Atomic force microscopy study of DNA deposited on poly l-ornithine-coated mica

Analyses of individual biomolecules, like DNA, or DNA–protein complexes, via atomic force microscopy, require ‘gentle’ methods to immobilize DNA on surfaces, which allow the ensemble of molecules to adopt conformations dictated primarily by their physical characteristics, and which possibly permit the use of a wide selection of buffers. We show that poly‐l ‐ornithine‐coated mica is a good substrate for fast, reliable deposition of DNA for wet or dry imaging. The surface firmly secures DNA, which retains the B‐form helical rise (0.34 nm bp^?1). The conformations of DNA that result are reminiscent of three‐dimensional random coils projected on to a plane. The contrast is good, especially in solution, and buffers with physiological concentrations of salt with or without divalent cations may be used. This is important for comparison of scanning probe microscopy results with those obtained by different techniques.     

原子力显微镜在多糖分子结构研究中的应用

评述原子力显微镜在多糖分子结构和功能研究的进展,AFM不仅可以在空气和液体中对多糖分子单分子和聚集体成像,得到单分子的直径、长度等量化信息和分子聚集体形貌特征。近年来AFM还用于在液体池中操纵单个多糖分子,获取单分子力学谱研究分子的弹性与构型转变的关系,在单分子水平上对多糖进行鉴定,用于细胞表面大分子黏附作用和细胞识别的研究等。AFM新技术的不断出现,必将在高分子科学的研究中起到越来越重要的作用。     

Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus

Chitosan has been reported to be a non-toxic, biodegradable antibacterial agent. The aim of this work was to elucidate the relationship between the molecular weight of chitosan and its antimicrobial activity upon two model microorganisms, one Gram-positive (Staphylococcus aureus) and one Gram-negative (Escherichia coli). Atomic force microscopy (AFM) imaging was used to obtain high-resolution images of the effect of chitosans on the bacterial morphology. The AFM measurements were correlated with viable cell numbers, which show that the two species reacted differently to the high- and low-molecular-weight chitosan derivatives. The images obtained revealed not only the antibacterial effects, but also the response strategies used by the bacteria; cell wall collapse and morphological changes reflected cell death, whereas clustering of bacteria appeared to be associated with cell survival. In addition, nanoindentation experiments with the AFM revealed mechanical changes in the bacterial cell wall induced by the treatment. The nanoindentation results suggested that despite little modification observed in the Gram-positive bacteria in morphological studies, cell wall damage had indeed occurred, since cell wall stiffness was reduced after chitooligosaccharide treatment.     

Atomic vacancy-induced friction on the graphite surface: observation by lateral force microscopy

Lateral force microscopy has been employed to investigate the frictional behaviour of atomic vacancies on the graphite surface. Such a study was only made possible by the controlled expansion of originally single‐atom vacancies into multiatom vacancies, employing oxygen plasma etching for this purpose. Enhanced friction was observed on the vacancy regions compared with pristine areas of graphite, the origin of which is examined and discussed.     

Atomic force microscopy investigation of the dependence of cellular elastic moduli on glutaraldehyde fixation

The atomic force microscope (AFM) has provided nanoscale analyses of surfaces of cells that exhibit strong adhesive and cell spreading properties. However, it is frequently reported that prior fixation is required for reliable imaging of cells with lower adhesive properties. In the present study, the AFM is used to assess the effects of fixation by glutaraldehyde on the elastic modulus of a human rhabdomyosarcoma transfectant cell line RDX2C2. Our results show a sharp increase in the elastic modulus for even mild fixation (0.5% glutaraldehyde for 60 s), accompanied by a dramatic improvement in imaging reproducibility. An even larger increase is seen in NIH-3T3 mouse fibroblasts, although in that case fixation is not typically necessary for successful imaging. In addition, our results suggest that treatment with glutaraldehyde restricts the content of the resulting images to features nearer to the cell surface.     

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 μm and 3–5.5 μm) with large RMS and R_(p–v) roughness values of 380 nm and 2.6 μm, 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 μm 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.     

