Standardized Nanomechanical Atomic Force Microscopy Procedure (SNAP) for Measuring Soft and Biological Samples

A previous study has illustrated that the alphaVbeta3 integrin served as the functional receptor for West Nile virus (WNV) entry into cells. Domain III (DIII) of WNV envelope protein (E) was postulated to mediate virus binding to the cellular receptor. In this study, the specificity and affinity binding of WNV E DIII protein to alphaVbeta3 integrin was confirmed with co-immunoprecipitation and receptor competition assay. Binding of WNV E DIII protein to alphaVbeta3 integrin induced the phosphorylation of focal adhesion kinase that is required to mediate ligand-receptor internalization into cells. A novel platform was then developed using the atomic force microscopy to measure this specific binding force between WNV E DIII protein and the cellular receptor, alphaVbeta3 integrin. The single protein pair-interacting force measured was in the range of 45 +/- 5 piconewtons. This interacting force was highly specific as minimal force was measured in the WNV E DIII protein interaction with alphaVbeta5 integrin molecules and heparan sulfate. These experiments provided an insight to quantitate virus-receptor interaction. Force measurement using atomic force microscopy can serve to quantitatively analyze the effect of candidate drugs that modulate virus-host receptor affinity.

Background Atomic force microscopy (AFM) is one of the main techniques used to characterize the mechanical properties of soft biological samples and biomaterials at the nanoscale. Despite efforts made by the AFM community to promote open-source data analysis tools, standardization continues to be a significant concern in a field that requires common analysis procedures. AFM-based mechanical measurements involve applying a controlled force to the sample and measure the resulting deformation in the so-called force-distance curves. These may include simple approach and retract or oscillatory cycles at various frequencies (microrheology). To extract quantitative parameters, such as the elastic modulus, from these measurements, AFM measurements are processed using data analysis software. Although open tools exist and allow obtaining the mechanical properties of the sample, most of them only include standard elastic models and do not allow the processing of microrheology data. In this work, we have developed an open-source software package (called PyFMLab, as of python force microscopy laboratory) capable of determining the viscoelastic properties of samples from both conventional force-distance curves and microrheology measurements. Methods PyFMLab has been written in Python, which provides an accessible syntax and sufficient computational efficiency. The software features were divided into separate, self-contained libraries to enhance code organization and modularity and to improve readability, maintainability, testability, and reusability. To validate PyFMLab, two AFM datasets, one composed of simple force curves and another including oscillatory measurements, were collected on HeLa cells. Results The viscoelastic parameters obtained on the two datasets analysed using PyFMLab were validated against data processing proprietary software and against validated MATLAB routines developed before obtaining equivalent results. Conclusions Its open-source nature and versatility makes PyFMLab an open-source solution that paves the way for standardized viscoelastic characterization of biological samples from both force-distance curves and microrheology measurements.

Micro-nano-scale mechanical properties are vital for engineering and biological materials. The elastic modulus is generally measured by processing the force-indentation curves obtained by atomic force microscopy (AFM). However, the measurement precision is largely affected by tip shape, tip wear, sample morphology, and the contact model. In such research, it has been found that the radius of the sharp tip increases due to wear during contact scanning, affecting elastic modulus calculations. For flat-ended tips, it is difficult to identify the contact condition, leading to inaccurate results. Our research team has invented a nano-spherical tip, obtained by implanting focused helium ions into a silicon microcantilever, causing it to expand into a silicon nanosphere. This nano-spherical tip has the advantages of sub-micro size and a smooth spherical surface. Comparative tests of the elastic modulus measurement were conducted on polytetrafluoroethylene (PTFE) and polypropylene (PP) using these three tips. Overall, the experimental results show that our nano-spherical tip with a consistent tip radius, symmetrical geometric shape, and resistance to wear and contamination can improve precision in elastic modulus measurements of polymer materials.

