domains laterally associated with one another, somewhat less than that seen in bipolar minifilaments. Analysis of images of the filaments decorated with an anti-FLAG antibody reveals head-to-tail association with mean distances between the antibodies of 75 ± 15 nm. We consider a set of molecular models to help interpret possible structures of the filaments.

Application of atomic force microscopy and scaling analysis of images to predict the effect of current density, temperature and leveling agent on the morphology of electrolytically produced copper

Atomic force microscopy (AFM) was used to study the morphology of electrodeposited Cu at current densities from 183 to 253 A m −2. Digital image analysis was employed to parameterize the morphological information encoded in AFM images and to extract information concerning the mechanism of the electrodeposition reaction. It has been shown how the limiting roughness, δ, the critical scaling length, L c and the aspect ratio, 4 δ/ L c, vary as a function of the deposition time and electrodeposition conditions, such as temperature, current density and the amount of organic additives. It has been demonstrated how laboratory experiments of short duration and the scaling analysis of AFM images can be used to predict roughness of the metal sample after 2 weeks of industrial electrorefining.

Effect of pore structure on anomalous behaviour of the lithium intercalation into porous V 2O 5 film electrode using fractal geometry concept

The effect of pore structure on anomalous behaviour of the lithium intercalation into porous V 2O 5 film electrode has been investigated in terms of fractal geometry by employing ac-impedance spectroscopy combined with N 2 gas adsorption method and atomic force microscopy (AFM). For this purpose, porous V 2O 5 film electrodes with different pore structures were prepared by the polymer surfactant templating method. From the analysis of N 2 gas adsorption isotherms and the triangulation analysis of AFM images, it was found that porous V 2O 5 surfaces exhibited self-similar scaling properties with different fractal dimensions depending upon amount of the polymer surfactant in solution and the spatial cut-off ranges. All the ac-impedance spectra measured on porous V 2O 5 film electrodes showed the non-ideal behaviour of the charge-transfer reaction and the diffusion reaction, which resulted from the interfacial capacitance dispersion and the frequency dispersion of the diffusion impedance, respectively. From the comparison between the surface fractal dimensions by using N 2 gas adsorption method and AFM, and the analysis of ac-impedance spectra by employing a constant phase element (CPE), it is experimentally confirmed that the lithium intercalation into porous V 2O 5 film electrode is crucially influenced by the pore surface irregularity and the film surface irregularity.

Interactive measurement and characterization of DNA molecules by analysis of AFM images

In the past few years, computer-based analysis of atomic-force microscopic images has acquired increasing importance for studying biomolecules such as DNA. On the one hand, fully automated methods do not allow analysis of complex shapes; on the other hand, manual methods are usually time consuming and inaccurate. The semiautomated approach presented in this report overcomes the drawbacks of both methods. Two kinds of images were analyzed: computer-generated filaments that modeled circular DNA molecules on a surface and real atomic-force microscopic images of DNA molecules adsorbed on an appropriate substrate surface. The algorithm was tested on a group of 140 simulated and 189 real plasmids with a nominal length of 913 nm. The accuracy of the length measurement was statistically evaluated on the ensemble of molecules, with particular attention to the influence of the noise. Mean contour lengths of 912 +/- 5 nm and 910 +/- 47 nm were found for simulated and real plasmids, respectively. The measured end-to-end distance of lambda-DNA molecules as a function of their contour length is reported, from which it is possible to estimate the stiffness of the DNA molecules adsorbed onto a surface; the value obtained for the DNA persistence length (42 +/- 5 nm) is consistent with values measured by other imaging techniques. An interactive algorithm for DNA molecule measurements based on the detection of the filament ridge line in a digitized image is presented. The simulation of artificial filaments combined with the experimental data demonstrates that the proposed method can be a valuable tool for the DNA contour length evaluation, especially in the case of complex shapes where the use of automatic methods is not possible.

Fine Surface Structure of Bovine Acrosome-Intact and Reacted Spermatozoa Observed by Atomic Force Microscopy

The atomic force microscope (AFM) provides nanometer resolution, topographic data of the natural surface structure of materials. We studied the topology of the surface structure of bovine sperm heads during the acrosome reaction by AFM. In addition, we numerically analyzed the areas of the median sagittal plane of the sperm heads. Bovine frozen-thawed spermatozoa were washed, capacitated by heparin, and incubated with lysophosphatidylcholine (LPC) to induce the acrosome reaction, smeared on a cover glass, air-dried, and observed with AFM using the dynamic force (tapping) mode. AFM analysis of spermatozoa showed the clear surface structure of acrosomes, equatorial segments, postacrosomal regions and necks. Although AFM images of spermatozoa capacitated by heparin had complete acrosomes, most spermatozoa treated with LPC had no acrosomal caps as shown by AFM. These observations coincided with those obtained by light microscopy after staining with naphthol yellow S and erythrosin B. Furthermore, numerical analysis of AFM images indicated that areas of the median sagittal plane of the anterior portions of acrosome-reacted sperm heads (2679 +/- 616 pixels) were approximately 40% less than those of intact heads (4535 +/- 174 pixels, P<0.05). These results indicate that AFM can usefully observe and numerically analyze the fine surface structures of bovine spermatozoa.