Atomic force microscopy studies of conditioner thickness distribution and binding interactions on the hair surface

The way in which common hair care products, such as conditioner, deposit onto and change hair properties is of interest in beauty care science, as these properties are closely tied to product performance. The binding interaction between conditioner and the hair surface is one of the important factors in determining the conditioner thickness distribution and consequently the proper functions of conditioner. In this study, atomic force microscopy was used to obtain the local conditioner thickness distribution, adhesive forces and effective Young's modulus mapping of various hair surfaces. The conditioner thickness was extracted by measuring the forces on the atomic force microscopy tip as it approached, contacted and pushed through the conditioner layer. The effective Young's moduli of various hair surfaces were calculated from the force distance curves using Hertz analysis. The intrinsic binding interactions between different silicones and the hair surface on the microscopic scale, as well as their effect on the effective Young's modulus of the hair, are also discussed. It was found that the effective Young's modulus of the hair is strongly affected by the binding of conditioner molecules on the hair surface.     

Atomic force microscopy analysis of enveloped and non-enveloped viral entry into, and egress from, cultured cells

Since its invention, the atomic force microscope has been used to image a wide variety of biological samples, including viruses. Viral entry into, and egress from, cultured cells has been extensively studied using numerous scientific techniques and to a limited extent using atomic force microscopy. One of the main structural differences that can exist between viruses is the absence, or presence, of an envelope and this factor has consequences for the mode of viral entry and egress. In this study, the entry into, and egress from, cultured cells of enveloped and non-enveloped viruses were investigated using atomic force microscopy. No significant cell surface changes were observed following infection with enveloped or non-enveloped viruses. Although roughness analysis of viral entry revealed cell smoothing post-infection, no differences between the roughness values of enveloped and non-enveloped viral entry were observed. Line analysis of viral entry revealed minor differences between cells infected with an enveloped rather than a non-enveloped virus. These differences may represent a distinction between the uptake processes of enveloped and non-enveloped viruses. Studies of viral egress revealed that infected cells were undergoing cytopathic changes. Whilst topographic, height and roughness differences clearly occurred between virally- and mock-infected cells, no significant differences were elucidated between enveloped and non-enveloped viral egress.     

Atomic force microscopy: influence of air drying and fixation on the morphology and viscoelasticity of cultured cells

The influence of fixation, air-drying and liquid-imaging on the morphology as well as on the viscoelasticity of malignant mesothelioma cells was studied by atomic force microscopy. In this study, dehydrated cells were more easily scanned and offered faster data recording than hydrated cells. However, the influence of fixation strength was more noticeable. Strong fixation induced flattening of the cytoplasm and loss of nuclear structure, resulting in a clearly visible cytoskeleton which could be easily seen as fibres orientated in the direction of the cell growth. By contrast, the morphology of hydrated cells was influenced to a lesser degree on fixation and showed an overall 'rounding' of the surface with vague, ill-defined structures. Nuclear areas of these samples were difficult to image.
Viscoelasticity measurements also exhibited large differences. Dehydrated cells were much harder and showed a uniform indentation profile over the whole cell that was independent of fixation. Indentation on hydrated cells was large and depended on the height of the measuring spot, the submembranous structure and, to a lesser extent, on fixation. To calculate an overall 'cellular' viscoelasticity, different methods were tested on these samples. Indentations of multiple, randomly chosen points, covering the whole cell, were measured and averaged to yield a mean indentation score. We avoided the thin and shadowed areas since it was shown that these regions were less suited for measuring. Using this design, large viscoelasticity differences were found, on which the influence of the external parameters could be shown. In another set-up, layered imaging was tried. However, long data acquisition times caused cellular activation and rearrangement, making this scanning mode unsatisfactory.     

Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001

A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy-dispersive X-ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al . (Search for past life on Mars — possible relic biogenic activity in martian meteorite ALH84001. Science , 1996, pp. 924–930) of NASA who used SEM imaging of gold-coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.     

Atomic force microscopy observations of acyl chains in phospholipids

The potential use of atomic force microscopy (AFM) to image the mode of assembly and to measure the corresponding lattice parameters of model systems consisting of ordered aggregates of cardiolipin molecules has been investigated. An unprecedented resolution of about 0·2 nm has been achieved on suitably prepared specimens. This enables the orientational order and the positional correlations of the individual molecules in the lattice to be defined, and submolecular details, such as the acyl chains and the polar groups, to be imaged. The structural parameters derived from AFMhave been compared with those obtained by transmission electron diffraction of the same specimen and found to be in excellent agreement. AFM turns out to be a powerful and probably a unique tool to reveal local phase variations in systems, such as biological membranes, that have non-homogeneous composition and organization.     

