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The role of lipid metabolic pathways in the pathophysiology of primary open-angle glaucoma (POAG) has been thoroughly elucidated, with pathways involved in lipid-related disorders such as hypercholesterolemia and hyperlipoprotein accumulation being of particular interest. The ABCA1/apoA-1 transduction pathway moderates reverse cholesterol transport (RCT), facilitating the transport of free cholesterol (FC) and phospholipids (PL) and preventing intracellular lipid aggregates in retinal ganglion cells (RGCs) due to excess FCs and PLs. A deficiency of ABCA1 transporters, and thus, dysregulation of the ABCA1/apoA-1 transduction pathway, may potentiate cellular lipid accumulation, which affects the structural and mechanical features of the cholesterol-rich RGC membranes. Atomic force microscopy (AFM) is a cutting-edge imaging technique suitable for imaging topographical surfaces of a biological specimen and determining its mechanical properties and structural features. The versatility and precision of this technique may prove beneficial in understanding the effects of ABCA1/apoA-1 pathway downregulation and decreased cholesterol efflux in RGCs and their membranes. In this protocol, ABCA1-/- RGC mouse models are prepared over the course of 3 days and are then compared with non-knockout ABCA1 RGC mouse models through AFM imaging of topographical surfaces to examine the difference in membrane dynamics of knockout vs. non-knockout models. Intracellular and extracellular levels of lipids are quantified through high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS).

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A direct focused He+beam direct machining is presented to fabricate solid-state nano-disk at the surface of a graphene multilayer micro-flake deposited on an Au/Ti/sapphire surface. At irradiation doses larger than 5.0 × 1017ions cm-2and with a beam size well below 1 nm, graphene disks down to 20 nm in diameter have been machined with for nano-disk down to 50 nm in diameter, a central hole for preparing the positioning of a rotation axle. The local heat generated by this irradiation is inducing a partial graphene amorphization and deformation, leading to a complete graphene nano-disk vaporization at doses larger than 5 × 1018ions cm-2. A dry transfer printing technique followed by a graphene surface cleaning was used to transfer the nano-disks from its initial surface to a fresh and clean surface. Tapping mode atomic force micrograph have been recorded to follow the vaporization as a function of the He+dose to confirm the graphene solid-state nano-disk fabrication limit to about 20 nm with this process.

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The morphology measurement of a surface can be done by using an atomic force microscope (AFM). However, it is difficult to ensure that the measurement does not introduce any damage to the sample surface. This paper proposes that phase shift, the phase change between the original surface and scanned area, can provide a characteristic signal of the tip-surface interaction. On a poly (methyl methacrylate) thin film, the present investigation explored the relationship between phase shift and nondestructive surface morphology measurement under the tapping mode of an AFM. The study showed that when the drive amplitude was doubled, the phase shift reached from 0.47° to 1.85°. Under this condition, wrinkles became observable. With the tip radius in the range of 15-20 nm, no phase shift appeared between a scanned area and the original surface after multiple measurements. In this case, the tip-surface energy dissipation was in the range of 10-35 eV, showing a nondestructive interaction of the surface with the AFM tip. When the tip radius was about 55 nm, under the same tip excitation parameters, the energy dissipation per tap varied from 60 to 110 eV, and a phase shift occurred in the range of 0.02-0.64°, while the surface plastic deformation was still extremely minor after multiple tip scanning. A higher phase shift was occurred on the softer surface attributed to multiple scanning under tapping mode. The study found that the phase shift characteristics was a more sensible measure to signify the transition from a nondestructive to a destructive surface morphology measurement by using the tapping mode of an AFM.

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High-speed atomic force microscopy (AFM) is a versatile method that can visualize proteins and protein systems on the nanometer scale and at a temporal resolution of 100 ms. The application to microtubules can not only reveal structural information with single-tubulin resolution but can also extract mechanical information and allows to study single motor proteins walking on microtubules, among others. This chapter provides a step-by-step guide from microtubule polymerization to successful observation with high-speed AFM.

