A method for calculating the spring constant of atomic force microscopy cantilevers with a nonrectangular cross section.
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Mathematics↗
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Analysis of the causes of variance in resistance measurements on metal-molecule-metal junctions formed by conducting-probe atomic force microscopy.
Alkanethiol tunnel junctions were studied using conducting-probe atomic force microscopy to determine causes of variability in measured resistance behavior. Measurements were made on Au/decanethiol/Au monolayer junctions, and effects of substrate roughness, tip chemistry, presence of solvent, extensive tip usage, applied load, and tip radius were examined. Resistance measurements yielded log-normal distributions under a variety of conditions, indicating that the origin of the variance is likely to be either changes in tunneling length or electronic overlap. Spreads in resistance values for a given tip were much less when flat, template-stripped Au substrates were used rather than rough, evaporated Au substrates. Chemical modification of tips with ethanethiol (C2) or butanethiol (C4) and performing measurements under cyclohexane were also found to reduce variance by a factor of about 2-4. Experiments performed with unmodified tips showed an increase in junction resistance over the course of hundreds of consecutive measurements, whereas junctions made with modified tips or under cyclohexane did not. Attempts to ascribe variance between tips to varying tip radii failed; however, decreases in resistance with increasing applied load on the tip contact were observed and could be interpreted in terms of conventional contact mechanics models.
Journal Article↗
Atomic force microscopy imaging of polycrystalline CuInSe2 thin films
In this study, atomic force microscopy (AFM) imaging has been used to study the structural properties of polycrystalline CuInSe2 films, which are widely used as absorber materials in thin film solar cell devices. This technique demonstrated an excellent capability for the reproducible imaging of these rough polycrystalline materials. AFM imaging in combination with statistical analysis revealed distinct differences in the structural properties (i.e. grain width and height distributions, root-mean-square (RMS) and peak to valley (R(p-v)) roughness values) as a function of the specific growth technique used and the bulk composition of the films. In the case of Cu-rich films, prepared by the H2Se/Ar treatment of Cu/In/Cu alloys, rough surface structures were in general observed. Statistical analysis revealed two distinct distribution of grains in these samples (1.0-2.5 &mgr;m and 3-5.5 &mgr;m) with large RMS and R(p-v) roughness values of 380 nm and 2.6 &mgr;m, respectively. In-rich films were characterized by the presence of much smaller, roughly circular clusters with a significant reduction in both the width and height distributions as well as RMS and R(p-v) roughness values. The most successful growth techniques, in terms of producing homogeneous and dense films, were in the cases of H2Se/Ar treated metallic InSe/Cu/InSe alloys and the coevaporation of all materials to form CuInSe2. Both these techniques produced absorber films with very narrow grain width and height distributions as well as small roughness values. It was possible to establish that high efficiency devices are associated with the use of absorber films with narrow width distributions between 0.5 and 2 &mgr;m and small RMS (> 300 nm) roughness values. These values are used as a figure of merit in our laboratories to evaluate the structural properties of our CuInSe2 thin films.
Journal Article↗
Cellular and molecular mechanics by atomic force microscopy: capturing the exocytotic fusion pore in vivo?
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Amylases↗
Atomic force microscopy of reovirus dsRNA: a routine technique for length measurements.
Atomic force microscopy (AFM) was used to image reovirus double stranded RNA (dsRNA) deposited from diluted buffer solution onto a chemically treated mica surface. This procedure allows AFM images of dsRNA molecules to be obtained with a quality close to that obtained with conventional electron microscopy. The length of the molecules were measured directly on a computer display using the digitally acquired images. The lengths of the molecules varied between 0.2 and 1.8 microns. Statistical analysis showed a multimodal distribution with clear maxima at 0.4, 0.65 and 1.05 microns. These data are in a good agreement with those obtained by electron microscopy and gel electrophoresis.
Microscopy, Scanning Tunneling↗
Atomic force microscopy of freeze-fracture replicas of rat atrial tissue.
Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.
Animals↗
Visualizing filamentous actin on lipid bilayers by atomic force microscopy in solution.
