Harnessing bifurcations in tapping-mode atomic force microscopy to calibrate time-varying tip-sample force measurements

Torsional harmonic cantilevers allow measurement of time-varying tip-sample forces in tapping-mode atomic force microscopy. Accuracy of these force measurements is important for quantitative nanomechanical measurements. Here we demonstrate a method to convert the torsional deflection signals into a calibrated force wave form with the use of nonlinear dynamical response of the tapping cantilever. Specifically the transitions between steady oscillation regimes are used to calibrate the torsional deflection signals.     

Cross-talk correction in atomic force microscopy

Commercial atomic force microscopes usually use a position-sensitive photodiode to detect the motion of the cantilever via laser beam deflection. This readout technique makes it possible to measure bending and torsion of the cantilever separately. A slight angle between the orientation of the photodiode and the plane of the readout laser beam, however, causes false signals in both readout channels. This cross-talk may lead to misinterpretation of the acquired data. We demonstrate this fault with images recorded in contact mode on periodically poled ferroelectric crystals and present a simple electronic circuit to compensate for it. This circuit can correct for cross-talk with a bandwidth of approximately 1 MHz suppressing the the false signal to <1%.     

Optical interference artifacts in contact atomic force microscopy images

Atomic force microscopy images are usually affected by different kinds of artifacts due to either the microscope design and operation mode or external environmental factors. Optical interferences between the laser light reflected off the top of the cantilever and the light scattered by the surface in the same direction is one of the most frequent sources of height artifact in contact (and occasionally non-contact) images. They are present when imaging highly reflective surfaces, or even when imaging non-reflective materials deposited onto reflective ones. In this study interference patterns have been obtained with a highly polished stainless steel planchet. The influence of these artifacts in surface roughness measurements is discussed, and a semi-quantitative method based on the fast Fourier transform technique is proposed to remove the artifacts from the images. This method improves the results obtained by applying the usual flattening routines.     

Adhesion artefacts in atomic force microscopy imaging

Artefacts that affect contrast and arise from adhesion forces in atomic force microscopy images of aramid fibres (both fresh and plasma-treated) are investigated. It is demonstrated that these stem not only from variations in the chemical composition of the surface but also from certain topographical features (which may appear hidden or enhanced in the images), resulting in changes in the lateral forces that are detected by the cantilever and are comparable to the vertical forces. It is also shown that both types of contribution to the forces can be uncoupled to yield images free from these artefacts, thus allowing more accurate quantitative measurements. These artefactual effects are also generally applicable to many other materials.     

Topographic and phase-contrast imaging in atomic force microscopy

Phase-contrast imaging in the tapping mode atomic force microscopy (AFM) is a powerful method in surface characterization. This method can provide fine details about rough surfaces, which are normally obscured in topographic imaging. To illustrate some of the capabilities of phase-contrast imaging, AFM studies of Pt/Ti/SiO2/Si and Pb(Zr0.52Ti0.48)O3 (PZT) films were carried out. Phase-contrast imaging revealed fine details of their microstructures, including grain boundaries, triple junctions and twinning, which could not be detected by topographic imaging. The studies showed that phase-contrast imaging is capable of providing superior information about surface characteristics when compared to the standard topographic imaging.     

Phase imaging atomic force microscopy in the characterization of biomaterials

The phase imaging atomic force microscopy is a powerful tool in surface characterization of the biomaterials, and the resulting phase image is able to detect chemical variation and reveal more detailed surface properties than the morphological image. However, the chemical‐ and morphological‐dependent phase images were still not distinguished well. In order to better understand actual occurring phase images, we examined non‐carious human maxillary incisor, microphase separated polyurethane and self‐assembling peptide nanofibres. We herein reported that phase image mainly plotted the morphological change: the phase peak corresponding to the morphological valley, and the morphological peak to the phase valley, and exhibited fine surface structures of materials. The chemical‐dependent phase contrast was generally masked by their inherent roughness. For the sample being very rough and having great phase separation, its chemical‐dependent phase contrast could be detected at the hard tapping mode (‘Amp. Ref. “set point ratio”’: ?0.4 to ?0.8), for the sample with medium roughness only at the light tapping mode (‘Amp. Ref.’: ?0.1 to ?0.4). These results will help us understand and determine actual occurring phase images of natural or fabricated biomaterials, even, other materials.     

Cross-sectional atomic force microscopy imaging of polycrystalline thin films

Atomic force microscopy (AFM) can be used to image cross-sections of thin-film samples. So far, however, it has mainly been used to study cross-sections of epitaxial systems or integrated circuits on crystalline substrates. In this paper, we show that AFM is a powerful tool to image fractured cross-sections of polycrystalline thin films deposited on crystalline and non-crystalline substrates, yielding unique information on the three-dimensional properties of the cross-sections, with a spatial resolution in the nm range. Original images of three different heterostructure systems are presented: Si(wafer)/SnO2/CdS/CdTe, glass/Mo/Cu(In,Ga)Se2,/CdS/ZnO, and glass/SnO2/WO3. We discuss the results by comparing AFM and scanning electron microscopy (SEM) images, and explain, for the different materials, why the AFM provides useful additional information.     