Atomic force microscopy and specular reflectance infrared spectroscopic studies of the surface structure of polypropylene treated with argon and oxygen plasmas

Isotactic polypropylene (PP) surfaces were modified with argon and oxygen plasmas using a radiofrequency (RF) glow discharge at 240 mTorr and 40 W. The changes in topography and surface structure were investigated by atomic force microscopy (AFM) in conjunction with specular reflectance of infrared (IR) microspectroscopy. Under our operating conditions, the AFM image analysis revealed that longer plasma treatment resulted in significant ablation on the PP surface, regardless of the kind of plasma employed, but the topography was dependent on the nature of the gases. Specular reflectance IR spectroscopic analysis indicated that the constant removal of surface material was an important ablative aspect when using either plasma, but the nature of the ablative behavior and the resultant aging effects were clearly dependent on the choice of plasma. The use of argon plasma resulted in a negligible aging effect; in contrast, the use of oxygen plasma caused a noticeable aging effect, which was due to reactions of trapped or isolated radicals with oxygen in air, and was partly responsible for the increased surface area caused by ablation. The use of oxygen plasma is believed to be an advantageous approach to modifying polymeric materials with functionalized surfaces, e.g., for surface grafting of unsaturated monomers and incorporating oxygen-containing groups onto PP.

Atomic Force Microscopy Study of the Structural Effects Induced by Echinomycin Binding to DNA

Atomic force microscopy (AFM) has been used to examine the conformational effects of echinomycin, a DNA bis-intercalating antibiotic, on linear and circular DNA. Four different 398 bp DNA fragments were synthesized, comprising a combination of normal and/or modified bases including 2,6-diaminopurine and inosine (which are the corresponding analogues of adenine and guanosine in which the 2-amino group that is crucial for echinomycin binding has been added or removed, respectively). Analysis of AFM images provided contour lengths, which were used as a direct measure of bis-intercalation. About 66 echinomycin molecules are able to bind to each fragment, corresponding to a site size of six base-pairs. The presence of base-modified nucleotides affects DNA conformation, as determined by the helical rise per base-pair. At the same time, the values obtained for the dissociation constant correlate with the types of preferred binding site available among the different DNA fragments; echinomycin binds to TpD sites much more tightly than to CpG sites. The structural perturbations induced when echinomycin binds to closed circular duplex pBR322 DNA were also investigated and a method for quantification of the structural changes is presented. In the presence of increasing echinomycin concentration, the plasmid can be seen to proceed through a series of transitions in which its supercoiling decreases, relaxes, and then increases.

Quantitative analysis and classification of AFM images of human hair.

The surface topography of human hair, as defined by the outer layer of cellular sheets, termed cuticles, largely determines the cosmetic properties of the hair. The condition of the cuticles is of great cosmetic importance, but also has the potential to aid diagnosis in the medical and forensic sciences. Atomic force microscopy (AFM) has been demonstrated to offer unique advantages for analysis of the hair surface, mainly due to the high image resolution and the ease of sample preparation. This article presents an algorithm for the automatic analysis of AFM images of human hair. The cuticular structure is characterized using a series of descriptors, such as step height, tilt angle and cuticle density, allowing quantitative analysis and comparison of different images. The usefulness of this approach is demonstrated by a classification study. Thirty-eight AFM images were measured, consisting of hair samples from (a) untreated and bleached hair samples, and (b) the root and distal ends of the hair fibre. The multivariate classification technique partial least squares discriminant analysis is used to test the ability of the algorithm to characterize the images according to the properties of the hair samples. Most of the images (86%) were found to be classified correctly.

In situ AFM studies of astrocyte-secreted apolipoprotein E- and J-containing lipoproteins.