The surface structure of actin filaments (F-actin) was visualized at high resolution, by atomic force microscopy (AFM) in aqueous solution, in large paracrystals prepared on positively charged lipid monolayers. The increased stability of these closely packed specimens allowed us to show that both the long pitch (38 nm) and the monomer (5.8 nm) can be directly resolved by AFM in the contact mode. The right-handed helical surface, distinguishable in high resolution images, was compared with reconstructed models based on electron microscopy. The height of the rafts, a measure of the actin filament diameter, was 10 +/- 1 nm, whereas the smaller inter-filament distance, 8 +/- 1 nm, was consistent with interdigitation of the filaments. The 10 +/- 1 nm F-actin diameter is in good agreement with the results of fibre X-ray diffraction. As such specimens are relatively easy to prepare without specialized equipment, this method may allow the study of the thin filaments in which F-actin-associated proteins are also present.
Actin Cytoskeleton↗
An atomic force microscopy approach for assessment of particle density applied to single spray-dried carbohydrate particles.
To evaluate an atomic force microscopy (AFM) approach for effective density analysis of single spray dried carbohydrate particles in order to investigate the internal structure of the particles. In addition, the AFM method was compared to an established technique, that is gas pycnometry. Resonant frequency AFM analysis was employed for determination of the mass of individual particles of spray-dried lactose, mannitol, and a mixture of sucrose/dextran (4:1). The effective particle density was calculated using the diameter of the spherical particles obtained from light microscopy. The apparent particle density was further analyzed with gas pycnometry. It was observed by microscopy that particles appeared either "solid" or "hollow." A solid appearance applied to an effective particle density close to the true density of the material, whereas a density around 1 g/cm(3) corresponded to a hollow appearance. However, carbohydrates, which crystallized during spray drying, for example, mannitol appeared solid but the average effective particle density was 0.95 g/cm(3), indicating a continuous but porous structure. AFM measurements of effective particle density corroborate the suggestion of differences in particle structure caused by the varying propensity of carbohydrates to crystallize during spray drying, resulting in mainly either amorphous hollow or crystalline porous particles.
Carbohydrates↗
[Effect of supporting substrates on the structure of DNA and DNA-trivaline complexes studied by atomic force microscopy].
Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA-TV complexes onto this surface. On mica, both purified DNA and DNA-TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA-TP displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.
Adsorption↗
Atomic force microscopy examination of conformations of polynucleotides in response to platinum isomers: significance of GC content at broken ends.
Atomic force microscopy is a technique that enables visualization of macromolecular conformations of polynucleotides at nanometer resolution. We investigated the results of interactions of cisplatin, a DNA binding anticancer drug, and its inactive counterpart, transplatin isomer, on the molecular conformation of polynucleotides: poly d(G-C). poly d(G-C) (polyGC) and poly d(A-T). poly d(A-T) (polyAT). We observed that polyAT exhibited an increased number of enlarged ends of molecules, which we attribute to unwound and/or collapsed regions of polyAT. PolyGC molecules did not show such ends unless cisplatin was added to the PolyGC polymers. Transplatin had the apparent effect of causing overlapping or stacking of the polymer molecules. Addition of exonuclease-III to these polymers removed the visible enlarged ends. The effects of cisplatin as compared to transplatin on the polyGC duplex polymers provide support for the presence of intrastrand covalent linkages, consistent with known N7 guanine interaction of the cis isomer on molecular conformation. Furthermore, our results indicate that the mechanism of interactions of DNA with cisplatin may be dependent on the GC content of the molecules. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 68-72, 2000.
Antineoplastic Agents↗
Nanostructure of native pectin sugar acid gels visualized by atomic force microscopy.