Improved estimation of contact compliance via atomic force microscopy using a calibrated cantilever as a reference sample

A method to improve accuracy of surface compliance determination by atomic force microscopy is presented, based on using calibrated cantilevers as the reference samples. During each work session, a 1-D compliance map of a reference cantilever is calculated from force–indentation curves along its axis, by the standard ‘indentation mode’. An independent measurement of local compliance on the reference cantilever is obtained by 2-D imaging in constant deflection and using analytical equations based on its known geometry and material properties, called ‘imaging mode’. A re-mapping of the apparent (‘indentation mode’) to the true (‘imaging mode’) compliance is thus obtained, which is applied on ‘indentation mode’ measurements of an unknown sample. This method demonstrates correction in the right direction for a polystyrene plate and a Teflon foil reference samples. The method is then applied on an unknown sample of flat agarose gel patterned with spots of polylysine protein.     

Atomic force microscopy of silica nanoparticles and carbon nanohorns in macrophages and red blood cells

The emerging interest in understanding the interactions of nanomaterial with biological systems necessitates imaging tools that capture the spatial and temporal distributions and attributes of the resulting nano–bio amalgam. Studies targeting organ specific response and/or nanoparticle-specific system toxicity would be profoundly benefited from tools that would allow imaging and tracking of in-vivo or in-vitro processes and particle-fate studies. Recently we demonstrated that mode synthesizing atomic force microscopy (MSAFM) can provide subsurface nanoscale information on the mechanical properties of materials at the nanoscale. However, the underlying mechanism of this imaging methodology is currently subject to theoretical and experimental investigation. In this paper we present further analysis by investigating tip-sample excitation forces associated with nanomechanical image formation. Images and force curves acquired under various operational frequencies and amplitudes are presented. We examine samples of mouse cells, where buried distributions of single-walled carbon nanohorns and silica nanoparticles are visualized.     

Atomic force microscopy of BHK-21 cells: an investigation of cell fixation techniques

The atomic force microscope (AFM) has been used to image a wide variety of biological samples, including cultured cells, in air. Whilst cultured cells have been prepared for AFM analysis using a variety of matrices and fixatives, a definitive study of sample preparation and its effects on cell morphology has not, as far as the authors are aware, previously been reported. Although a considerable number of cell fixatives exist, no single fixative is ideal for all investigations. Prior to the performance of specialised techniques, such as atomic force microscopy of cultured cells in air, the cell fixation method must be investigated and optimised. The fixative abilities of 2% paraformaldehyde-lysine-periodate, 0.25% glutaraldehyde, paraformaldehyde-glutaraldehyde, 4% phosphate-buffered formal saline, 1% formaldehyde, methanol:acetone, formal saline, 4% paraformaldehyde and ethanol:acetic acid were assessed in this study. A qualitative assessment system was used to evaluate the efficacy of the above fixatives using conventional fixation criteria (i.e. the presence of fibroblastic morphology consistent with optical microscopy and the absence of fixation artifacts). The optimal fixative was identified as 4% paraformaldehyde, which was capable of providing optically consistent images of BHK-21 (fibroblastic) cells, whose heights remained within the measurement capability of the AFM instrument used in this study.     

Atomic and magnetic force microscopy imaging of thin-film recording heads

Investigations on the use of the scanning probe microscope (SPM) in the atomic force microscopy (AFM) mode for topography imaging and the magnetic force microscopy (MFM) mode for magnetic imaging are presented for a thin-film recording head. Results showed that the SPM is suitable for imaging the surface profile of the recording head, determining the width of the pole gap region, and mapping the magnetic field patterns of the recording head excited under current bias conditions of different polarity. For the cobalt sputter-coated tips used in MFM imaging, it was found that the magnetic field patterns obtained under different polarities of the current bias to the recording head were similar. This can be explained by the nature of the thin-film MFM tip, in which the direction of the tip magnetic moment can follow the stray magnetic field of the sample as the current bias to the recording head reverses in direction.     