The three-dimensional shapes and sizes of plasma lipoproteins and astrocyte-secreted lipoproteins (ASLPs) were characterized with the aid of in situ atomic force microscopy (AFM), which has the unique ability to study three-dimensional nanostructures under physiological conditions. Apolipoprotein E (apoE) and apolipoprotein J (apoJ) are the two most abundant apolipoproteins produced in the central nervous system (CNS). This study revealed that ASLPs containing apoE3, apoE4, or apoJ significantly differ from high density lipoprotein particles, thought to be their closest analogs in plasma, in aggregation properties, size, and shape. ASLPs were found to be significantly flatter and smaller than their plasma counterparts. Plasma lipoproteins were able to form ordered arrays on a mica surface at high concentration, but ASLPs did not. Rather, they formed amorphous aggregates at similar concentrations. Comprehensive quantitative characterization of particle size and shape was facilitated by two advances in AFM image analysis: (1) automated analysis through image-recognition algorithms, and (2) correction for the finite size of the AFM probe based on geometric modeling. This study and the developed AFM methodologies open the way to further in situ AFM studies of the lipoproteins in general and more specifically of CNS lipoproteins.

DNA-Directed Synthesis of Generation 7 and 5 PAMAM Dendrimer Nanoclusters

A novel nanostructure was constructed using two different generations of polyamidoamine (PAMAM) dendrimers and three sets of complementary oligonucleotides (34, 50, and 66 bases in length). The oligonucleotides were covalently conjugated to partially acetylated generation 5 and 7 PAMAM dendrimers, and these conjugates were characterized by agarose gel electrophoresis. The agarose gel electrophoresis appearance of these covalently linked oligonucleotide dendrimers was also compared to electrostatically bound oligonucleotide−dendrimer complexes. Equimolar amounts of the G5 and G7 conjugates were then hybridized together to allow for the DNA-directed self-assembly of supramolecular clusters. Dynamic light scattering (DLS) analysis indicated that the overall size of the DNA-linked dendrimer clusters tended to increase according to the length of the oligonucleotide used ranging from 30 to 50 nm, which agreed with the diameter of dendrimer nanoclusters predicted by molecular modeling. The DNA-linked novel dendrimer nanoclusters were also examined with tapping-mode atomic force microscopy (AFM) to distinguish the DNA-linked structure from a nonlinked simple G7/G5 dendrimer mixture. AFM image analysis suggested that the distance between the DNA-linked dendrimers was significantly larger than what was seen after simple mixing of G7/G5 dendrimers. The mixture showed a few dendrimers physically in contact with an interdendrimer distance of 8−10 nm. The interdendrimer distance of the nanoclusters linked with the 50-base-long oligonucleotide pairs was measured to be 21 ± 2 nm, which is in agreement with the theoretical length of the oligonucleotides duplex. These results suggest that PAMAM dendrimers can be self-assembled via complementary oligonucleotides to form supramolecular nanoclusters.

UV-induced graft polymerization of polypropylene-g-glycidyl methacrylate membrane in the vapor phase

UV-induced graft polymerization of glycidyl methacrylate (GMA) to a polypropylene (PP) membrane was carried out in the vapor phase with benzophenone (BP) as a photoinitiator. Attenuated total reflection Fourier transform infrared spectroscopy, atomic force microscopy (AFM), and scanning electron microscopy (SEM) were utilized to characterize the copolymer. The degree of grafting increased with increasing reaction time, increased UV irradiation source intensity, and increased immersion concentration of the BP solution. The optimum synthetic condition for the PP-g-GMA membrane was obtained with a reaction time of 2 hrs, a UV irradiation source intensity of 450W, and an immersion concentration of the BP solution of 0.5 mol/L. The pure water flux decreased upon increasing the degree of grafting and increasing the amount of diethylamino functional group introduced. The analysis of AFM and SEM images shows that the graft chains and diethylamino groups of PP-g-GMA grew on the PP membrane surface, resulting in a change in surface morphology.

Atomic force microscopic studies of novel arborescent block and linear triblock polystyrene–polyisobutylene copolymers

This paper reports the investigation of the nanostructured surface morphology of novel arborescent polyisobutylene- block-polystyrene (PIB–PS) copolymers, in comparison with linear PS–PIB–PS triblock copolymers, using atomic force microscopy (AFM) in tapping mode. Arborescent PIB–PS samples displayed interesting new phase morphologies, which changed dramatically upon annealing but remained irregular. Linear PS–PIB–PS samples showed morphologies similar to those previously found by transmission electron microscopy (TEM) in cryomicrotomed bulk samples, ranging from spherical/cylindrical to lamellar nanometer-sized discreet PS phases dispersed in a continuous PIB matrix. Annealing the samples resulted in more ordered structures. Three-dimensional AFM image and section analysis indicated a height difference between PIB and PS in the block copolymers, which became more prominent during annealing. This feature was verified on compression moulded and protein coated samples. The arborescent PIB–PS materials displayed thermoplastic elastomeric behaviour with a tensile strength between 7 and 10 MPa and elongation ranging from 1000% to 1830%. In comparison, linear triblock samples had a tensile strength between 7 and 20 MPa and elongation ranging from 380% to 640%. Block copolymers with irregular elastomeric midsegments may emerge as a new class of TPEs.