Height and phase shift images of high methoxyl sugar acid gels (HMSAG) of pectin were obtained by atomic force microscopy in the tapping mode. Images revealed that pores in these gels were fluid and flattened out when measured as a function of time. These images revealed for the first time the structure of adsorbed sugar on pectin in the hydrated native gels and how the pectin framework is organized within these gels. Segmentation of images revealed that the underlying pectin framework contained combinations of rods, segmented rods, and kinked rods connected end to end and laterally. The open network of strands was similar to pectin aggregates from 5 mM NaCl solution imaged earlier by electron microscopy (Fishman et al., Arch. Biochem. Biophys. 1992, 294, 253). Area measurements revealed that the ratio of bound sugar to pectin was in excess of 100 to 1 (w/w). Furthermore, images indicated relatively small differences in the organization of native commercial citrus pectin, orange albedo pectin, and lime albedo pectin gels at optimal pH as determined in this study. The findings are consistent with earlier gel strength measurements of these gels. In addition, values of gel strength were consistent with values of molar mass and viscosity of the constituent pectins in that they increased in the same order. Finally, we demonstrated the advantage of simultaneous visualization of height and phase shift images for observing and quantitating the nanostructure of relatively soft gels which are fully hydrated with a buffer.
Citrus aurantiifolia↗
Non-Hertzian approach to analyzing mechanical properties of endothelial cells probed by atomic force microscopy.
Detailed measurements of cell material properties are required for understanding how cells respond to their mechanical environment. Atomic force microscopy (AFM) is an increasingly popular measurement technique that uniquely combines subcellular mechanical testing with high-resolution imaging. However, the standard method of analyzing AFM indentation data is based on a simplified "Hertz" theory that requires unrealistic assumptions about cell indentation experiments. The objective of this study was to utilize an alternative "pointwise modulus" approach, that relaxes several of these assumptions, to examine subcellular mechanics of cultured human aortic endothelial cells (HAECs). Data from indentations in 2- to 5-microm square regions of cytoplasm reveal at least two mechanically distinct populations of cellular material. Indentations colocalized with prominent linear structures in AFM images exhibited depth-dependent variation of the apparent pointwise elastic modulus that was not observed at adjacent locations devoid of such structures. The average pointwise modulus at an arbitrary indentation depth of 200 nm was 5.6+/-3.5 kPa and 1.5+/-0.76 kPa (mean+/-SD, n=7) for these two material populations, respectively. The linear structures in AFM images were identified by fluorescence microscopy as bundles of f-actin, or stress fibers. After treatment with 4 microM cytochalasin B, HAECs behaved like a homogeneous linear elastic material with an apparent modulus of 0.89+/-0.46 kPa. These findings reveal complex mechanical behavior specifically associated with actin stress fibers that is not accurately described using the standard Hertz analysis, and may impact how HAECs interact with their mechanical environment.
Algorithms↗
Atomic force microscopy: a new way to look at chromatin.
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Animals↗
Structure of branched DNA molecules: gel retardation and atomic force microscopy studies.
DNA heteroduplexes as models for slipped strand DNA have been analyzed by polyacrylamide gel migration and atomic force microscopy (AFM). All heteroduplexes containing one hairpin or loop have reduced electrophoretic mobilities compared with that expected for their molecular weights. The retarded gel mobility correlates with the formation of a sharp kink detected by AFM. Increasing the hairpin length from 7 bp to 50 bp results in a monotonous decrease in gel mobility of heteroduplexes. This secondary retardation effect appears to depend only on the hairpin size since the AFM data show no dependence of the kink angle on the hairpin length. Heteroduplex isomers with a loop or hairpin in opposite strands migrate with distinct mobilities. Analysis of gel migration of heteroduplexes with altered hairpin orientations as well as of truncated heteroduplexes indicates that the difference in mobility is due to an inherent curvature in one of the long arms. This is confirmed by the end-to-end distance measurements from AFM images. In addition, significant variation of the end-to-end distances is consistent with a dynamic structure of heteroduplexes at the three-way junction. Double heteroduplexes containing one hairpin in each of the complementary strands also separate in a gel as two isomers. Their appearance in AFM showed a complicated pattern of flat representations of the three-dimensional structure and may indicate a certain degree of interaction between complementary parts of the hairpins that are several helical turns apart.
Base Sequence↗
Frequency modulation atomic force microscopy reveals individual intermediates associated with each unfolded I27 titin domain.
