Atomic Force Microscopy Instrument Research Articles

Exploring the Challenges of Characterising Surface Topography of Polymer-Nanoparticle Composites.

Nanomechanical testing plays a crucial role in evaluating surfaces containing nanoparticles. Testing verifies surface performance concerning their intended function and detects any potential shortcomings in operational standards. Recognising that nanostructured surfaces are not always straightforward or uniform is essential. The chemical composition and morphology of these surfaces determine the end-point functionality. This can entail a layered surface using materials in contrast to each other that may require further modification after nanomechanical testing to pass performance and quality standards. Nanomechanical analysis of a structured surface consisting of a poly-methyl oxazoline film base functionalised with colloidal gold nanoparticles was demonstrated using an atomic force microscope (AFM). AFM nanomechanical testing investigated the overall substrate architecture's topographical, friction, adhesion, and wear parameters. Limitations towards its potential operation as a biomaterial were also addressed. This was demonstrated by using the AFM cantilever to apply various forces and break the bonds between the polymer film and gold nanoparticles. The AFM instrument offers an insight to the behaviour of low-modulus surface against a higher-modulus nanoparticle. This paper details the bonding and reaction limitations between these materials on the application of an externally applied force. The application of this interaction is highly scrutinised to highlight the potential limitations of a functionalised surface. These findings highlight the importance of conducting comprehensive nanomechanical testing to address concerns related to fabricating intricate biomaterial surfaces featuring nanostructures.

Enhanced two consecutive samples based de-modulation technique for atomic force microscopy application

This article investigates robust amplitude detectors suitable for atomic force microscopy (AFM) while discussing better alternatives. An AFM instrument’s measurement unit is responsible for providing the amplitude information obtained from the tip of a cantilever beam to identify the surface smoothness of a test material. Therefore, two efficient approaches are suggested to leverage Lyapunov’s theory while adhering to better noise suppression and DC-offset rejection capabilities. Nevertheless, an enhanced two samples-based Lyapunov’s demodulation approach is proposed to detect the amplitude information rapidly. Consequently, the modifications applied to the conventional method help reduce the tuning efforts and structural complications. The proposed solution remains structurally simpler and useful for high- and low-frequency probes. Furthermore, the extensive design guidelines for all techniques and the simulation results are presented. Different amplitude signals are synthetically generated from several rough pseudo-test surfaces for early verification and sent to a real-time digital controller to judge the proposal’s efficacy.

The adsorption and inhibition efficiency of 2-amino-4-methoxy-6-methyl-1,3,5-triazine for corrosion of mild steel in hydrochloric acid solution

PurposeThis study aims to prevent mild steel (MS) against corrosion in 0.5 M HCl solution, 2-amino-4-methoxy-6-methyl-1,3,5-triazine was used. The effectiveness of the compound as a corrosion inhibitor was studied via electrochemical, surface and theoretical calculation techniques.Design/methodology/approachFor concentrations ranging from 0.5 to 10.0 mM, almost similar polarization resistances were obtained from electrochemical impedance spectroscopy (EIS) and linear polarization resistance tests. It also investigated inhibitive activity of 2-amino-4-methoxy-6-methyl-1,3,5-triazine on the steel surface using scanning electron and atomic force microscope instruments. Langmuir adsorption is the best matched isotherm for the adsorption of the inhibitor to the steel surface.FindingsEIS method was used to determine inhibition efficiency, which was determined to be 95.7% for 10.0 mM inhibitor containing acid solution. Density functional theory’s predictions for quantum chemistry agreed well with the other experimental results.Originality/valueThe methods used in this study are effective and applicable; the used organic inhibitor is 2-amino-4-methoxy-6-methyl-1,3,5-triazine; and protective effectiveness is important, which is crucial for the task of MS corrosion prevention.

