Using fractal dimensions to characterize atomic force microscope images of epoxy resin fracture surfaces

Atomic force microscopy was used to obtain images of the fracture surface of a tri-ethylene-tetramine and 4,4 (methyl thylidene) epoxy resin. Images were obtained in the mirror, mist, and hackle regions of each sample. Fractal dimensions were calculated from the images using the box dimension and contour analysis method. The box dimension fractal dimension increment for all regions on the fracture surfaces were determined to average 0.26 ± 0.06 and the contour analysis fractal dimension increment were determined to average 0.46 ± 0.05. The box dimension technique is shown to provide the “true” fractal dimension of the surface. The fractal dimension measurements for all three regions indicated that the fracture surface was self-affine and possibly self-similar.     

Membrane deformation of living glial cells using atomic force microscopy

Using atomic force microscopy (AFM) it has been possible to detect actin filaments that are beneath the cell membrane of living cells despite the fact that the AFM tip is applied to the surface of the cell. To determine whether the AFM tip actually penetrates or deforms the cell membrane we determined whether an intracellularly trapped fluorescent indicator was lost from cells during AFM. Using epi-fluorescence illumination to monitor the presence of fluo-3 in the cell, we found that AFM did not cause dye leakage from the cell. Further, force–distance curves indicated that standard tips did not penetrate the membrane while sharper Supertips^TM did. In addition, the physiology of cells was found to be unaffected by AFM with standard tips since volume regulatory signal transduction mechanisms were intact in such studies. Thus, traditional AFM tips deform the cell membrane in order to reveal the presence of subcellular structures.     

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.     

Ultrastructure of Trypanosoma cruzi revisited by atomic force microscopy

Most advances in atomic force microscopy (AFM) have been accomplished in recent years. Previous attempts to use AFM to analyze the organization of pathogenic protozoa did not significantly contribute with new structural information. In this work, we introduce a new perspective to the study of the ultrastructure of the epimastigote form of Trypanosoma cruzi by AFM. Images were compared with those obtained using field emission scanning electron microscopy of critical point dried cells and transmission electron microscopy of negative stained detergent-extracted and air-dried cells. AFM images of epimastigote forms showed a flagellum furrow separating the axoneme from the paraflagellar rod (PFR) present from the emergence of the flagellar pocket to the tip of the flagellum. At high magnification, a row of periodically organized structures, which probably correspond to the link between the axoneme, the PFR and the flagellar membrane were seen along the furrow. In the origin of the flagellum, two basal bodies were identified. Beyond the basal bodies, small periodically arranged protrusions, positioned at 400 nm from the flagellar basis were seen. This structure was formed by nine substructures distributed around the flagellar circumference and may correspond to the flagellar necklace. Altogether, our results demonstrate the importance of the application of AFM in the structural characterization of the surface components and cytoskeleton on protozoan parasites.     

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.     

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.     

Imaging surface and submembranous structures with the atomic force microscope: a study on living cancer cells, fibroblasts and macrophages

Atomic force microscopy (AFM) has been used to image a wide variety of cells. Fixed and dried-coated, wet-fixed or living cells were investigated. The major advantage of AFM over SEM is the avoidance of vacuum and electrons, whereas imaging can be done at environmental pressure and in aqueous conditions. Evidence of the successful application of AFM in biological imaging is provided by comparing results of AFM with SEM and/or TEM. In this study, we investigated surface and submembranous structures of living and glutaraldehyde-fixed colon carcinoma cells, skin fibroblasts and liver macrophages by AFM. Special attention was paid to the correct conditions for the acquisition of images of the surface of these cells, because quality SEM examinations have already been abundantly presented.
AFM imaging of living cells revealed specific structures, such as the cytoskeleton, which were not observed by SEM. Membrane structures, such as ruffles, lamellipodia, microspikes and microvilli, could only clearly be observed after fixing the cells with 0.1% glutaraldehyde. AFM images of living cells were comparable to SEM images of fixed, dried and coated cells, but contained a number of artefacts due to tip–sample interaction. In addition, AFM imaging allowed the visualization of cytoplasmic submembranous structures without the necessity for further preparative steps, allowing us: (i) to follow cytoskeletal changes in fibroblasts under the influence of the microfilament disrupting agent latrunculin A; (ii) to study particle phagocytosis in macrophages. Therefore, in spite of the slow image acquisition of the AFM, the instrument can be used for high-resolution real-time studies of dynamic changes in submembranous structures.     