In this study, we apply a dynamic atomic force microscopy (AFM) technique, frequency modulation (FM) detection, to the mechanical unfolding of single titin I27 domains and make comparisons with measurements made using the AFM contact or static mode method. Static mode measurements revealed the well-known force transition occurring at 100-120 pN in the first unfolding peak, which was less clear, or more often absent, in the subsequent unfolding peaks. In contrast, some FM-AFM curves clearly resolved a force transition associated with each of the unfolding peaks irrespective of the number of observed unfolded domains. As expected for FM-AFM, the frequency shift response of the main unfolding peaks and their intermediates could only be detected when the oscillation amplitudes used were smaller than the interaction lengths being measured. It was also shown that the forces measured for the dynamical interaction of the FM-AFM technique were significantly lower than those measured using the static mode. This study highlights the potential for using dynamic AFM for investigating biological interactions, including protein unfolding and the detection of novel unfolding intermediates.
Aluminum Silicates↗
Morphology of cultured human epidermal melanocytes observed by atomic force microscopy.
The objective of this study was to image the surface structure of cultured human epidermal melanocytes using atomic force microscopy (AFM). Epidermis obtained from human foreskins was treated with 0.5% dispase. Cell suspensions of the epidermis were prepared and seeded in six-well plates, in which sheets of mica had been placed. Samples for AFM were fixed on mica and scanning AFM images were captured by contacting and tapping modes operated under normal atmospheric pressure and temperature. Human epidermal melanocytes exhibited rounded, oval, triangular or quadrangular perikarya from which eight to 10 thick dendrites arose. These dendrites first bifurcated near the soma and then divided profusely into daughter branches, which spread out in all directions. We observed string-like long thin projections, growth cones and shorter thicker projections, which arose from the dendritic shafts, in which groups of melanosomes were arrayed. In addition to such structures, the most striking feature was the presence of filopodia arising from the melanocyte dendrite tips and the melanocyte cell body, many of which contained melanosomes. The termini of dendrites formed unbranched terminal protrusions (approximately 1,500-2,000 nm wide) consisting of two to three melanosomes wrapped in an arc, with their filopodia extending outwards. The tips of these structures also appeared to be squeezed and finally pinched off by the melanocyte to form a pouch filled with numerous melanosomes. We conclude that secondary and tertiary branches and subordinate branches might take part in transferring melanosomes into keratinocytes in addition to the transfer through the tips of the dendritic shafts. The melanin granules were expelled by exocytosis.
Cells, Cultured↗
Real-time monitoring of polymer swelling on the nanometer scale by atomic force microscopy.
The swelling of a polymer surface has been monitored in real time on the nanometer scale by atomic force microscopy (AFM). After modification by oxygen plasma treatment, poly(p-phenylene terephthalamide) (PPTA) displays a characteristic nanostructured surface morphology consisting of high-lying features alternating with topographically depressed areas. Selective swelling of the least cross-linked, depressed areas after the adsorption of ambient water or water from saturated humid atmospheres was observed by tapping mode AFM operated in the attractive interaction regime. The swollen areas could be distinguished from the nonswollen ones by local variations in the sample indentation made by the AFM tip when imaging in the tapping mode repulsive interaction regime. Monitoring the swelling of the plasma-treated polymer surface provided a means to reveal the nanometer-scale heterogeneity that this type of treatment creates on the polymer surface, which is something that would not be possible otherwise. Measurement of AFM tip-sample adhesion forces evidenced rapid water adsorption onto the oxygen plasma-treated surface, supporting the idea of water-induced swelling. This high hydrophilicity was interpreted as arising from the incorporation of polar oxygen functionalities, as demonstrated by X-ray photoelectron spectroscopy (XPS).
Microscopy, Atomic Force↗
Atomic force microscopy studies of living cells: visualization of motility, division, aggregation, transformation, and apoptosis.
Atomic force microscopy, in contact mode, has been used to image living mammalian cells in culture at both low and high resolutions. The method is shown practical for revealing cytoskeletal features beneath the cell membrane and their restructuring during a variety of cellular activities. Among the processes that we have visualized are locomotion, tissue formation, cell division, transformation by viruses, and cell death. We show that some processes that occur well within cells can, nonetheless, be observed using the atomic force probe. At high resolution, features on the cell surface on the order of 0.5 micron and their changes with time, can be recorded.
Animals↗