High-resolution imaging of chromosome-related structures by atomic force microscopy

An atomic force microscope (AFM) was combined with a conventional optical microscope. The optical microscope proved to be very convenient for locating objects of interest. In addition, the high-resolution AFM image can be compared directly with the traditional optical image. The instrument was used to study chromosome structures. High-resolution chromosome images revealed details of the 30-nm chromatide structure, confirming earlier electron microscopic observations. Chromosomes treated with trypsin revealed a banding pattern in height which is very similar to the optical image observed after staining with Giemsa. Furthermore, it is shown that the AFM can be used to locate DNA probes on in situ hybridized chromosomes. Images of the synaptonemal complex isolated from rat spermatocytes revealed details that improve the understanding of the three-dimensional structure of this protein.     

Drift-free atomic force microscopy measurements of cell height and mechanical properties

Atomic force microscopy (AFM) is used to study the morphological and mechanical properties of living cells. However, experiments performed over minutes to hours are subject to significant instrumental drift. The main sources of drift are the cantilever's geometrical asymmetry and bimorphic construction. We developed a simple software Stick-and-Move (SaM) routine for AFM that eliminates drift by continuously referencing the sample position to the substrate while acquiring force-distance curves. Control experiments show no drift over 15 min at an acquisition rate of 0.1 Hz. As a proof of concept, we applied the SaM to study the response of rat astrocytes to osmotic stress, observing dimensional and constitutive changes during volume regulation.     

Force-gradient-induced mechanical dissipation of quartz tuning fork force sensors used in atomic force microscopy

We have studied the dynamics of quartz tuning fork resonators used in atomic force microscopy taking into account the mechanical energy dissipation through the attachment of the tuning fork base. We find that the tuning fork resonator quality factor changes even in the case of a purely elastic sensor-sample interaction. This is due to the effective mechanical imbalance of the tuning fork prongs induced by the sensor-sample force gradient, which in turn has an impact on dissipation through the attachment of the resonator base. This effect may yield a measured dissipation signal that can be different from the one exclusively related to the dissipation between the sensor and the sample. We also find that there is a second-order term in addition to the linear relationship between the sensor-sample force gradient and the resonance frequency shift of the tuning fork that is significant even for force gradients usually present in atomic force microscopy, which are in the range of tens of N/m.     

Three-dimensional imaging of undercut and sidewall structures by atomic force microscopy

Sidewall surface roughness is an important parameter in electronic device manufacture. At present, no high resolution technique exists to quantitatively characterize this property for undercut structures created by semiconductor processing techniques. We developed a three-dimensional atomic force microscope (3D-AFM) to measure the surface roughness of undercut sidewalls with nanometer precision. Decoupled from the positional scanner, the 3D-AFM probe had a variable tilt up to 40° off the normal. Nonorthogonal scans resolved the sidewall surface roughness, base width, and acute critical angle for undercut structures, including a metal overhang and the transmission line of a photonic device. Compatible with standard cantilevers, the 3D-AFM demonstrates great potential for characterizing the sidewalls of soft materials such as photoresist.     

Method of imaging low density lipoproteins by atomic force microscopy

This short paper reports a simple method to image low density lipoproteins (LDL) using atomic force microscopy (AFM). This instrument allows imaging of biological samples in liquid and presents the advantage of needing no sample preparation such as staining or fixation that may affect their general structure. Dimensions (diameter and height) of individual LDL particles were successfully measured. AFM imaging revealed that LDL have a quasi-spherical structure on the x and y axis with an oblate spheroid structure in the z axis (i.e., height). LDLs were found to have an average diameter of 23 +/- 3 nm. The obtained mean height was 10 +/- 2 nm.     

Combined atomic force microscopy and scanning tunneling microscopy imaging of cross-sectioned GaN light-emitting diodes

Cross-sectional scanning tunneling microscopy (STM) was combined with atomic force microscopy (AFM) over the same area to characterize a cross-sectioned GaN light emitting diode. Because GaN is typically grown on a non-native substrate and also forms a wurtzite crystal structure, a cryogenic cleaving technique was developed to generate smooth surfaces. The depletion region surrounding the p-n junction was clearly identified using STM. Furthermore, by imaging under multiple sample biases, distinctions between the n-doped and p-doped GaN could be made.     

Time-lapse imaging of In Vitro myogenesis using atomic force microscopy

Myoblast therapy relies on the integration of skeletal muscle stem cells into distinct muscular compartments for the prevention of clinical conditions such as heart failure, or bladder dysfunction. Understanding the fundamentals of myogenesis is hence crucial for the success of these potential medical therapies. In this report, we followed the rearrangement of the surface membrane structure and the actin cytoskeletal organization in C2C12 myoblasts at different stages of myogenesis using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). AFM imaging of living myoblasts undergoing fusion unveiled that within minutes of making cell–cell contact, membrane tubules appear that unite the myoblasts and increase in girth as fusion proceeds. CLSM identified these membrane tubules as built on scaffolds of actin filaments that nucleate at points of contact between fusing myoblasts. In contrast, similarly behaving membrane tubules are absent during cytokinesis. The results from our study in combination with recent findings in literature further expand the understanding of the biochemical and membrane structural rearrangements involved in the two fundamental cellular processes of division and fusion.     

