Assembling and imaging of his-tag green fluorescent protein on mica surfaces studied by atomic force microscopy and fluorescence microscopy

The adsorption of his-tag green fluorescent protein (GFPH(6)) onto the mica surfaces has been studied by atomic force microscopy (AFM) and laser confocal fluorescence microscopy. By controlling the adsorption conditions, separated single GFPH(6) and GFPH(6) monolayer can be adsorbed and formed on mica surfaces. In present experiments, based on the AFM measurement, we found that the adsorbed GFPH(6) was bound on the mica surface with its beta-sheets. The formed GFPH(6) monolayer on mica surfaces was flat, uniform, and stable. Some applications of the formed monolayer have been demonstrated. The formed monolayer can be used as a substrate for DNA imaging and AFM mechanical lithography.     

Stick–slip motion in the atomic force microscope

Measurements of atomic friction in the atomic force microscope frequently show periodic variations at the lattice spacing of the surface being scanned, which have the saw‐tooth wave form characteristic of “stick–slip” motion. Simple models of this behaviour have been proposed, in which the “dynamic element” of the system is provided by the elastic stiffness and inertia of the cantilever which supports the tip of the microscope, in its lateral, i.e., torsional mode of vibration. These models have been successful in predicting the observed motion, but only by assuming that the cantilever is heavily damped. However, the source of this damping in a highly elastic cantilever is not explained. To resolve the paradox, it is shown in this note that it is necessary to introduce the elastic stiffness of the contact into the model. The relationship between the contact stiffness, the cantilever stiffness and the amplitude of the periodic friction force is derived in order for stick–slip motion at lattice spacing to be achieved. This revised version was published online in July 2006 with corrections to the Cover Date.     

Calibration of the scanning (atomic) force microscope with gold particles

Scanning force microscopy (SFM) holds great promise for biological research. Two major problems that have confronted imaging with the scanning force microscope have been the distortion of the image and overestimation in measurements of lateral size due to the varying geometry and characteristics of the scanning tip. In this study, spherical colloidal gold particles (10, 20 and 40 nm in diameter) were used to determine (1) tip parameters (size, shape and semivertical angle); (2) the distortion of the image caused by the tip; and (3) the overestimation or broadening of lateral dimensions. These gold particles deviate little in size, are rigid and have a size similar to biological macromolecules. Images of the colloidal gold particles by SFM were compared with those obtained by electron microscopy (EM). The height of the gold particles as measured by SFM and EM was comparable and was little affected by the tip geometry. The measurements of the lateral dimensions of colloidal gold, however, showed substantial differences between SFM and EM in that SFM resulted in an overestimate of the lateral dimensions. Moreover, the distortion of images and broadening of lateral dimensions were specific to the SFM tip used. The calibration of the SFM tip with mica provided little clue as to the type of distortion and the amount of lateral broadening observed when the larger gold particles were scanned. The SFM image also depended on the orientation of the tip with respect to the specimen. Our results suggest that quantitative SFM imaging requires calibration to identify and account for both the distortions and the magnitude of lateral broadening caused by the cantilever tip. Calibration with gold particles is fast and nondestructive to the tip. The raw imaging data of the specimen can be corrected for the tip effect and true structural information can be derived. In summary, we present a simple and practical method for the calibration of the SFM tip using gold particles with a size in the range of biomacromolecules that allows: (1) selection of a cantilever tip that produces an image with minimal distortion; (2) quantitative determination of tip parameters; (3) reconstruction of the shape of the tip at different heights from the tip apex; (4) appreciation of the type of distortion that may be introduced by a specific tip and quantification of the overestimation of the lateral dimensions; and (5) calculation of the true structure of the specimen from the image data. The significance is that such calibration will permit quantitative and accurate imaging with SFM.     

原子力显微镜的力曲线分析与转化

原子力显微镜测定的力曲线需转化为力位移曲线来应用.力位移曲线是以任意点为零点的,当研究粘附或者分子模型对比时,需要知道针尖样品间的作用力或确切的零点位置,这时需将其转化为力-距离曲线.本文首先从力曲线的测定原理得出了典型的力曲线,之后从理论上分析了力曲线、力位移曲线和力-距离曲线间的转化,从中得出了转化过程中需要的两个重要参量:灵敏度和零距离,并提出了确定方法.最后,利用MATLAB实现了曲线的自动转化.     