#6669 ANALYSIS OF KIDNEY BIOPSIES AND URINE SEDIMENT USING SCANNING ELECTRON MICROSCOPY (SEM) AND ATOMIC FORCE MICROSCOPY (AFM)

Abstract Background and Aims The classification of renal diseases is dependent on the evaluation of kidney biopsies using light microscopy techniques. Ultrastructural details (e.g. podocyte foot processes, amyloid fibrils, Fabri's intracellular inclusions) need transmission electron microscopy (TEM) which has high costs, requires specialized personnel and is time consuming. Therefore, alternative techniques are highly desirable. Here we report our experience with scanning electron microscopy (SEM) and atomic force microscopy (AFM). Method Paraffin-embedded kidney sections (3μm thick) were examined after dewaxing and Jones-methenamine methane staining for type IV collagen fibers. Dried urine sediments were prepared using standard methods for urine cytological analysis. Analysis was then performed using atomic force microscopy (nGauge system) on dried specimens and with Scanning Electron Microscopy (Zeiss) after gold-spattering of the surface. Reference images were obtained by standard microscopy. Results The combination of SEM and thin paraffin sections allows the observation of ultrastructural details in kidney biopsies with a very simple and fast preparation. AFM is even faster than SEM, as it does not require any preparation of the surface of the sample. An important advantage of SEM is the possibility to view the sample at low magnification, which makes it easier to identify the location of the glomeruli. It is thus possible to switch from small magnifications (100x) to very extreme magnifications (10000x). Notably, the interpretation of the images requires knowledge of the artifacts induced by paraffin inclusion. Specifically, structures and cells containing lipids (for example foamy cells) appear empty. Chromatin is also condensed in the nuclei around the periphery of the nucleus and in spike structures whit large empty areas. The recognition of nuclei in SEM and AFM is important for the subsequent interpretation of the images. Podocyte pedicles fusion can be identified relatively easily in SEM and in AFM. Both techniques add relevant ultrastructural details of urine sediment cells, from epithelial cells to isolated tubular cells. Also, casts show many ultrastructural features which are not evident in classical optical microscopy. It is also possible to measure the thickness of the basement membrane with great precision both in SEM and with AFM. The major limitation of this approach is the limited experience of pathologists with these methods and the absence of an atlas of normal and pathological kidney. Conclusion Considering the affordable costs of SEM and AFM instruments and the simple preparation of samples for their analysis, these should be considered a valid alternative to TEM. SEM and AFM are alternative methods, simple and relatively cheap and very fast to obtain ultrastructural details of renal biopsies. We thank ANED and AST for the great support given to research.

A cantilever-based, ultrahigh-vacuum, low-temperature scanning probe instrument for multidimensional scanning force microscopy.

Cantilever-based atomic force microscopy (AFM) performed under ambient conditions has become an important tool to characterize new material systems as well as devices. Current instruments permit robust scanning over large areas, atomic-scale lateral resolution, and the characterization of various sample properties using multifrequency and multimodal AFM operation modes. Research of new quantum materials and devices, however, often requires low temperatures and ultrahigh vacuum (UHV) conditions and, more specifically, AFM instrumentation providing atomic resolution. For this, AFM instrumentation based on a tuning fork force sensor became increasingly popular. In comparison to microfabricated cantilevers, the more macroscopic tuning forks, however, lack sensitivity, which limits the measurement bandwidth. Moreover, multimodal and multifrequency techniques, such as those available in cantilever-based AFM carried out under ambient conditions, are challenging to implement. In this article, we describe a cantilever-based low-temperature UHV AFM setup that allows one to transfer the versatile AFM techniques developed for ambient conditions to UHV and low-temperature conditions. We demonstrate that such a cantilever-based AFM offers experimental flexibility by permitting multimodal or multifrequency operations with superior force derivative sensitivities and bandwidths. Our instrument has a sub-picometer gap stability and can simultaneously map not only vertical and lateral forces with atomic-scale resolution, but also perform rapid overview scans with the tip kept at larger tip–sample distances for robust imaging.