Schistosoma mansoni, an intravascular parasitic worm and the causative agent of schistosomiasis, relies on its tegument (outer layer) for survival and host interaction. This study explored the morphology and mechanical properties of S. mansoni tegument using Atomic Force Microscopy (AFM). Notably, we employed the PeakForce Quantitative Nanomechanical Mapping (PF-QNM) mode in air, enabling simultaneous acquisition of 3D topography and mechanical property contrasts (adhesion, elastic modulus). Additionally, nanoindentation (AFM contact mode) was performed on female worm tegument for elastic modulus measurement. Both techniques revealed an elastic modulus range of fractions or units of GPa for the tegument. Interestingly, mechanical property maps, particularly adhesion contrast, displayed a recurring pattern of light and dark bands. We also measured the depth of annular furrows on the female tegument, finding an average of 128 ± 10 nm. These findings establish AFM, particularly PF-QNM, as a valuable tool to characterize S. mansoni tegument properties, offering insights for future investigations into parasite biology and its response to immunological or pharmacological challenges.

P8. Responsiveness of patient derived intervertebral disc cells to mechanobiological treatment

This article addresses the compensation for the dynamics-coupling effects in piezo-actuators used for positioning in atomic force microscopes (AFMs). Piezo-actuators are used to position the AFM probe (relative to the sample) both parallel to the sample surface (x-y-axes) and perpendicular to the sample surface (z-axis). During AFM operation, such as nanofabrication and imaging of soft biological samples, the probe-sample distance (in the z-axis) needs to be precisely controlled to maintain the probe-sample interaction at a desired value; otherwise, large variation of the probe-sample distance results in distortions of the fabricated parts (in nanofabrication) and can cause sample damage (in imaging soft biological samples). In this article, we show that dynamics-coupling from the x-y-axes (the scanning axes) to the z-axis, referred to as x-to-z dynamics-coupling, can generate significant variations in the probe-sample distance when operating AFM at high speed, i.e., when the sample is scanned at high speed. We present an inversion-based approach to compensate for these dynamics-coupling effects. Additionally, for applications where the x-y-axes movement is repetitive (as in AFM scanning operations), an iterative approach is proposed to further reduce the coupling-caused positioning errors. Convergence of the iterative approach is investigated and experimental results show that the coupling-caused errors can be reduced to the noise level using the proposed approach. Thus, the main contribution of this article is the development of an approach to substantially reduce the coupling-caused positioning errors and thereby, enable high-speed high-precision positioning of piezoscanners used in AFMs.

