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 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.     

The molecular machines of DNA repair: scanning force microscopy analysis of their architecture

The application of scanning force microscope (SFM, also called atomic force microscope or AFM) imaging to study the architecture of proteins and their functional assemblies on DNA has provided new and exciting information on the mechanism of vital cellular processes. Rapid progress in molecular biology has resulted in the identification and isolation of proteins and protein complexes that function in specific DNA transactions. These proteins and protein complexes can now be analysed at the single molecule level, whereby the functional assemblies are often described as nanomachines. Understanding how they work requires understanding their structure and functional arrangement in three dimensions. The SFM is uniquely suited to provide three‐dimensional structural information on biomolecules at nanometre resolution. In this review we focus on recent applications of SFM to reveal detailed information on the architecture and mechanism of action of protein machinery involved in safeguarding genome stability through DNA repair processes.     

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 of a hydrated bacterial surface protein

The protein surface layer of the bacterium Deinococcus radiodurans (HPI layer) was examined with an atomic force microscope (AFM). The measurements on the air-dried, but still hydrated layer were performed in the attractive imaging mode in which the forces between tip and sample are much smaller than in AFM in the repulsive mode or in scanning tunnelling microscopy (STM). The results are compared with STM and transmission electron microscopy (TEM) data.     

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.     

Atomic force microscopy of purple membranes

The surface structure of purple membranes was imaged using an atomic force probe mounted in a scanning tunnelling microscope. One of the two different membrane surfaces showed protruding, disc-shaped features forming an hexagonal lattice with about 6 nm centre to centre spacing. These are identified as the cytoplasmic surfaces of trimers of bacteriorhodopsin molecules and are correlated with the structural information on bacteriorhodopsin obtained from numerous earlier electron microscope and diffraction studies.     

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 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.     

Atomic force microscopy in the photochemistry of chalcones

The application of atomic force microscopy (AFM) to photodimerization of crystalline chalcones provides new insights into the detailed mechanisms of solid-state reactions on the molecular level. Well-directed long-range transport phenomena are found which reach far beyond the crystal lattice distances. Reactions occur in the surface region where the light is absorbed. Characteristic features are built up that depend on crystal structure and crystal face. This could not be foreseen by previous theories based solely on a topochemical postulate/principle. There is now a much more intimate correlation of crystal structure with solid-state reactivity and this is directly studied and proven experimentally by AFM. Even solid-state reactions which are in opposition to topochemistry can be studied and understood on a molecular basis. The three-dimensional resolution of undisturbed insulating surfaces which is obtained by AFM is not available by any other technique.     

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.     

Imaging the native structure of the chaperone protein GroEL without fixation using atomic force microscopy

Most sample preparation methods for scanning probe or electron microscopy require that biomolecules, such as proteins, be fixed. Fixation destroys the molecular functionality and can possibly affect the true molecular structure. Here we report sample preparation conditions that allow the imaging of an unfixed protein, GroEL, under in-vivo conditions, by atomic force microscopy. Under these conditions, the protein should maintain its native structure and biological activity. The typical toroidal shape with pore of the GroEL complex was easily visible in the images. Images of a single complex show dimensions that agree well with crystallographic data. Under in-vivo conditions, it should be possible to study the biological activity and function of proteins.     

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.     

原子力显微镜在DNA研究中的应用

原子力显微镜作为一种新型的表面表征手段已经得到了各个方面的应用，本文探索了AFM在DNA表面结构中的研究方法，讨论了AFM在研究DNA中优势。     

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: 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 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 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.