Very-high-frequency probes for atomic force microscopy with silicon optomechanics

Atomic force microscopy (AFM) has been consistently supporting nanosciences and nanotechnologies for over 30 years and is used in many fields from condensed matter physics to biology. It enables the measurement of very weak forces at the nanoscale, thus elucidating the interactions at play in fundamental processes. Here, we leverage the combined benefits of micro/nanoelectromechanical systems and cavity optomechanics to fabricate a sensor for dynamic mode AFM at a frequency above 100 MHz. This frequency is two decades above the fastest commercial AFM probes, suggesting an opportunity for measuring forces at timescales unexplored thus far. The fabrication is achieved using very-large-scale integration technologies derived from photonic silicon circuits. The probe’s optomechanical ring cavity is coupled to a 1.55 μm laser light and features a 130 MHz mechanical resonance mode with a quality factor of 900 in air. A limit of detection in the displacement of 3 × 10−16 m/√Hz is obtained, enabling the detection of the Brownian motion of the probe and paving the way for force sensing experiments in the dynamic mode with a working vibration amplitude in the picometer range. When inserted in a custom AFM instrument embodiment, this optomechanical sensor demonstrates the capacity to perform force-distance measurements and to maintain a constant interaction strength between the tip and sample, an essential requirement for AFM applications. Experiments indeed show a stable closed-loop operation with a setpoint of 4 nN/nm for an unprecedented subpicometer vibration amplitude, where the tip–sample interaction is mediated by a stretched water meniscus.

AFM nanoindentation-based mechanical investigation of 3D confinement effects on nanoarchitectonic arrays of Ag nanodisks

Ag nanostructures have been widely used in the development of emergent micro electro-mechanical systems (MEMS) and nano electro-mechanical systems (NEMS). Dimensional confinement effects on their mechanical properties are a key issue in the field of MEMS/NEMS for understanding their mechanical performances and potential lifespans. In this work, we report a systematic study of 3D confinement effects on the mechanical resistance of Ag nanodisks fabricated by interference laser lithography (ILL). Nanodisks with thicknesses (t) between 20 and 150 nm and radii (r) between 125 and 900 nm have been studied. Mechanical properties have been studied by atomic force microscopy (AFM)-assisted nanoindentation. The results showed a strong influence of substrate effects on the mechanical response of the nanodisks, generally being dominant at the considered scales. Lateral confinement effects have been observed for low indentation depths (<0.1 t), at which the substrate effects become smaller. Confinement effects depend more on relative size effects (r/t ratios) than on absolute length scales. The nanodisks showed greater susceptibility to plastic deformation (lower mechanical resistance) as r decreased; this became clearly appreciable when r was comparable to t. Complementary finite element analysis showed similar tendencies, which could be explained considering pure dimensional confinement effects. This study shows the potential capabilities of modern AFM instruments for understanding confinement effects on the mechanical behavior of solids.

The improvement of CoZrTaB thin films on different substrates for flexible device applications

In this paper, the CoZrTaB thin films are fabricated using sputtering on different substrates and characterized comprehensively. The CoZrTaB thin films have been fabricated in the single-layer and multi-layer structures with SiO2 as the insulator layer, of which the vibrating sample magnetometer results suggest that the saturation field and the coercivity change of the multi-layer structure can be minimized while the lamination structure can suppress the eddy current at high working frequency. Compared with the continuously sputtered 400 nm CoZrTaB thin film, the multi-layer thin film shows a hysteresis loop with a single domain and small coercivity. The surface roughness of each CoZrTaB thin film and two kinds of polyimide substrates is extracted by the atomic force microscope instrument. Besides, material characterization such as X-ray powder diffraction and Rutherford backscattering spectrometry have been conducted to acquire the information of phase and element ratio of the CoZrTaB thin film. Also, the different process conditions that could affect the magnetic properties are investigated and verified. A comparison is made with the previously reported results, sustaining the improvement of the CoZrTaB thin film on different substrates.

Aberration corrected STEM techniques to investigate polarization in ferroelectric domain walls and vortices

Ferroelectric domain wall (DW) based nano-electronics is an emerging new field of research. It is only recently with advancements in electron and atomic force microscopy instrumentation that the complex nature of these 2D entities can be probed. In this Research Update, the advances in aberration corrected scanning transmission electron microscopy applied to ferroelectric topological defects are summarized. We discuss sub-atomic imaging and diffraction techniques used to observe changes in polarization, chemical composition, charge density, and strain at DWs and vortices. We further highlight the current achievements in mapping the 3D nature of ferroelectric polar skyrmions and in situ biasing. This Review will focus on both the fundamental physics of DW and polar vortex formation and their dynamics. Finally, we discuss how electron spectroscopy can be used to relate the quantified structural distortions of polar topological entities to changes in their oxidation state and band structure.

