Scanning force microscopy on live cultured cells: Imaging and force-versus-distance investigations

Extensive measurements with the scanning force microscope on living cells in their native liquid environment are described with the purpose of critically assessing the extent of the interaction between the SFM tip and the (soft) cell materials and the effect of such interaction on topographic information. Images are obtained under various force conditions and systematically correlated with force-versus-distance curves. As a result, detailed indications about tip indentation are given, thickness estimates deduced and identification of submembranous cytoplasmic structures suggested.     

Application of atomic force microscopy to the study of natural and model soil particles

The structure and surface chemistry of soil particles has extensive impact on many bulk scale properties and processes of soil systems and consequently the environments that they support. There are a number of physiochemical mechanisms that operate at the nanoscale which affect the soil's capability to maintain native vegetation and crops; this includes soil hydrophobicity and the soil's capacity to hold water and nutrients. The present study used atomic force microscopy in a novel approach to provide unique insight into the nanoscale properties of natural soil particles that control the physiochemical interaction of material within the soil column. There have been few atomic force microscopy studies of soil, perhaps a reflection of the heterogeneous nature of the system. The present study adopted an imaging and force measurement research strategy that accounted for the heterogeneity and used model systems to aid interpretation. The surface roughness of natural soil particles increased with depth in the soil column a consequence of the attachment of organic material within the crevices of the soil particles. The roughness root mean square calculated from ten 25 microm(2) images for five different soil particles from a Netherlands soil was 53.0 nm, 68.0 nm, 92.2 nm and 106.4 nm for the respective soil depths of 0-10 cm, 10-20 cm, 20-30 cm and 30-40 cm. A novel analysis method of atomic force microscopy phase images based on phase angle distribution across a surface was used to interpret the nanoscale distribution of organic material attached to natural and model soil particles. Phase angle distributions obtained from phase images of model surfaces were found to be bimodal, indicating multiple layers of material, which changed with the concentration of adsorbed humic acid. Phase angle distributions obtained from phase images of natural soil particles indicated a trend of decreasing surface coverage with increasing depth in the soil column. This was consistent with previous macroscopic determination of the proportions of organic material chemically extracted from bulk samples of the soils from which specimen particles were drawn. Interaction forces were measured between atomic force microscopy cantilever tips (Si(3)N(4)) and natural soil and model surfaces. Adhesion forces at humic acid free specimen surfaces (Av. 20.0 nN), which are primarily hydrophilic and whose interactions are subject to a significant contribution from the capillary forces, were found to be larger than those of specimen surfaces with adsorbed humic acid (Av. 6.5 nN). This suggests that adsorbed humic acid increased surface hydrophobicity. The magnitude and distribution of adhesion forces between atomic force microscopy tips and the natural particle surfaces was affected by both local surface roughness and the presence of adsorbed organic material. The present study has correlated nanoscale measurements with established macroscale methods of soil study. Thus, the research demonstrates that atomic force microscopy is an important addition to soil science that permits a multiscale analysis of the multifactorial phenomena of soil hydrophobicity and wetting.     

Study of morphological and mechanical features of multinuclear and mononuclear SW480 cells by atomic force microscopy

This article studies the morphological and mechanical features of multinuclear and mononuclear SW480 colon cancer cells by atomic force microscopy to understand their drug‐resistance. The SW480 cells were incubated with the fullerenol concentrations of 1 mg/ml and 2 mg/ml. Morphological and mechanical features including the height, length, width, roughness, adhesion force and Young's modulus of three multinuclear cell groups and three mononuclear cell groups were imaged and analyzed. It was observed that the features of multinuclear cancer cells and mononuclear cancer cells were significantly different after the treatment with fullerenol. The experiment results indicated that the mononuclear SW480 cells were more sensitive to fullerenol than the multinuclear SW480 cells, and the multinuclear SW480 cells exhibited a stronger drug‐resistance than the mononuclear SW480 cells. This work provides a guideline for the treatments of multinuclear and mononuclear cancer cells with drugs.     

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.     

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.     

Evaluation of surface topography changes in three NiTi file systems using rotary and reciprocal motion: An atomic force microscopy study

Aim: To evaluate the surface topography changes in three nickel‐titanium (NiTi) file systems using either rotary or reciprocal motion using atomic force microscopy (AFM), and to determine the effect of scanning area on the AFM results in this study. Methodology: Five points on a F2 Protaper file, R25 Reciproc file, and a Primary file from WaveOne systems were scanned preoperatively in 1 × 1 and 5 × 5 µm² with an AFM device that can scan an intact (not sectioned) file. One standardized resin block was used for each instrument, according to the manufacturer's recommendations. Points were re‐scanned postoperatively using the same AFM and settings. Root‐mean‐square (RMS) and roughness average (Ra) values were obtained. The preoperative and postoperative surface topographies were compared separately in terms of RMS and Ra values. The surface topography change scores were analyzed using Kruskal–Wallis and Mann–Whitney U tests using a 0.10 significance level. Results: There were no significant differences preoperatively among the NiTi file systems in 1 × 1 or 5 × 5 µm² areas. Postoperatively, the WaveOne Primary had more surface irregularities (significant for 5 × 5 µm² scan in Ra evaluation). Conclusions: Three‐dimensional AFM images of instrument surfaces showed topographic irregularities preoperatively and postoperatively. AFM results differ depending on the scanning area and file used. Microsc. Res. Tech. 77:177–182, 2014. © 2013 Wiley Periodicals, Inc.     