Error analysis and regression mode of the V‐grooved sample in the atomic force microscope simulation measurement mode by the molecular mechanics

Based on the molecular mechanics, this study uses the two‐body potential energy function to construct a trapezoidal cantilever nano‐scale simulation measurement model of contact mode atomic force microscopy (AFM) under the constant force mode to simulate the measurement the nano‐scale V‐grooved standard sample. We investigate the error of offset distance of the cross‐section profile when using the probes with different trapezoidal cantilever probe tip radii (9.5, 8.5, and 7.5 Å) to scan the peak of the V‐grooved standard sample being reduced to one‐tenth (1/10) of its size, and use the offset error to inversely find out the regression equation. We analyze how the tip apex as well as the profile of the tip edge oblique angle and the oblique edge angle affects the offset distance. Furthermore, a probe with a larger radius of 9.5 nm is used to simulate and measure the offset error of scan curve, and acquire the regression equation. By the conversion proportion coefficient of size (ω), and revising the size‐reduced regression equation during the small size scale, a revised regression equation of a larger size scale can be acquired. The error is then reduced, further enhancing the accuracy of the AFM scanning and measurement. SCANNING 31: 147–159, 2009. © 2009 Wiley Periodicals, Inc.     

Characterization of the tip-loading force-dependent tunneling behavior in alkanethiol metal–molecule–metal junctions by conducting atomic force microscopy

We report on a tip-loading force-dependent tunneling behavior through alkanethiol self-assembled monolayers formed in metal–molecule–metal junctions, using conducting atomic force microscopy. The metal–molecule contacts were formed by placing a conductive tip in a stationary point contact on alkanethiol self-assembled monolayers under a controlled tip-loading force. Current–voltage characteristics in the alkanethiol junctions are simultaneously measured, while varying the loading forces. Tunneling current through the alkanethiol junctions increases and decay coefficient β_N decreases, respectively, with increasing tip-loading force, which results from enhanced intermolecular charge transfer in a tilted molecular configuration under the tip-loading effect.     

Modulus characterization of cells with submicron colloidal probes by atomic force microscope

Colloidal probes have been increasingly demanded for the characterization of cellular modulus in atomic force microscope because of their well-defined geometry and large contact area with cell. In this work, submicron colloidal probes are prepared by scanning electron microscope/focused ion beam and compared with sharp tip and micron colloidal probe, in conjunction with loading velocity and indentation depth on the apparent elastic modulus. NIM and cartilage cells are used as specimens. The results show that modulus value measured by sharp tip changes significantly with loading velocity while remains almost stable by colloidal probes. Also, submicron colloidal probe is superior in characterizing the modulus with increasing indentation depth, which could help reveal the mechanical details of cellular membrane and the modulus of the whole cell. To test the submicron colloidal probe further, the modulus distribution map of cell is scanned with submicron colloidal probe of 50 nm radius during small and large indentation depths with high spatial resolution. The outcome of this work will provide the effective submicron colloidal probe according to the effect of loading velocity and indentation depth, characterizing the mechanical properties of the cells.     

Investigation of the morphological and fractal behavior at nanoscale of patterning lines by scratching in an atomic force microscope

In this work, the topographical effect of the scratching trajectory and the feed direction on the formation of lithographed lines on the (001) InP surface was investigated using an atomic force microscope (AFM) tip-based nanomachining approach. Nanoscratching tests were carried out using the sharp face of a diamond AFM tip in contact mode. From the topographic maps obtained by AFM, several morphological and fractal parameters were obtained and analyzed. Surface morphology presented a surface smoothing for surfaces with scratches produced in [011] and [001] directions. The height parameters confirmed this behavior because scratches in [001] direction exhibited lower roughness. Moreover, this scratch direction promoted the height distribution most symmetrical and platykurtic. The other morphological parameters revealed that this direction provided a more irregular surface (smaller S_mc and S_xp), peak distribution, denser and pointed, smaller portion of material in the core, less deep furrows, higher spatial frequency components, and high isotropy. Fractal parameters revealed that FRE90 has the highest spatial complexity, it is dominated by higher spatial frequencies, and has the lowest surface percolation. Furthermore, all samples exhibited high topographic uniformity.     