Studying local mechanical properties of metallic alloys and composites and their defect structures such as grain boundaries, cracks, and dislocations allows a better understanding and design of these structural materials. With a nanoindenting atomic force microscope (NI-AFM), which uses diamond tips, such measurements are possible on a nanometer scale. Measurements on the microstructure of superalloys are presented, where different precipitates with sizes in the range of 100 nm are characterized by their different hardness values. In addition, this new technique was used to characterize the nanohardness of the plastic zone and stress fields of cracks and grain boundaries. Therefore, crack tips of in-situ loaded bending specimens of intermetallic alloys (NiAl) were investigated. The measurements show a change in nanohardness and elasticity from the distance of interfaces. In material science the high-resolution capabilities of the atomic force microscopy (AFM) are used for studying metallographic sections, where different phases produce topographic contrasts in the image. A extremly small height difference in the microstructure is sufficient for a height contrast in the image, which allows a quantitative determination of particle size distributions and volume contents of precipitates [1, 2]. The plastic deformation behavior of metals is strongly influenced by the distribution and movement of defects as dislocations, grain boundaries and cracks. In-situ loading experiments in an AFM on specimens loaded in 4-point bending demonstrate the good imaging properties of an AFM for such defects [3]. Mechanical properties such as Young’s modulus of elasticity and hardness values were obtainable in the past by various indentation techniques, where a load displacement curve is recorded during the indentation process. These techniques were mainly applied for studying the mechanical properties of thin films. A combination of a nanoindenting instrument with an AFM now allows the local mechanical properties of different phases and particles in metallic alloys to be measured, since the indents can be positioned very exactly with the AFM. Such a combined nanoindenting AFM (NI-AFM) requires a special force measurement technique. Piezo hysteresis and creep of the used piezo scanner have to be eliminated. In addition, indenting should be performed by a tip which is centrally fixed and not at the end of a bending beam (cantilever) [4], because otherwise the location of the indent changes during loading. These properties are fulfilled by a capacitive transducer, which is used for load generation and measurement instead of the standard laser deflection unit. A diamond tip is used for the indentation experiments and topographic imaging in a conventional AFM mode. The resulting force indentation curves contain two pieces of information: the universal hardness H and the reduced modulus of elasticity Er. The possibilities of the NI-AFM for studying local mechanical properties are demonstrated on two different examples: first the hardness of the superalloy CMSX-6 was characterized; second, local changes of mechanical properties near a crack tip in a NiAl single crystal were examined. 1 Experimental setup and sample preparation The nanoindentation measurements shown in this paper were performed with an add-on-force transducer from Hysitron Inc. This transducer, mounted on a conventional AFM, controls the z movement of the tip (indentation, applied load, and feedback signal) while the piezos of the AFM control the lateral x-y movement. Further technical details on the transducer are described in [5]. Specimens of the superalloy CMSX-6 were examined with a Nanoscope II AFM from Digital Instruments. Since the x-y movement is achieved by scanning the whole specimen, only small specimens with a maximum diameter of 20 mm can be analyzed. Crack tips in NiAl single crystals were investigated with a bending apparatus specially constructed for the Explorer AFM from TopoMetrix. (This bending apparatus is described in [6].) Because the whole transducer assembly is mounted on the x-y scanner, the combined system is more sensitive to mechanical noise. However, large specimens can be examined with

This paper addresses a relatively simple method of measuring Young's modulus of electroplated nickel using Atomic Force Microscope. Thin layer of nickel to be measured is electroplated onto the tip side of AFM silicon cantilever, whose Young's modulus and the geometric dimensions are defined from manufacturer. The resonant frequency and the quality factor of the electroplated AFM cantilever are measured by the tapping mode of AFM and its spring constant is calculated using Sader's method. The spring constant of the electroplated cantilever is also calculated by using the laminar composite beam theory. Comparing two spring constants, Young's modulus of the electroplated nickel is determined. The measured elastic modulus of nickel in each time step is in the range of between and the average elastic modulus is with relative uncertainty of less than 5%

Unique features of diatoms are their intricate cell covers (frustules) made out of hydrated, amorphous silica. The frustule defines and maintains cell shape and protects cells against grazers and pathogens, yet it must allow for cell expansion during growth and division. Other siliceous structures have also evolved in some chain-forming species as means for holding neighboring cells together. Characterization and quantification of mechanical properties of these structures are crucial for the understanding of the relationship between form and function in diatoms, but thus far only a handful of studies have addressed this issue. We conducted micro-indentation experiments, using atomic force microscopy (AFM), to examine local variations in elastic (Young's) moduli of cells and linking structures in the marine, chain-forming diatom Lithodesmium undulatum. Using a fluorescent tracer that is incorporated into new cell wall components we tested the hypothesis that new siliceous structures differ in elastic modulus from their older counterparts. Results show that the local elastic modulus is a highly dynamic property. Elastic modulus of stained regions was significantly lower than that of unstained regions, suggesting that newly formed cell wall components are generally softer than the ones inherited from the parent cells. This study provides the first evidence of differentiation in local elastic properties in the course of the cell cycle. Hardening of newly formed regions may involve incorporation of additional, possibly organic, material but further studies are needed to elucidate the processes that regulate mechanical properties of the frustule during the cell cycle.