Effects of 3-D Confinement on the Mechanical Behavior of Ag Nanodisks: Novel Insights into Nanomaterials Mechanics

Ag nanostructures have been widely used for the development of emergent Micro Electro-Mechanical Systems (MEMS) and Nano Electro-Mechanical Systems (NEMS). The dimensional confinement effects on the mechanical properties are a key issue in the MEMS/NEMS area for understanding their mechanical performance and potential lifespan. In this work, we report a systematic study about the 3-D confinement effects on the mechanical resistance of Ag nanodisks fabricated by Interference Laser Lithography (ILL). Nanodisks with thicknesses (t) between 20 and 150 [nm], and radiuses (r) between 125 and 900[nm], were studied. Mechanical properties were studied by Atomic Force Microscopy (AFM)-assisted nanoindentation. Results showed a strong influence of substrate effects on the nanodisks mechanical response, being generally dominant at the considered scales. Lateral confinement effects were observed for low indentations depths (<0.1 t), where the substrate effects become smaller. Confinement effects depended more on relative size effects (r/t ratios) than absolute length scales. The nanodisks presented more susceptibility to plastic deformation (lower mechanical resistance) as r decreased; being clearly appreciable when r becomes comparable with t. Complementary finite element analysis showed similar tendencies, which could be explained considering pure dimensional confinement effects. This study shows the potential capabilities of modern AFM instruments for understanding confinement effects on the solids’ mechanical behavior.

Atomic Force Microscopy (AFM) As a Surface Mapping Tool in Microorganisms Resistant Toward Antimicrobials: A Mini-Review.

The worldwide emergence of antimicrobial resistance (AMR) in pathogenic microorganisms, including bacteria and viruses due to a plethora of reasons, such as genetic mutation and indiscriminate use of antimicrobials, is a major challenge faced by the healthcare sector today. One of the issues at hand is to effectively screen and isolate resistant strains from sensitive ones. Utilizing the distinct nanomechanical properties (e.g., elasticity, intracellular turgor pressure, and Young’s modulus) of microbes can be an intriguing way to achieve this; while atomic force microscopy (AFM), with or without modification of the tips, presents an effective way to investigate such biophysical properties of microbial surfaces or an entire microbial cell. Additionally, advanced AFM instruments, apart from being compatible with aqueous environments—as often is the case for biological samples—can measure the adhesive forces acting between AFM tips/cantilevers (conjugated to bacterium/virion, substrates, and molecules) and target cells/surfaces to develop informative force-distance curves. Moreover, such force spectroscopies provide an idea of the nature of intercellular interactions (e.g., receptor-ligand) or propensity of microbes to aggregate into densely packed layers, that is, the formation of biofilms—a property of resistant strains (e.g., Staphylococcus aureus, Pseudomonas aeruginosa). This mini-review will revisit the use of single-cell force spectroscopy (SCFS) and single-molecule force spectroscopy (SMFS) that are emerging as powerful additions to the arsenal of researchers in the struggle against resistant microbes, identify their strengths and weakness and, finally, prioritize some future directions for research.

In situ AFM Observation of the Movements of Isolated Isotactic Poly(methyl methacrylate) Chains in a Precursor Film of an Oligo(methyl methacrylate) Droplet Spreading on Mica.

Atomic force microscopy (AFM) is a powerful tool to observe polymer chains at the molecular level. In this study, we show that the movements of isolated linear polymer chains in a precursor film of a droplet of an oligomer spreading on a substrate could be visualized in situ at the molecular level by AFM for the first time. The system was an isotactic poly(methyl methacrylate) (it-PMMA) solubilized in an oligo(MMA) matrix (it-PMMA/oligo(MMA) = 1/10,000 w/w) spreading on mica under high humidity. Because of the limited resolution of the AFM instrument, condensed linear polymer chains could not be visualized, but a small amount of it-PMMA chains that were solubilized as isolated chains in the oligo(MMA) matrix could be visualized in the precursor film, the contrast of which came from a large difference in glass transition temperature (Tg) of it-PMMA and oligo(MMA). The it-PMMA chains in the precursor film spread in the radial direction of the droplet with vigorously changing chain conformations. The spreading rate of it-PMMA chains under 72% relative humidity was ∼1/30 of the spreading rate of the oligo(MMA) matrix, which was estimated based on the decrease in the volume of the macroscopic droplet. The spreading of the it-PMMA chains and droplet strongly depended on humidity and was suppressed with the decrease in humidity, most likely because of the increase in friction with the substrate. The difference in the spreading rate of it-PMMA and oligo(MMA) further increased under low humidity. The dynamic molecular information of a precursor film by AFM should help to elucidate the wetting dynamics on a substrate.