New direct observations of asphalts and asphalt binders by scanning electron microscopy and atomic force microscopy

Observations made using AFM and SEM have been combined in order to study the structure of asphalts. Fluorescence microscopy was used to aid in understanding the structural changes occurring when polymer is added to the asphalts.   With the atomic force microscope we are able to study the structure of the asphalts without any pre-preparation. Despite very low resolution, our study reveal ed a network of asphaltene molecules with regard to asphalt gel. The same result is obtained by SEM observation but with a much better resolution. SEM observation, however, needs an adequate preparation method.   In the presence of polymer we observed a rearrangement of the initial asphaltene association which leads to the assumption that polymer can aggregate the asphaltene phase.     

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.     

A convenient and rapid sample repositioning approach for atomic force microscopy

A novel repositioning approach is described for repeated observations of a specimen at a close proximal location in the atomic force microscope. The approach is similar to keystone architecture, whereby the repositioning is achieved by forming a male structured base for the specimen, and a corresponding female counterpart as the frame. For the combination of an acrylic acid frame and a metal base, 90% translation shifts are less than 10 µm, and almost all angular disorientations are within +3° to ?3°. Nanometre‐scale surface features can be relocated easily and reliably even after 40 imaging–removal–imaging cycles, dipping the specimen in solutions or heating up to 500 °C.     

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.     

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.     

Long-term structural changes of plasmid DNA studied by atomic force microscopy

Long-term stability of plasmid DNA (pDNA) conformations is critical in many research areas, especially those concerning future gene therapy. Despite its importance, the time-evolution of pDNA structures has rarely been studied at a molecular resolution. Here, the time-evolution of pDNA solutions spanning four years was observed with atomic force microscopy (AFM). The AFM data show that the pDNA molecules changed over time from isolated supercoiled structures, to aggregated supercoiled structures, to thin, branched network structures, and finally to wider, branched network structures. Additional topographical analysis of the AFM data suggests that the actions of residual proteins could be the main mechanism for the structural changes in our laboratory-prepared pDNA.     

Visualization of cellular DNA crosslinks by atomic force microscopy

Cellular DNA crosslinks are a type of DNA damage induced by toxic chemicals or high‐energy radiation. If damaged DNA is not rapidly repaired, cells will die or mutate. To evaluate the types of DNA damage and their influence on vital cell activities, it is necessary to be able to detect DNA crosslinks. To date, indirect methods such as alkaline elution, potassium chloride–sodium dodecyl sulfate assay and comet assay have been used to detect DNA damage. Direct morphological observation, on the other hand, may be a useful tool to differentiate the types of DNA damage. In this report, atomic force microscopy (AFM) has been employed to visualize the breakage and crosslinking of cellular DNA strands in cells treated with formaldehyde and hydrogen peroxide. Our results showed that toxic chemical‐induced crosslinking of cellular DNA occurred in a dose‐dependent manner. DNA conglomerates were observed with high concentrations of formaldehyde, and the AFM observations were consistent with those of a comet assay. Our experiments demonstrate that AFM is an efficient method to differentiate the types of DNA damage. SCANNING 31: 75–82, 2009. © 2009 Wiley Periodicals, Inc.     

Capturing the elasticity and morphology of live fibroblast cell cultures during degradation with atomic force microscopy

Atomic force microscopy, in a liquid environment, was used to capture in vitro the morphological and mechanical changes that cultured fibroblasts undergo as time elapses from the completion of the cell culture. Topography images illustrated that initially, the nucleus had a height of 1.18 ± 0.2 μm, and after 48 h it had decreased to 550 ± 60 nm; similarly, the cell membrane exhibited significant shrinkage from 34 ± 4 to 23 ± 2 μm. After each image scan, atomic force microscopy indentation was performed on the centre of the nucleus, to measure the changes in the cell elasticity. Examination of the force‐distance curves indicated that the membrane elastic modulus at the nucleus remained the same within the time frame of 48 h, even though the cell morphology had significantly changed.     