An integrated approach to the study of living cells by atomic force microscopy

We describe a technique for studying living cells with the atomic force microscope (AFM) in tapping mode using a thermostated, controlled-environment culture system. We also describe the integration of the AFM with bright field, epifluorescence and surface interference microscopy, achieving the highest level of integration for the AFM thus far described. We succeeded in the continuous, long-term imaging of relatively flat but very fragile cytoplasmic regions of COS cells at a lateral resolution of about 70 nm and a vertical resolution of about 3 nm. In addition, we demonstrate the applicability of our technology for continuous force volume imaging of cultured vertebrate cells.
The hybrid instrument we describe can be used to collect simultaneously a diverse variety of physical, chemical and morphological data on living vertebrate cells. The integration of light microscopy with AFM and steady-state culture methods for vertebrate cells represents a new approach for studies in cell biology and physiology.     

Atomic force microscopy investigation of morphological changes in living keratinocytes treated with HgCl(2) at not cytotoxic doses

Morphological changes of normal human keratinocyte cells have been monitored by means of atomic force microscopy after the exposure at a mercury solution containing HgCl(2) at 10(-7) M. The measurements have been carried out in contact mode in a thermostated liquid cell, to reproduce a cellular environment similar to the physiologic one. Remarkable alterations of the cellular morphology and volume have been revealed after few minutes from starting the exposure experiment, although the HgCl(2) concentration is several orders of magnitudes lower than the cytotoxic value (10(-4) M). The atomic force microscopy technique results to be a powerful mean to investigate modifications induced in the cell morphology by external chemical agents.     

Climbing the steps of viral atomic force microscopy: visualization of Dengue virus particles

In recent years, the application of atomic force microscopy (AFM) to biological systems has highlighted the potential of this technology. AFM provides insights into studies of biological structures and interactions and can also identify and characterize a large panel of pathogens, including viruses. The Flaviviridae family contains a number of viruses that are important human and animal pathogens. Among them, Dengue virus causes epidemics with fatal outcomes mainly in the tropics. In this study, Dengue virus is visualized for the first time using the in air AFM technique. Images were obtained from a potassium-tartrate gradient-purified virus. This study enhances the application of AFM as a novel tool for the visualization and characterization of virus particles. Because flavivirus members are closely related, studies of the morphologic structure of the Dengue virus can reveal strategies that may be useful to identify and study other important viruses in the family, including the West Nile virus.     

Comparative study of lithium fluoride and graphite by atomic force microscopy (AFM)

The atomic force microscope (AFM) offers the possibility to image the topography of insulating as well as conductive surfaces. Highly oriented pyrolytic graphite (HOPG) was chosen as an example for a layered material and compared to single crystalline lithium fluoride (LiF). Both materials are easily prepared and inert at ambient pressure. Furthermore they are well characterized by Helium atom scattering experiments and other techniques. On HOPG atomic resolution has been achieved. Distortions can be observed which we interpret as a frictional effect. In addition we performed large area scans where we seldomly observed dislocations. For the first time we present measurements on LiF, showing steps of one unit cell height. On larger areas the surface of LiF showed terraces, separated by steps of variable heights, ranging from a few ångströms to 100 Å. We used a static method to get information about the distance dependence of the force between lever and sample. By slowly expanding and retracting the sample piezo and simultaneous measurement of the lever deflection, plots were recorded, showing the force as a function of sample position. The results were compared with theoretical calculations. We could determine the tip radius and found differences between LiF and HOPG being characteristic for the samples.     

Atomic force and scanning electron microscopy of atmospheric particles

Atomic force microscopy (AFM) and scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) have been used for both morphological and elemental mass analysis study of atmospheric particles. As part of the geometrical particle analysis, and in addition to the traditional height profile measurement of individual particles, AFM was used to measure the volume relative to the projection area for each particle separately, providing a particle shape model. The element identification was done by the EDS analysis, and the element mass content was calculated based on laboratory calibration with particles of known composition. The SEM-EDS mass measurements from two samples collected at 150 and 500 m above the surface of the Mediterranean Sea were found to be similar to mass calculations derived from the AFM volume measurements. The AFM results show that the volume of most of the aerosols that were identified as soluble marine sulfate and nitrate aerosol particles can be better estimated using cylindrical shapes than spherical or conical geometry.