Origin of Ubiquitous Stripes at the Graphite-Water Interface.

The investigation of solid-liquid interfaces is pivotal for understanding processes like wetting, corrosion, and mineral dissolution and growth. The graphite-water interface constitutes a prime example for studying the water structure at a seemingly hydrophobic surface. Surprisingly, in a large number of atomic force microscopy (AFM) experiments, well-ordered stripes have been observed at the graphite-water interface. Although many groups have reported on the observation of stripes at this interface, fundamental properties and, in particular, the origin of the stripes are still under debate. Proposed origins include contamination, interplanar stacking of graphene layers, formation of methanol-water nanostructures, and adsorption of nitrogen molecules. Especially, the latter interpretation has received considerable attention because of its potential impact on explaining the long-range nature of the hydrophobic interaction. In this study, we demonstrate that these stripes readily form when using standard plastic syringes to insert the water into the AFM instrument. In contrast, when clean glass syringes are used instead, no such stripes form even though nitrogen was present. We, therefore, conclude that contaminations from the plastic syringe rather than nitrogen constitute the origin of the stripes we observe. We provide high-resolution AFM data that reveal detailed structural insights into the arrangement of the stripes. The rich variability of our data suggests that the stripes might be composed of several different chemical species. Still, we cannot rule out that the stripes observed in the literature might originate from other sources; our study offers a rather straightforward explanation for the origin of the stripes. In the view of these results, we propose to carefully reconsider former assignments.

Instrument-Dependent Factors Affecting the Precision in the Atomic Force Microscopy Stiffness Measurement of Nanoscale Liposomes.

The mechanical strength (stiffness) of liposomes affects their cellular uptake efficiency and drug release in drug delivery processes. We recently developed a tip shape evaluation method for improving the precision of liposome stiffness measurement by quantitative imaging (QI)-mode atomic force microscopy (AFM). The present study applied our method to the widely-used AFM instruments equipped for intermittent contact (IC)-mode force curve measurements, and examined instrument-dependent factors that affect the liposome stiffness measurements. We demonstrated that the evaluation of the tip shape for cantilever selection can be applicable to the IC mode as well as the QI mode. With the cantilever selection, the improved precision of the liposome stiffness was obtained when the stiffness of each liposome was determined from the slope in the force-deformation curve by the IC-mode force curve measurement. Further, the stiffness values were found to be similar to that measured by QI-mode measurements. These results indicate that our developed method can be widely used via IC-mode force curve measurements as well as via QI mode. It was also revealed that spatial drift of the cantilever position was instrument-dependent factors which could affect the precision of liposome stiffness measurements in the case of IC-mode force curve measurement. Therefore, in case of stiffness measurement by IC-mode force curve measurement, it is vital to obtain force-deformation curves immediately after imaging a liposome for the precise stiffness measurement of liposomes. These findings will promote the usage of the AFM stiffness measurement method for the characterization of lipid nanoparticle-based drug delivery systems.

Effects of different polyaniline emeraldine compositions in electrodepositing ginsenoside encapsulated poly(lactic-co-glycolic acid) microcapsules coating: Physicochemical characterization and in vitro evaluation.