Investigation of nanoparticles by atomic and lateral force microscopy

Metallic nanoparticles have been produced on vitreous carbon substrates by means of thermal evaporation. From pictures of the particles, made by a high-resolution scanning electron microscope (HRSEM), a semispherical shape is suggested due to the total mass of deposited material. Atomic force microscopy (AFM) has been applied to this sample in order to get direct topographic information. The AFM has been operated with normal and super tips, the latter having a smaller cone angle and radius, thus following more precisely the contours of an object. Simultaneously lateral-force microscopic (LFM) images have been recorded. Major differences between the contents of HRSEM- and AFM-images are considered, emphasizing the important influence of the tips' geometry. Both the AFM and LFM line scans have been compared with and have qualitatively agreed with those calculated under simplifying assumptions.     

Nanostructural analysis by atomic force microscopy followed by light microscopy on the same archival slide

Integrated information on ultrastructural surface texture and chemistry increasingly plays a role in the biomedical sciences. Light microscopy provides access to biochemical data by the application of dyes. Ultrastructural representation of the surface structure of tissues, cells, or macromolecules can be obtained by scanning electron microscopy (SEM). However, SEM often requires gold or coal coating of biological samples, which makes a combined examination by light microscopy and SEM difficult. Conventional histochemical staining methods are not easily applicable to biological material subsequent to such treatment. Atomic force microscopy (AFM) gives access to surface textures down to ultrastructural dimensions without previous coating of the sample. A combination of AFM with conventional histochemical staining protocols for light microscopy on a single slide is therefore presented. Unstained cores were examined using AFM (tapping mode) and subsequently stained histochemically. The images obtained by AFM were compared with the results of histochemistry. AFM technology did not interfere with any of the histochemical staining protocols. Ultrastructurally analyzed regions could be identified in light microscopy and histochemical properties of ultrastructurally determined regions could be seen. AFM-generated ultrastructural information with subsequent staining gives way to novel findings in the biomedical sciences. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Cooperativity and intermediate structures of single-stranded DNA binding-assisted RecA-single-stranded DNA complex formation studied by atomic force microscopy

The formation of a complex between RecA protein and single-stranded (ss) DNA was studied systematically by atomic force microscopy (AFM) by varying incubation time and the molecular ratio of RecA protein to single-stranded DNA binding (SSB) protein. New intermediate structures, such as small circular, tangled, and protruded structures in the absence of SSB and sharply turned structures in the presence of SSB, were clearly identified at the early stage of complex formation. These structures have probably resulted from competitive binding of RecA and SSB to DNA. After long incubation, only fully covered RecA-ssDNA and totally RecA-free SSB-ssDNA complexes were present regardless of RecA concentrations. Together with intermediate structures which consisted of only two parts, that is, ssDNA covered by SSB and by RecA proteins, the observation suggested strong neighbor cooperative binding of RecA to ssDNA assisted by SSB.     

Effect of aging on the morphology of bitumen by atomic force microscopy

Effect of aging on the morphology of bitumen was investigated. Two bitumens were aged according to the thin film oven test (TFOT), pressure aging vessel (PAV) test and ultraviolet (UV) radiation, respectively. The morphology of the binders before and after aging was characterized by atomic force microscopy. The physical properties and chemical compositions of the binders were also measured. The results showed that aging affected the bitumen morphology significantly. Aging increased the overall surface stiffness of the bitumen and made the bitumen surface more solid-like. The extent of these changes was dependent on aging conditions. TFOT decreased the contrast between the dispersed domains and the matrix, which contributed to the single-phase trend of the binders. The effect of PAV aging on morphology of the binders was dependent on the base bitumen. In one case, it further accelerated the single-phase trend of bitumen in comparison with that after TFOT. In the other case, it caused the phase separation of bitumen. In both cases, PAV aging increased the surface roughness of the binders obviously. As a result of UV aging, the contrast between the matrix phase and dispersed phase was increased due to the difference in sensitivity to UV radiation of the bitumen molecules, which caused or further promoted the phase separation in the binders. Regardless of the aging procedure carried out, a strong correlation was observed between the changes in morphology and physical properties as well as chemical compositions of the binders before and after aging.     

Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy

Nucleosome is a fundamental structural unit of chromatin, and the exposure from or occlusion into chromatin of genomic DNA is closely related to the regulation of gene expression. In this study, we analyzed the molecular dynamics of poly-nucleosomal arrays in solution by fast-scanning atomic force microscopy (AFM) to obtain a visual glimpse of nucleosome dynamics on chromatin fiber at single molecule level. The influence of the high-speed scanning probe on nucleosome dynamics can be neglected since bending elastic energy of DNA molecule showed similar probability distributions at different scan rates. In the sequential images of poly-nucleosomal arrays, the sliding of the nucleosome core particle and the dissociation of histone particle were visualized. The sliding showed limited fluctuation within ∼50 nm along the DNA strand. The histone dissociation occurs by at least two distinct ways: a dissociation of histone octamer or sequential dissociations of tetramers. These observations help us to develop the molecular mechanisms of nucleosome dynamics and also demonstrate the ability of fast-scanning AFM for the analysis of dynamic protein–DNA interaction in sub-seconds time scale.     

Bituminous emulsions and their characterization by atomic force microscopy

We present a new method for observing oil-in-water emulsions with a continuous water phase and a dispersed bitumen phase. The fine polydispersed bitumen micelles were adsorbed to an atomically smooth mica substrate and imaged in solution by atomic force microscopy in a liquid cell. The height of the adsorbed bitumen sheet in wet and dry states can be measured and the homogeneity of film formation by coalescence can be determined. Localization of surfactant onto and between bitumen micelles is also visualized.     

The comparison between force volume and peakforce quantitative nanomechanical mode of atomic force microscope in detecting cell's mechanical properties

Atomic force microscope (AFM) has been widely used in the biological field owing to its high sensitivity (subnanonewton), high spatial resolution (nanometer), and adaptability to physiological environments. Nowadays, force volume (FV) and peakforce quantitative nanomechanical (QNM) are two distinct modes of AFM used in biomechanical research. However, numerous studies have revealed an extremely confusing phenomenon that FV mode has a significant difference with QNM in determining the mechanical properties of the same samples. In this article, for the case of human benign prostatic hyperplasia cells (BPH) and two cancerous prostate cells with different grades of malignancy (PC3 and DU145), the differences were compared between FV and QNM modes in detecting mechanical properties. The results show measured Young's modulus of the same cells in FV mode was much lower than that obtained by QNM mode. Combining experimental results with working principles of two modes, it is indicated that surface adhesion is highly suspected to be a critical factor resulting in the measurement difference between two modes. To further confirm this conjecture, various weight ratios of polydimethylsiloxane (PDMS) were assessed by two modes, respectively. The results show that the difference of Young's modulus measured by two modes increases with the surface adhesion of PDMS, confirming that adhesion is one of the significant elements that lead to the measurement difference between FV and QNM modes.     

Scanning tunnelling and atomic force microscopy performed with the same probe in one unit

The technique demonstrated here provides features of both scanning tunnelling microscopy (STM) and atomic force microscopy (AFM). The metallic probe acts to record current variations and sense forces from the same sample area simultaneously. Thus, separate images may be recorded, in registry. The collected data allows real space correlations between some electrical properties and the geometric structure of a sample surface. The same tip is used since the geometry and condition of the tip can effect the data recordings. Platinum alloys, tungsten and graphite tips have been employed successfully. An AFM lever can respond to surface contact forces, within the elastic limits of the sample, while electric current is sensed by the tip of the lever. The usefulness of this experimental procedure is tested here by an application to semiconducting samples of Ag-doped CdTe in air and in paraffin oil media.     

Using the atomic force microscope to observe and study the ultrastructure of the living BIU-87 cells of the human bladder cancer

In this study, the ultrastructure of living BIU-87 cells of human bladder cancer was mapped using atomic force microscopy to reveal the dynamic change of single cancerous cell division. Simultaneously, the feasibility and functional reliability of the atomic force microscope (AFM) were established and a laboratory model using AFM to study living cancerous cells was created. In this experiment, BIU-87 cells of human bladder cancer were cultured by conventional methods and grown in gelatin-treated dishes. A thermostat was used for preserving the cell's living temperature. Scanning of these cells using AFM was carried out in physiologic condition. The AFM images of the ultrastructure of living BIU-87 cells as well as those of the cell's membrane and cytoskeleton were very clear. The dynamic phenomenon of single cell division was observed. It was concluded that the AFM was able to observe and depict the ultrastructure of living cells of human bladder cancer directly and in real time. This experimental model is expected to play an important role in elucidating the cancerous mechanism of bladder normal cells at the atomic or nanometer level.