Local raster scanning for high-speed imaging of biopolymers in atomic force microscopy

A novel algorithm is described and illustrated for high speed imaging of biopolymers and other stringlike samples using atomic force microscopy. The method uses the measurements in real-time to steer the tip of the instrument to localize the scanning area over the sample of interest. Depending on the sample, the scan time can be reduced by an order of magnitude or more while maintaining image resolution. Images are generated by interpolating the non-raster data using a modified Kriging algorithm. The method is demonstrated using physical simulations that include actuator and cantilever dynamics, nonlinear tip-sample interactions, and measurement noise as well as through scanning experiments in which a two-axis nanopositioning stage is steered by the algorithm using simulated height data.     

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.     

Identification and ultrastructural imaging of photodynamic therapy-induced microfilaments by atomic force microscopy

Atomic force microscopy (AFM) is an emerging technique for imaging biological samples at subnanometer resolution; however, the method is not widely used for cell imaging because it is limited to analysis of surface topology. In this study, we demonstrate identification and ultrastructural imaging of microfilaments using new approaches based on AFM. Photodynamic therapy (PDT) with a new chlorin-based photosensitizer DH-II-24 induced cell shrinkage, membrane blebbing, and reorganization of cytoskeletons in bladder cancer J82 cells. We investigated cytoskeletal changes using confocal microscopy and atomic force microscopy. Extracellular filaments formed by PDT were analyzed with a tandem imaging approach based on confocal microscopy and atomic force microscopy. Ultrathin filaments that were not visible by confocal microscopy were identified as microfilaments by on-stage labeling/imaging using atomic force microscopy. Furthermore, ultrastructural imaging revealed that these microfilaments had a stranded helical structure. Thus, these new approaches were useful for ultrastructural imaging of microfilaments at the molecular level, and, moreover, they may help to overcome the current limitations of fluorescence-based microscopy and atomic force microscopy in cell imaging.     

基于几何成像模型的鱼眼镜头图像校正算法和技术研究

鱼眼镜头是一种具有广视场角的摄像机镜头,广泛应用于大场景视频监控领域,但其成像存在严重的非线性径向畸变,违反人类的视觉习惯,且为测量和模式识别等应用带来了不便.为了实现鱼眼镜头图像的校正,提出了一种基于几何成像模型的鱼眼镜头校正算法和技术,通过研究鱼眼镜头成像模型,根据鱼眼镜头类型采用了相应的几何模型校正图像;以“等距投影”模型为例提出了校正公式和实现方法.最后针对工程应用的实时性要求,提出了基于缓存的快速优化方法以及在保证一定精度的前提下快速实现双线性插值的优化方法.研究结果表明,这种新方法可以高效地完成鱼眼镜头图像校正,比传统方法适用面更广,更易实现.     

Distinction of heterogeneity on Au nanostructured surface based on phase contrast imaging of atomic force microscopy

The discrimination of the heterogeneity of different materials on nanostructured surfaces has attracted a great deal of interest in biotechnology as well as nanotechnology. Phase imaging through tapping mode of atomic force microscopy (TMAFM) can be used to distinguish the heterogeneity on a nanostructured surface. Nanostructures were fabricated using anodic aluminum oxide (AAO). An 11-mercaptoundecanoic acid (11-MUA) layer adsorbed onto the Au nanodots through self-assembly to improve the bio-compatibility. The Au nanostructures that were modified with 11-MUA and the concave surfaces were investigated using the TMAFM phase images to compare the heterogeneous and homogeneous nanostructured surfaces. Although the topography and phase images were taken simultaneously, the images were different. Therefore, the contrast in the TMAFM phase images revealed the different compositional materials on the heterogeneous nanostructure surface.     

Energy dissipation and dynamic response of an amplitude-modulation atomic-force microscopy subjected to a tip-sample viscous force

In a common environment of atomic force microscopy (AFM), a damping force occurs between a tip and a sample. The influence of damping on the dynamic response of a cantilever must be significant. Moreover, accurate theory is very helpful for the interpretation of a sample's topography and properties. In this study, the effects of damping and nonlinear interatomic tip-sample forces on the dynamic response of an amplitude-formulation AFM are investigated. The damping force is simulated by using the conventional Kelvin-Voigt damping model. The interatomic tip-sample force is the attractive van der Waals force. For consistance with real measurement of a cantilever, the mathematical equations of the beam theory of an AM-AFM are built and its analytical solution is derived. Moreover, an AFM system is also simplified into a mass-spring-damper model. Its exact solution is simple and intuitive. Several relations among the damping ratio, the response ratio, the frequency shift, the energy dissipation and the Q-factor are revealed. It is found that the resonant frequencies and the phase angles determined by the two models are almost same. Significant differences in the resonant quality factors and the response ratios determined by using the two models are also found. Finally, the influences of the variations of several parameters on the error of measuring a sample's topography are investigated.