Atomic force microscopy characterisation of scratch deformation in long and short chain isotactic polypropylenes and ethylene-propylene copolymers

Surface damage induced during the scratch process in two different grades of high isotactic (high crystallinity) polypropylenes, long and short chain, has been examined by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Atomic force microscopy enabled qualitative and quantitative prediction of the extent of scratch damage, by analysing AFM images before and after scratch deformation. Surface deformation induced by the scratch test on long and short chain high isotactic polypropylenes involved the formation of zigzag scratch tracks. Scratch tracks were regular in long chain and irregular/nonuniform in short chain high isotactic polypropylenes. The line profile and section analysis of AFM images indicated that short chain high isotactic polypropylene is more resistant to mechanically induced surface damage than long chain high isotactic polypropylene. Furthermore, a comparison of scratch damage behaviours of high isotactic polypropylenes, low isotactic polypropylenes, and ethylene-propylene copolymers indicated that the resistance to scratch deformation follows the sequence: high isotactic (high crystallinity) polypropylenes > low isotactic (low crystallinity) polypropylenes > ethylene-propylene copolymers.

An investigation of the capacitance dispersion on the fractal carbon electrode with edge and basal orientations

Capacitance dispersion on the fractal carbon electrode with edge and basal orientations was investigated using atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and a.c.-impedance spectroscopy. For this purpose, four types of as-received pyrolytic graphite electrode, as-received, mechanically polished, and as-activated glassy carbon electrodes were prepared with different surface irregularities and amounts of edge orientations. The apparent self-similar fractal dimensions of the carbon electrodes were determined from the analyses of AFM images based upon triangulation method. The amounts of edge orientations on the surface of the carbon electrodes were qualitatively estimated from the XPS analysis of surface acidic functional groups that were preferably formed on the edge planes by the heat treatment of the carbon electrodes. The values of the constant phase element exponent α determined from the apparent self-similar fractal dimensions did not accord with those α determined from the measured impedance spectra. Instead, they were closely related to the amounts of the edge orientations. From the results, it is indicated that the contribution of surface inhomogeneity is much higher than the contribution of the surface irregularity to the capacitance dispersion on the carbon electrode.

Formation and optical characterisation of colloidal gold monolayers

We study the deposition of charge-stabilised gold nanocolloids on silicon substrates, which have been derivatised with (aminopropyl)triethoxysilane. Atomic force microscopy (AFM) and spectroscopic ellipsometry are employed to investigate the nanocrystal monolayers ex situ. Analysis of AFM images provides evidence that the formation of colloidal monolayers is governed by random sequential adsorption. The results indicate that the ionic strength of the suspension influences the spatial distribution of the colloids. In situ reflectometry also shows the effect of the (tunable) electrostatic interactions between the gold particles on the deposition kinetics. Especially in the saturation regime these interactions dominate the self-assembly.

A study on ionic diffusion towards self-affine fractal electrode by cyclic voltammetry and atomic force microscopy

Ionic diffusion towards a self-affine fractal electrode was investigated experimentally using both cyclic voltammetry and atomic force microscopy (AFM). For this purpose, three kinds of self-affine fractal Pt film electrodes were first prepared by dc sputtering of Pt on such substrate materials with different roughnesses as polished Al 2O 3, etched Ni and unpolished Al 2O 3. Then, the surface morphologies of the electrode specimens were examined by using AFM and cyclic voltammograms (CVs) were measured on the electrode specimens in a 30 wt.% glycerol+70 wt.% (0.01 M K 4[Fe(CN) 6]+0.5 M Na 2SO 4) solution at various scan rates. Finally, the fractal dimensions of the electrode surfaces were determined from analyses of AFM images and the power relation between peak current and scan rate in the CVs. All the fractal dimensions determined from the CVs were much smaller than the self-affine fractal dimensions determined by the perimeter–area method. Assuming that the self-affine fractal surface can have a self-similar scaling property, the apparent self-similar fractal dimensions of the self-affine fractal electrodes were determined by a triangulation method. These values agreed well with the fractal dimensions determined from the CVs. From the above results, it is concluded that ionic diffusion towards a self-affine fractal electrode should be described in terms of the apparent self-similar fractal dimension rather than the self-affine fractal dimension.