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The phase information reflects the energy dissipation of the probe and sample interactions in tapping mode atomic force microscopes (TM AFMs). In this paper, we use the method of tune test in TM AFM to study the contribution of external environment to energy dissipation by changing the probe position and ambient humidity. Finally, the theoretical and experimental quality factors of air viscous damping, squeeze film damping and liquid bridge force are obtained to characterise energy dissipation. The analytically predicted values of the model established on squeeze film damping, viscous damping and liquid bridge force comparing to the experimental results in this paper is rational. And the comparative analysis results show that the main mechanism of energy dissipation is different at different probe positions and different relative humidness. This result is of great significance for understanding the mechanism of phase imaging experimentally and theoretically.

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Atomic force microscopy (AFM) is an important tool for measuring a variety of nanoscale surface properties, such as topography, viscoelasticity, electrical potential and conductivity. Some of these properties are measured using contact methods (static contact or intermittent contact), while others are measured using noncontact methods. Some properties can be measured using different approaches. Conductivity, in particular, is mapped using the contact-mode method. However, this modality can be destructive to delicate samples, since it involves continuously dragging the cantilever tip on the surface during the raster scan, while a constant tip-sample force is applied. In this paper we discuss a possible approach to develop an intermittent-contact conductive AFM mode based on Fourier analysis, whereby the measured current response consists of higher harmonics of the cantilever oscillation frequency. Such an approach may enable the characterization of soft samples with less damage than contact-mode imaging. To explore its feasibility, we derive the analytical form of the tip-sample current that would be obtained for attractive (noncontact) and repulsive (intermittent-contact) dynamic AFM characterization, and compare it with results obtained from numerical simulations. Although significant instrumentation challenges are anticipated, the modelling results are promising and suggest that Fourier-based higher-harmonics current measurement may enable the development of a reliable intermittent-contact conductive AFM method.

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Atomic force microscopy (AFM) can characterize nanomaterial elasticity. However, some one-dimensional nanomaterials, such as DNA, are too small to locate with an AFM tip because of thermal drift and the nonlinearity of piezoelectric actuators. In this study, we propose a novel approach to address the shortcomings of AFM and obtain the radial Young's modulus of a DNA duplex. The elastic properties are evaluated by combining physical calculations and measured experimental results. The initial elasticity of the DNA is first assumed; based on tapping-mode scanning images and tip⁻sample interaction force simulations, the calculated elastic modulus is extracted. By minimizing the error between the assumed and experimental values, the extracted elasticity is assigned as the actual modulus for the material. Furthermore, tapping-mode image scanning avoids the necessity of locating the probe exactly on the target sample. In addition to elasticity measurements, the deformation caused by the tapping force from the AFM tip is compensated and the original height of the DNA is calculated. The results show that the radial compressive Young's modulus of DNA is 125⁻150 MPa under a tapping force of 0.5⁻1.3 nN; its original height is 1.9 nm. This approach can be applied to the measurement of other nanomaterials.

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In this work PeakForce tapping (PFT) imaging was demonstrated with carbon nanotube atomic force microscopy (CNT-AFM) probes; this imaging mode shows great promise for providing simple, stable imaging with CNT-AFM probes, which can be difficult to apply. The PFT mode is used with CNT-AFM probes to demonstrate high resolution imaging on samples with features in the nanometre range, including a Nioprobe calibration sample and gold nanoparticles on silicon, in order to demonstrate the modes imaging effectiveness, and to also aid in determining the diameter of very thin CNT-AFM probes. In addition to stable operation, the PFT mode is shown to eliminate "ringing" artefacts that often affect CNT-AFM probes in tapping mode near steep vertical step edges. This will allow for the characterization of high aspect ratio structures using CNT-AFM probes, an exercise which has previously been challenging with the standard tapping mode.