Comparative study of the conditions required to image live human epithelial and fibroblast cells using atomic force microscopy

Successful imaging of living human cells using atomic force microscopy (AFM) is influenced by many variables including cell culture conditions, cell morphology, surface topography, scan parameters, and cantilever choice. In this study, these variables were investigated while imaging two morphologically distinct human cell lines, namely LL24 (fibroblasts) and NCI H727 (epithelial) cells. The cell types used in this study were found to require different parameter settings to produce images showing the greatest detail. In contact mode, optimal loading forces ranged between 2-2.8 x 10(-9) and 0.1-0.7 x 10(-9) (N) for LL24 and NCI H727 cells respectively. In tapping (AC) mode, images of LL24 cells were obtained using cantilevers with a spring constant of at least 0.32 N/m, while NCI H727 cells required a greater spring constant of at least 0.58 N/m. To obtain tapping mode images, cantilevers needed to be tuned to resonate at higher frequencies than their resonance frequencies to obtain images. For NCI H727 cells, contact mode imaging produced the clearest images. For LL24 cells, contact and tapping mode AFM produced images of comparable quality. Overall, this study shows that cells with different morphologies and surface topography require different scanning approaches and optimal conditions must be determined empirically to achieve images of high quality.     

Using atomic force microscopy to probe food biopolymer functionality

Atomic force microscopes (AFMs) generate images by “feeling” rather than “looking” at samples. This permits a magnification range spanning that associated with both the light and electron microscopes, but under the “natural” imaging conditions normally associated with light microscopes. Molecules and molecular interactions can be imaged at molecular or submolecular resolution in gaseous or liquid environments. By careful design of experiments it is possible to use AFM to probe how food biopolymers determine the structure and texture of food products. This approach will be illustrated through studies on food polysaccharides and proteins.     

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.     

Stereoscopic display of atomic force microscope images using anaglyph techniques

This paper describes the use of a standard stereo-pair image display method for presenting the three-dimensional relief information found in atomic force microscope (AFM) images. The method makes use of commercially available image processing software packages. The techniques are illustrated on AFM images of the cuticle structure of a human hair fibre.     

Atomic force microscopy study on chlorpromazine‐induced morphological changes of living HeLa cells In Vitro

Chlorpromazine (CPZ)‐induced morphological changes of living human cervical carcinoma cells were investigated by atomic force microscopy in near physiological condition. The results showed that the cell morphology changed visibly with time in the presence of CPZ (>21 µM). The cell membrane shrank gradually and detached finally from the substrate. After being treated with CPZ for 50 min, the cell volume increased by about 27.6% while its projective area (cell adhered to the substrate) decreased by about 12%. The mechanism was also discussed. SCANNING 31: 259–265, 2009. © 2010 Wiley Periodicals, Inc.     

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.     

The mechanism of G-banding detected by atomic force microscopy

The morphologic changes occurring in human chromosomes during G-banding by trypsin treatment on the same metaphase were followed with the aid of an atomic force microscope (AFM). It was found that trypsin treatment alone caused a pattern of collapse in the chromosomes that was clearly dependent on the duration of trypsinization. The progressive pattern of collapse first indicated the loss of internal differentiation between chromatids, then bands, and finally all internal structures, except for edges running around the chromosomes' perimeter. When stained with Giemsa, the collapsed chromosomes partly regained their original form, and transverse ridges appeared that correspond to G-positive band regions. However, the treatment of fixed chromosomes with trypsin for 42 s diminished the chromosomal edges, and the z-dimensions could not be measured even with the subsequent application of Giemsa.     