Comparative study of two types of parallel kinematic flexure scanners for atomic force microscopy

A little consideration will show that a scanner is one of the most critical components of any atomic force microscope (AFM), and properly designing a scanner remains a challenging aspect in the minds of developers. We closely examined two types of flexure-based parallel kinematic scanners (push–pull and push-only configurations) as they have been applied to AFM. The custom-fabricated scanners have been installed on a commercial AFM while keeping other parameters identical except for the scanners. The results show that intrinsically there is no significant difference in performance of both scanner designs. However, it was found that preloading conditions more critically affect the performance of the push–pull scanner than the push-only scanner. In addition, the Prandtl–Ishlinskii model has been applied to model the obtained hysteresis curves for both scanners. The application of the inverse of the Prandtl–Ishlinskii model improved the linearity of the measured hysteresis. Although both scanners possess similar characteristics and can operate at higher speeds than commercial scanners in reduced scan areas, simpler operating requirements and the monolithic construction make the push-only scanner a preferred choice for AFM.     

Effects of Galla chinensis on the surface topography of initial enamel carious lesion: an atomic force microscopy study

To investigate the effect of Galla chinensis on the surface topography of initial enamel carious lesion, atomic force microscope (AFM) was used, and it was a new AFM application in enamel de‐/remineralization research. Bovine sound enamel slabs were demineralized to produce initial carious lesion in vitro. Then, the lesions were exposed to a pH‐cycling regime for 12 days. Each daily cycle included 4×1 min applications with one of three treatments: negative control group: deionized water; positive control group: 1 g/L aqueous solutions of NaF; experimental group: 4 g/L aqueous solutions of G. chinensis extract (GCE). The surface topography and roughness were investigated on the enamel slabs before and after pH‐cycling by AFM. 3D AFM images revealed the surface topographical changes of GCE‐treated enamel. Significant difference existed before and after the pH‐cycling among the groups. AFM offers a powerful tool for enamel de‐/remineralization research. The surface roughness results provide the evidences to remineralization of carious lesion, and indicate the potential of G. chinensis in promoting the remineralization. G. chinensis may become one more promising agent for caries prevention. SCANNING 31: 195–203, 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.     

Calibration of step heights and roughness measurements with atomic force microscopes

In this paper we present a method for the vertical calibration of a metrological atomic force microscope (AFM), which can be applied to most AFM systems with distance sensors. A thorough analysis describes the physical z-coordinate of an imaged surface as a function of the observed and uncorrected z-coordinate and the horizontal position. The three most important correction terms in a Taylor expansion of this function are identified and estimated based on series of measurements on a calibrated step height and a flat reference surface. Based on this calibration a number of step heights are calibrated by the AFM with measured values consistent with reference values, where available. Relative standard uncertainty of about 0.5% is achieved for step heights above 200 nm. For step heights below 50 nm, the standard uncertainty is about 0.5 nm. While a calibration of step heights done by AFM and interference microscopy can be compared directly as demonstrated here, this is not straightforward for roughness measurement. To asses this, the exact same area on an important applied surface (a hip joint prosthesis) was measured by both AFM and interference microscopy. Similarities in the images were seen; however, the calculated roughness was significantly different (R_q=3 and 1.5 nm). Applying a low-pass filter with a cut-off wavelength of λ_c=1.5 μm, the appearance of the images and the calculated roughness become almost identical. This strongly suggests that the two methods are consistent, and that the observed differences in shape and roughness in the nanometer range can be explained by the limited lateral resolution of the interference microscope.     

Mechanical test and friction-mapping on recycled polypropylene beads using atomic force microscopy

Mechanical tests at sub-micron scales using force microscopy are often used for the characterization of materials. Here we report the mechanical, tribologic, and morphological characterization of recycled polypropylene beads using force spectroscopy and lateral-force microscopy. The compression-elastic moduli calculated using the Hertzian model for polypropylene beads was between 0.448 ± 0.010 and 1.044 ± 0.057 GPa. The grain size analysis revealed a significant correlation between the grain size and measured compression-elastic moduli. Friction-maps of recycled polypropylene beads obtained using lateral-force microscopy were also reported for 25 μm² scanning areas.     

Second harmonic atomic force microscopy imaging of live and fixed mammalian cells

Higher harmonic contributions in the movement of an oscillating atomic force microscopy (AFM) cantilever are generated by nonlinear tip–sample interactions, yielding additional information on structure and physical properties such as sample stiffness. Higher harmonic amplitudes are strongly enhanced in liquid compared to the operation in air, and were previously reported to result in better structural resolution in highly organized lattices of proteins in bacterial S-layers and viral capsids [J. Preiner, J. Tang, V. Pastushenko, P. Hinterdorfer, Phys. Rev. Lett. 99 (2007) 046102]. We compared first and second harmonics AFM imaging of live and fixed human lung epithelial cells, and microvascular endothelial cells from mouse myocardium (MyEnd). Phase–distance cycles revealed that the second harmonic phase is 8 times more sensitive than the first harmonic phase with respect to variations in the distance between cantilever and sample surface. Frequency spectra were acquired at different positions on living and fixed cells with second harmonic amplitude values correlating with the sample stiffness. We conclude that variations in sample stiffness and corresponding changes in the cantilever–sample distance, latter effect caused by the finite feedback response, result in second harmonic images with improved contrast and information that is not attainable in the fundamental frequency of an oscillating cantilever.