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Peritubular dentine (PTD) and intertubular dentine (ITD) were investigated by 3D correlative Focused Ion Beam (FIB)-Scanning Electron Microscopy (SEM)-Energy Dispersive Spectroscopy (EDS) tomography, tapping mode Atomic Force Microscopy (AFM) and scattering-type Scanning Near-Field Optical Microscopy (s-SNOM) mapping. The brighter appearance of PTD in 3D SEM-Backscattered-Electron (BSE) imaging mode and the corresponding higher grey value indicate a greater mineral concentration in PTD (~160) compared to ITD (~152). However, the 3D FIB-SEM-EDS reconstruction and high resolution, quantitative 2D map of the Ca/P ratio (~1.8) fail to distinguish between PTD and ITD. This has been further confirmed using nanoscale 2D AFM map, which clearly visualised biopolymers and hydroxyapatite (HAp) crystallites with larger mean crystallite size in ITD (32 ± 8 nm) than that in PTD (22 ± 3 nm). Correlative microscopy reveals that the principal difference between PTD and ITD arises primarily from the nanoscale packing density of the crystallites bonded together by thin biopolymer, with moderate contribution from the chemical composition difference. The structural difference results in the mechanical properties variation that is described by the parabolic stiffness-volume fraction correlation function introduced here. The obtained results benefit a microstructure-based mechano-chemical model to simulate the chemical etching process that can occur in human dental caries and some of its treatments.

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The nonlinear vibration behavior of a Tapping mode atomic force microscopy (TM-AFM) microcantilever under acoustic excitation force has been modeled and investigated. In dynamic AFM, the tip-surface interactions are strongly nonlinear, rapidly changing and hysteretic. First, the governing differential equation of motion and boundary conditions for dynamic analysis are obtained using the modified couple stress theory. Afterwards, closed-form expressions for nonlinear frequency and effective nonlinear damping ratio are derived utilizing perturbation method. The effect of tip connection position on the vibration behavior of the microcantilever are also analyzed. The results show that nonlinear frequency is size dependent. According to the results, an increase in the equilibrium separation between the tip and the sample surface reduces the overall effect of van der Waals forces on the nonlinear frequency, but its effect on the effective nonlinear damping ratio is negligible. The results also indicate that both the change in the distance between tip and cantilever free end and the reduction of tip radius have significant effects on the accuracy and sensitivity of the TM-AFM in the measurement of surface forces. The hysteretic behavior has been observed in the near resonance frequency response due to softening and hardening of the forced vibration response.

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We explore the contact problem of a flat-end indenter penetrating intermittently a generalized viscoelastic surface, containing multiple characteristic times. This problem is especially relevant for nanoprobing of viscoelastic surfaces with the highly popular tapping-mode AFM imaging technique. By focusing on the material perspective and employing a rigorous rheological approach, we deliver analytical closed-form solutions that provide physical insight into the viscoelastic sources of repulsive forces, tip-sample dissipation and virial of the interaction. We also offer a systematic comparison to the well-established standard harmonic excitation, which is the case relevant for dynamic mechanical analysis (DMA) and for AFM techniques where tip-sample sinusoidal interaction is permanent. This comparison highlights the substantial complexity added by the intermittent-contact nature of the interaction, which precludes the derivation of straightforward equations as is the case for the well-known harmonic excitations. The derivations offered have been thoroughly validated through numerical simulations. Despite the complexities inherent to the intermittent-contact nature of the technique, the analytical findings highlight the potential feasibility of extracting meaningful viscoelastic properties with this imaging method.

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Tapping mode atomic force microscopy (AFM), also known as amplitude modulated (AM) or AC mode, is a proven, reliable, and gentle imaging mode with widespread applications. Over the several decades that tapping mode has been in use, quantification of tip-sample mechanical properties such as stiffness has remained elusive. Bimodal tapping mode keeps the advantages of single-frequency tapping mode while extending the technique by driving and measuring an additional resonant mode of the cantilever. The simultaneously measured observables of this additional resonance provide the additional information necessary to extract quantitative nanomechanical information about the tip-sample mechanics. Specifically, driving the higher cantilever resonance in a frequency modulated (FM) mode allows direct measurement of the tip-sample interaction stiffness and, with appropriate modeling, the set point-independent local elastic modulus. Here we discuss the advantages of bimodal tapping, coined AM-FM imaging, for modulus mapping. Results are presented for samples over a wide modulus range, from a compliant gel (∼100 MPa) to stiff materials (∼100 GPa), with the same type of cantilever. We also show high-resolution (subnanometer) stiffness mapping of individual molecules in semicrystalline polymers and of DNA in fluid. Combined with the ability to remain quantitative even at line scan rates of nearly 40 Hz, the results demonstrate the versatility of AM-FM imaging for nanomechanical characterization in a wide range of applications.