The atomic force microscope (AFM) is a probe-based microscope that uses nanoscale and structural imaging where high resolution is desired. AFM has also been used in mechanical, electrical, and thermal engineering applications. This unique technique provides vital local material properties like the modulus of elasticity, hardness, surface potential, Hamaker constant, and the surface charge density from force versus displacement curve. Therefore, AFM was used to measure both the diameter and mechanical properties of the collagen nanostraws in human costal cartilage. Human costal cartilage forms a bridge between the sternum and bony ribs. The chest wall of some humans is deformed due to defective costal cartilage. However, costal cartilage is less studied compared to load bearing cartilage. Results show that there is a difference between chemical fixation and non-chemical fixation treatments. Our findings imply that the patients' chest wall is mechanically weak and protein deposition is abnormal. This may impact the nanostraws' ability to facilitate fluid flow between the ribs and the sternum. At present, AFM is the only tool for imaging cells' ultra-structure at the nanometer scale because cells are not homogeneous. The first layer of the cell is called the cell membrane, and the layer under it is made of the cytoskeleton. Cancerous cells are different from normal cells in term of cell growth, mechanical properties, and ultra-structure. Here, force is measured with very high sensitivity and this is accomplished with highly sensitive probes such as a nano-probe. We performed experiments to determine ultra-structural differences that emerge when such cancerous cells are subject to treatments such as with drugs and electric pulses. Jurkat cells are cancerous cells. These cells were pulsed at different conditions. Pulsed and non-pulsed Jurkat cell ultra-structures were investigated at the nano meter scale using AFM. Jurkat cell mechanical properties were measured under different conditions. In addition, AFM was used to measure the charge density of cell surface in physiological conditions. We found that the treatments changed the cancer cells' ultra-structural and mechanical properties at the nanometer scale. Finally, we used AFM to characterize many non-biological materials with relevance to biomedical science. Various metals, polymers, and semi-conducting materials were characterized in air and multiple liquid media through AFM - techniques from which a plethora of industries can benefit. This applies especially to the fledging solar industry which has found much promise in nanoscopic insights. Independent of the material being examined, a reliable method to measure the surface force between a nano probe and a sample surface in a variety of ionic concentrations was also found in the process of procuring these measurements. The key findings were that the charge density increases with the increase of the medium's ionic concentration.

Carbon nanotube yarns (CNTYs) have received more consideration recently due to their excellent specific mechanical, electrical and thermal properties, making them promising materials for different applications. Until now, the axial properties of the yarn have been thoroughly investigated; however, the transverse or radial properties, orthogonal to the fiber axis, remain relatively unknown due to the challenges associated with their measurement. In this study, the transverse or radial response of the CNTY including its elastic modulus was determined using Atomic Force Microscopy (AFM) and Raman Spectroscopy. Determining transverse properties in fibrous materials presents challenges owing to their geometry, inherent anisotropy, whereby mechanical characteristics exhibit directional disparities; i.e., the properties in the transverse direction may be several orders of magnitude smaller than those in the axial direction. To overcome these difficulties, AFM was utilized to perform nanoindentation experiments, where a tipless flexible cantilever probe was used to apply a controlled force to the CNTY surface. The resulting indentation depth was then analyzed to determine the transversal elastic modulus. Preliminary findings indicate that the transverse elastic modulus of the CNTYs ranges from 10–54 kPa for strain levels below 3%. Complementary Raman spectroscopy provided insight into the bulk-scale mechanical behavior of CNTYs. Incremental compressive loading between microscope slides induced nonlinear upshifts in the 2D Raman band (from ~2686.6 to 2691.4 cm−1), indicating nanoscale tube realignment, inter-tube densification, and compaction. From lateral diameter measurements under load, a stress–strain curve was constructed, revealing three distinct regimes: one with an initial elastic modulus of 3.12 MPa (0.3–11.2% strain), another one with an elastic modulus increasing to 8.46 MPa (11.2–14.4%), and finally one with an elastic modulus peaking at 16.86 MPa beyond 14.4% strain. Together, these methods delineate the hierarchical and anisotropic nature of CNTYs, validating the importance of multiscale mechanical characterization for their deployment in piezoresistive sensors and multifunctional composites. This study establishes a robust framework for quantifying the transverse mechanical response of CNTYs.