Cytoskeletal response of microvessel endothelial cells to an applied stress force at the submicrometer scale studied by atomic force microscopy

Cytoskeleton fibers form an intricate three-dimensional network to provide structure and function to microvessel endothelial cells. During accommodation to blood flowing, stress fiber bundles become more prominent and align with the direction of blood flow. This network either mechanically resists the applied shear stress (lateral force) or, if deformed, is dynamically remodeled back to a preferred architecture. However, the detailed response of these stress fiber bundles to applied lateral force at submicrometer scales are as yet poorly understood. In our in vitro study, the tip, topography probe in lateral force microscopy of atomic force microscopy, acted as a tool for exerting quantitative vertical and lateral force on the filaments of the cytoskeleton. Moreover, the authors developed a formula to calculate the value of lateral force exerted on every point of the filaments. The results show that cytoskeleton fibers of healthy tight junctions in rat cerebral microvessel endothelial cells formed a cross-type network, and were reinforced and elongated in the direction of scanning under lateral force of 15-42 nN. Under peroxidation (H(2)O(2) of 300 micromol/L), the cytoskeleton remodeled at intercellular junctions, and changed over the meshwork structures into a dense bundle, that redistributed the stress. Once mechanical forces were exerted on an area, the cells shrank and lost morphologic tight junctions. It would be useful in our understanding of certain pathological processes, such as cerebral ischemia/reperfusion injury, which maybe caused by biomechanical forces and which are overlooked in current disease models.     

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.     

Modeling the clay minerals–enzyme binding by fusion fluorescent proteins and under atomic force microscope

In the present study, binding of cellulase protein to different clay minerals were tested using fluorescent–protein complex and microscopic techniques. Cellulase gene (Cel5H) was cloned into three fluorescent vectors and expressed as fusion enzymes. Binding of Cel5H–mineral particles was confirmed by confocal microscopy, and enzyme assay. Among the Cel5H–fusion enzymes, green–fusion enzyme showed higher intensity compared with other red and yellow fusion–proteins. Intensity of fusion–proteins was dependent on the pH of the medium. Confocal microscopy revealed binding of the all three fusion proteins with different clay minerals. However, montmorillonite displayed higher binding capacity than kaolinite clay. Likewise, atomic force microscopy (AFM) image profile analysis showed proteins appeared globular molecules in free‐state on mica surface with an average cross sectional diameter of 110 ± 2 nm and rough surface of montmorillonite made protein appear flattened due to structural alteration. Even surface of kaolinite also exerted some strain on protein molecular conformation after binding to surface. Our results provide further evidence for 3D visualization of enzyme–soil complex and encourage furthering study of the force involved interactions. Therefore, our results indicate that binding of proteins to clay minerals was external and provides a molecular method to observe the interaction of clay minerals–enzyme complex.     

Atomic force microscopy as a tool for study of human hair

Atomic force microscopy (AFM) is a newly developed microscopic technique that offers high-resolution power, less intrusive measurement, and requires little sample pretreatment for elucidating structures of biological materials in three dimensions and in their natural environment. In this study, AFM has been used not only as an imaging technique for examining human hair structure at high resolution, but also as a tool for quantitative assessment of the effect of treatment in 10 mM phosphate buffered saline of pHs 3.0, 7.0, and 11.0 and heating on human hair structure. It is observed that the hair cuticle is a sensitive indicator of external influences on hair structure, and that its height can be used as a parameter for quantitative assessment. The experimental results obtained show that the swelling of hair caused by the incubation in the buffer decreases with the increase of the pH values and that, depending on the duration of heating, the hair undergoes structural expansion and shrinkage. This study demonstrates that AFM can be used as a valuable alternative to conventional microscopic techniques for hair research.