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Tapping mode (TM) atomic force microscopy (AFM) in a liquid environment is widely used to measure the contours of biological specimens. The TM triggers the AFM probe approximately at the resonant frequencies and controls the tip such that it periodically touches the specimen along the scanning path. The AFM probe and its tip produce a hydrodynamic pressure on the probe itself and press the specimen. The tip to specimen size ratio is known to affect the measurement accuracy of AFM, however, few studies have focused on the hydrodynamic pressure caused by the effects of specimen size. Such pressure affects the contour distortion of the biological specimen. In this study, a semi-analytical method is employed for a semicircular specimen to analyze the vorticity and pressure distributions for specimens of various sizes and at various tip locations. Changes in pressure distribution, fluid spin motion, and specimen deformation are identified as the tip approaches the specimen. The results indicate the following: the specimen surface experiences the highest pressure when the specimen diameter equals the tip width; the vorticity between tip and specimen is complex when the tip is close to the specimen center line; and the specimen inflates when the tip is aligned with the specimen center line.

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Adaptive multiloop-mode (AMLM) imaging to substantially increase (over an order of magnitude) the speed of tapping-mode (TM) imaging is tested and evaluated through imaging three largely different heterogeneous polymer samples in experiments. It has been demonstrated that AMLM imaging, through the combination of a suite of advanced control techniques, is promising to achieve high-speed dynamic-mode atomic force microscopy imaging. The performance, usability, and robustness of the AMLM in various imaging applications, however, is yet to be assessed. In this work, three benchmark polymer samples, including a PS-LDPE sample, an SBS sample, and a Celgard sample, differing in feature size and stiffness of two orders of magnitude, are imaged using the AMLM technique at high-speeds of 25 Hz and 20 Hz, respectively. The comparison of the images obtained to those obtained by using TM imaging at scan rates of 1 Hz and 2 Hz showed that the quality of the 25 Hz and 20 Hz AMLM imaging is at the same level of that of the 1 Hz TM imaging, while the tip-sample interaction force is substantially smaller than that of the 2 Hz TM imaging.

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Bottom up self-assembly of functional materials at liquid-liquid interfaces has recently emerged as method to design and produce novel two-dimensional (2D) nanostructured membranes and devices with tailored properties. Liquid-liquid interfaces can be seen as a "factory floor" for nanoparticle (NP) self-assembly, because NPs are driven there by a reduction of interfacial energy. Such 2D assembly can be characterized by reciprocal space techniques, namely X-ray and neutron scattering or reflectivity. These techniques have drawbacks, however, as the structural information is averaged over the finite size of the radiation beam and nonperiodic isolated assemblies in 3D or defects may not be easily detected. Real-space in situ imaging methods are more appropriate in this context, but they often suffer from limited resolution and underperform or fail when applied to challenging liquid-liquid interfaces. Here, we study the surfactant-induced assembly of SiO2 nanoparticle monolayers at a water-oil interface using in situ atomic force microscopy (AFM) achieving nanoscale resolved imaging capabilities. Hitherto, AFM imaging has been restricted to solid-liquid interfaces because applications to liquid interfaces have been hindered by their softness and intrinsic dynamics, requiring accurate sample preparation methods and nonconventional AFM operational schemes. Comparing both AFM and grazing incidence X-ray small angle scattering data, we unambiguously demonstrate correlation between real and reciprocal space structure determination showing that the average interfacial NP density is found to vary with surfactant concentration. Additionally, the interaction between the tip and the interface can be exploited to locally determine the acting interfacial interactions. This work opens up the way to studying complex nanostructure formation and phase behavior in a range of liquid-liquid and complex liquid interfaces.

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Using atomic force microscopy, we monitored the nanoscale surface morphology of human teeth at the dentin-enamel junction after performing successive demineralization steps with an acidic soft drink. Subsequently, we studied the remineralization process with a paste containing calcium and phosphate ions. Repeated atomic force microscopy imaging of the same sample areas on the sample allowed us to draw detailed conclusions regarding the specific mechanism of the demineralization process and the subsequent remineralization process. The about 1-µm-deep grooves that are caused by the demineralization process were preferentially filled with deposited nanoparticles, leading to smoother enamel and dentine surfaces after 90 min exposure to the remineralizing agent. The deposited material is found to homogeneously cover the enamel and dentine surfaces in the same manner. The temporal evolution of the surface roughness indicates that the remineralization caused by the repair paste proceeds in two distinct successive phases.