Contact mode atomic force microscopy (AFM) is the most frequently used AFM imagingmode in biology. It is about 5–10 times faster than oscillating mode imaging (inconventional AFM setups), and provides topographs of biological samples withsub-molecular resolution and at a high signal-to-noise ratio. Unfortunately, contact modeimaging is sensitive to the applied force and intrinsic force drift: inappropriate force appliedby the AFM tip damages the soft biological samples. We present a methodologythat automatically searches for and maintains high resolution imaging forces. Wefound that the vertical and lateral vibrations of the probe during scanning arevaluable signals for the characterization of the actual applied force by the tip.This allows automated adjustment and correction of the setpoint force during anexperiment. A system that permanently performs this methodology steered the AFMtowards high resolution imaging forces and imaged purple membrane at molecularresolution and live cells at high signal-to-noise ratio for hours without an operator.

Atomic force microscopy (AFM) is a part of the scanning probe microscopy family. It provides a platform for high-resolution topographical imaging, surface analysis as well as nanomechanical property mapping for stiff and soft samples (live cells, proteins, and other biomolecules). AFM is also crucial for measuring single-molecule interaction forces and important parameters of binding dynamics for receptor-ligand interactions or protein-protein interactions on live cells. However, performing AFM measurements and the associated data analytics are tedious, laborious experimental procedures requiring specific skill sets and continuous user supervision. Significant progress has been made recently in artificial intelligence (AI) and deep learning (DL), extending into microscopy. In this review, we summarize how researchers have implemented machine learning approaches so far to improve the performance of atomic force microscopy (AFM), make AFM data analytics faster, and make data measurement procedures high-throughput. We also shed some light on the different application areas of AFM that have significantly benefited from applications of machine learning frameworks and discuss the scope and future possibilities of these crucial approaches.

In this study, the in situ hardness and elastic modulus measurements of arc-evaporated Cr0.49Al0.51N, Al0.66Ti0.34N, and Al0.65Ti0.33Y0.02N films are investigated using a high-temperature nanoindentation tester. It is found that the hardness of Cr0.49Al0.51N initially increases from 30.2 ± 2.3 GPa at room temperature (RT) and reaches a maximum of 33.2 ± 2.4 GPa at 600 °C before decreasing to 27.9 ± 2.1 GPa at 800 °C. However, the results for elastic modulus versus temperature follow a degraded trend, with 405 ± 23 GPa for RT, decreasing to 280 ± 18 GPa at 800 °C. Increases in the hardness and the elastic modulus can be achieved for both Al0.66Ti0.34N and Al0.65Ti0.33Y0.02N films as their temperatures increase. The addition of Y effectively promotes the hardness and the elastic modulus of Al0.66Ti0.34N at RT–300 °C. However, both hardness and elastic modulus change slightly as Y is incorporated at 300–800 °C.

A calibrated method of force sensing is demonstrated in which the buckled shape ofa long flexible metallic nanowire, referred to as a ‘nanoneedle’, is interpretedto determine the applied force. An individual needle of 157 nm diameter by15.6 µm length is grown on an atomic force microscope (AFM) cantilever with a desired orientation(by the method of Yazdanpanah et al 2005 J. Appl. Phys. 98 073510). Using ananomanipulator the needle is buckled in the chamber of a scanning electron microscope(SEM) and the buckled shapes are recorded in SEM images. Force is determined as afunction of deflection for an assumed elastic modulus by fitting the shapes using thegeneralized elastica model (De Bona and Zelenika 1997 Proc. Inst. Mech. Eng. C 211509–17). In this calibration the elastic modulus (68.3 GPa) was determined using anauxiliary AFM measurement, with the needle in the same orientation as in theSEM. Following this calibration the needle was used as a sensor in a differentorientation than the AFM coordinates to deflect a suspended PLLA polymer fiber fromwhich the elastic modulus (2.96 GPa) was determined. The practical value ofthe sensing method does depend on the reliability and ruggedness of the needle.In this study the same needle remained rigidly secured to the AFM cantileverthroughout the entire SEM/AFM calibration procedure and the characterization of thenanofiber.