Influence of Additives on Copper Electrodeposition on Physical Vapor Deposited (PVD) Copper Substrates

Cross-sectional scanning electron microscopy (SEM), secondary-ion mass spectrometry (SIMS), atomic force microscopy (AFM), and electrochemical measurements are utilized to examine the effect of additives on the morphology and nature of Cu electrodeposited on Cu substrates. The additives used here include 4,5-dithiaoctane-1,8-disulfonic acid (SPS), polyethylene glycol (PEG), and SIMS results show that SPS is always included into Cu electrodeposits, while PEG is never included. is included in deposits only when SPS is present. SEM measurements show that one consequence of this inclusion is the formation of an internal grain structure, which is not evident in the surface morphology measured by AFM alone. The growth of deposits is discussed in terms of cross-sectional and scaling analysis of AFM images. © 2003 The Electrochemical Society. All rights reserved.

Native surface structure of natural soil particles determined by combining atomic force microscopy and X-ray photoelectron spectroscopy

A procedure was developed for handling natural soil particles and probing their native surface structure by atomic force microscopy (AFM) under water. This procedure was used to investigate the nanometer scale organisation of organic matter at the surface of sand particles taken from three soil horizons. The latter were selected for the contrasted properties of their organic matter, namely Podzol E and Bh horizons and a Cambisol A–B horizon. The presence of an adsorbed layer was visualised at the surface of Podzol Bh and Cambisol particles in the form of aggregated structures that interacted with the AFM probe. Surface analysis by X-ray photoelectron spectrometry (XPS) confirmed the carbonaceous nature of this adsorbed layer. Displacement of organic matter by the scanning probe was directly evidenced for Podzol Bh sand particles. Such displacement was not observed for Cambisol particles. A dramatic effect of drying on the concentration, nanometer scale distribution and properties of the adsorbed organic matter was clearly demonstrated by combining AFM imaging and XPS analysis. The procedure developed here gives access to direct, nanoscale information of the surface structure of sand particles and offers promising prospects for the characterisation of other environmentally-relevant particles in native conditions.

Micro-Phase Separation in Binary Mixed Langmuir Monolayers of n-Alkyl Fatty Acids and a Perfluoropolyether Derivative

Micro-phase separation in binary mixed Langmuir monolayers of cadmium salts of n-alkyl fatty acids (CH3(CH2)n-2COOH; Cn (n = 18, 20, 22, 24)) and a perfluoropolyether surfactant (F(CF(CF3)CH2O)3CF(CF3)COOH, PFPE) is studied by film balance measurement and atomic force microscopy (AFM). At different temperatures, mixtures of Cn/PFPE in chloroform were spread onto the aqueous Cd2+ subphase and deposited on silicon wafers. AFM images showed that Cn and PFPE separate into microscopic domains of condensed phase and a surrounding matrix of expanded phase, respectively, in their mixed monolayers. The morphological feature of phase-separated structures was characterized by characteristic length (λ) expressing the periodicity of two-phase distribution and fractal dimension (D) of Cn domains reflecting the complexity of domain shape, which were determined through AFM image analyses. It was found that the monolayer morphologies systematically vary with alkyl chain length of Cn and temperature of the water surface; c...

Study of the surface morphology of polyisobutylene-based block copolymers by atomic force microscopy

This paper reports the investigation of the nanostructured surface morphology of linear polystyrene-block-polyisobutylene-block-polystyrene (SIBS) triblock copolymers and novel arborescent SIBS block copolymers by Atomic Force Microscopy (AFM) in the tapping mode. Thin films spin coated from toluene onto silicon wafers were studied. The nanostructured morphology of the block copolymers varied with the hard polystyrene (PS) and soft polyisobutylene (PIB) segment composition, ranging from spherical to lamellar nanometer-sized discreet PS phases dispersed in a continuous PIB matrix. Annealing the samples resulted in well developed/ordered structures. The arborescent blocks had irregularly distributed PS phases in the PIB matrix. Annealing had a dramatic effect on the morphology which still remained irregular. Three-dimensional AFM image and section analysis indicated the presence of a height difference between PIB (high-lying plateaus or hills) and PS (low-lying plateaus or valleys) in the block copolymers, which became more prominent during annealing. It is theorized that the rubbery PIB chains are able to relax, thereby protruding from the surface, anchored by the physically crosslinked PS phases.