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Tapping mode atomic force microscopy (AFM) provides phase images in addition to height and amplitude images. Although the behavior of tapping mode AFM has been investigated using mathematical modeling, comprehensive understanding of the behavior of tapping mode AFM still poses a significant challenge to the AFM community, involving issues such as the correct interpretation of the phase images. In this paper, the cantilever's dynamic behavior in tapping mode AFM is studied through a three dimensional finite element method. The cantilever's dynamic displacement responses are firstly obtained via simulation under different tip-sample separations, and for different tip-sample interaction forces, such as elastic force, adhesion force, viscosity force, and the van der Waals force, which correspond to the cantilever's action upon various different representative computer-generated test samples. Simulated results show that the dynamic cantilever displacement response can be divided into three zones: a free vibration zone, a transition zone, and a contact vibration zone. Phase trajectory, phase shift, transition time, pseudo stable amplitude, and frequency changes are then analyzed from the dynamic displacement responses that are obtained. Finally, experiments are carried out on a real AFM system to support the findings of the simulations.

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Bimodal force microscopy has expanded the capabilities of atomic force microscopy (AFM) by providing high spatial resolution images, compositional contrast and quantitative mapping of material properties without compromising the data acquisition speed. In the first bimodal AFM configuration, an amplitude feedback loop keeps constant the amplitude of the first mode while the observables of the second mode have not feedback restrictions (bimodal AM). Here we study the conditions to enhance the compositional contrast in bimodal AM while imaging heterogeneous materials. The contrast has a maximum by decreasing the amplitude of the second mode. We demonstrate that the roles of the excited modes are asymmetric. The operational range of bimodal AM is maximized when the second mode is free to follow changes in the force. We also study the contrast in trimodal AFM by analyzing the kinetic energy ratios. The phase contrast improves by decreasing the energy of second mode relative to those of the first and third modes.

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Several atomic force microscopy (AFM) tests have been carried out on both smooth (polyimide) and rough (polyurethane) surfaces so that to obtain the best results; subsequently, the optimization of experiments performed is presented. A special emphasis has been put on the effect of tip geometry, image pre-processing procedure, scanning area, resolution, pixel size, and cantilever oscillation amplitude in tapping mode, as well as on the quality of the topographical images and 3D surface texture parameters. After viewing the scanning tip and finding out its sharpness, degradation, and contamination, a simultaneous calibration in X, Y, and Z directions, lateral calibration of SPM scanners and detection of lateral non-linearity, hysteresis, creep, and cross-coupling effects has been made. We have established the following experimental parameters: proper scanning resolution (512 × 512 pixels), adequate pixel size (between 2.9 and 19.5 nm) and suitable intermittent contact region (moderate tapping) for which the AFM images present good contrast and resolution. Using these parameters, the values of 3D texture parameters remain constant. These kinds of measurements are extremely important to conduct further AFM experiments on polyimide and polyurethane surfaces under optimal conditions, thus avoiding unwanted artifacts on the morphological images or unrealistic values for the 3D surface texture parameters that might occur.

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We present a simulation environment, dForce, which can be used for a better understanding of dynamic force microscopy experiments. The simulator presents the cantilever-tip dynamics for two dynamic AFM methods, tapping mode AFM and bimodal AFM. It can be applied for a wide variety of experimental situations in air or liquid. The code provides all the variables and parameters relevant in those modes, for example, the instantaneous deflection and tip-surface force, velocity, virial, dissipated energy, sample deformation and peak force as a function of time or distance. The simulator includes a variety of interactions and contact mechanics models to describe AFM experiments including: van der Waals, Hertz, DMT, JKR, bottom effect cone correction, linear viscoelastic forces or the standard linear solid viscoelastic model. We have compared two numerical integration methods to select the one that offers optimal accuracy and speed. The graphical user interface has been designed to facilitate the navigation of non-experts in simulations. Finally, the accuracy of dForce has been tested against numerical simulations performed during the last 18 years.