Even though drug-eluting stent (DES) has prominently reduced restenosis, however, its complication of delayed endothelialization has caused chronic side effect. A coating of ginseng-based biodegradable polymer could address this issue due to its specific therapeutic values. However, deposition of this type of stable coating on metallic implant often scarce. Therefore, in this study, different polyaniline (PANI) emeraldine compositions were adopted to electrodeposit ginsenoside encapsulated poly(lactic-co-glycolic acid) microcapsules coating. The coating surfaces were analyzed using attenuated total reflectance-Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, contact angle, and atomic force microscopy instruments. A month coating stability was then investigated with an evaluation of in vitro human umbilical vein endothelial cell analyses consisted of cytotoxicity and cells attachment assessments. The 1.5 mg PANI emeraldine has assisted the formation of stable, uniform, and rounded microcapsules coating with appropriate wettability and roughness. Less than 1.5 mg PANI emeraldine was not enough to drive the formation of microcapsules coating while greater than 1.5 mg caused the deposition of melted microcapsules. The similar coating also has promoted greater cells proliferation and attachment compared to other coating variation. Therefore, the utilization of electrodeposition to deposit a drug-based polymer coating could be implemented to develop DES, in accordance to stent implantation which ultimately aims for enrich endothelialization.

A novel approach to study the interactions between polymeric stabilized micron-sized oil droplets by optical tweezers

Abstract The well understanding of interaction forces between single dispersed droplets is crucial to the understanding of emulsion stabilization mechanism. Recently, many studies have reported the direct quantitative measurements of interaction forces between 20-200 μm single droplet coated polymers by atomic force microscope (AFM). These studies have revealed many important results about the relationship of the interaction forces and the droplet deformation. However, these studies of the quantitative relationship between the measured interaction forces and the separation distance of the front end of the droplet have rarely been reported. Optical tweezer instrument can make it possible to establish the quantitative relationship between the measured force and the separation distance of the front end of the droplet, which will make better understanding of the interaction mechanisms between droplets. Due to the differences of the measuring mechanism between atomic force microscopy (AFM) and optical tweezers, the theory model of AFM measurements cannot be fitted with the force measurement by optical tweezers. We have made an exhaustive comparison of the measuring differences between AFM and optical tweezer instrument in this work. Moreover, we built a numerical model to derive the repulsive pressure through the measured force curve in order to quantify the measured force of two micron-sized oil droplet coated polymers by optical tweezers. Furthermore, the novel method can be extended to other micron-sized emulsion systems, and these findings will be a vital progress on quantitative force measurements between micron-sized droplets.

Scanning Probe-Directed Assembly and Rapid Chemical Writing Using Nanoscopic Flow of Phospholipids.

Nanofluidic systems offer a huge potential for discovery of new molecular transport and chemical phenomena that can be employed for future technologies. Herein, we report on the transport behavior of surface-reactive compounds in a nanometer-scale flow of phospholipids from a scanning probe. We have investigated microscopic deposit formation on polycrystalline gold by lithographic printing and writing of 1,2-dioleoyl-sn-glycero-3-phosphocholine and eicosanethiol mixtures, with the latter compound being a model case for self-assembled monolayers (SAMs). By analyzing the ink transport rates, we found that the transfer of thiols was fully controlled by the fluid lipid matrix allowing to achieve a certain jetting regime, i.e., transport rates previously not reported in dip-pen nanolithography (DPN) studies on surface-reactive, SAM-forming molecules. Such a transport behavior deviated significantly from the so-called molecular diffusion models, and it was most obvious at the high writing speeds, close to 100 μm s-1. Moreover, the combined data from imaging ellipsometry, scanning electron microscopy, atomic force microscopy (AFM), and spectroscopy revealed a rapid and efficient ink phase separation occurring in the AFM tip-gold contact zone. The force curve analysis indicated formation of a mixed ink meniscus behaving as a self-organizing liquid. Based on our data, it has to be considered as one of the co-acting mechanisms driving the surface reactions and self-assembly under such highly nonequilibrium, crowded environment conditions. The results of the present study significantly extend the capabilities of DPN using standard AFM instrumentation: in the writing regime, the patterning speed was already comparable to that achievable by using electron beam systems. We demonstrate that lipid flow-controlled chemical patterning process is directly applicable for rapid prototyping of solid-state devices having mesoscopic features as well as for biomolecular architectures.

Metasurface-enhanced optical lever sensitivity for atomic force microscopy

We proposed the integration of metasurfaces on atomic force microscopy (AFM) cantilevers to improve the optical lever sensitivity. The metasurface positioned at the back of an AFM cantilever enables anomalous reflection of the laser beam and provides a nonlinear relationship between the incidence and reflection angles following the generalized Snell’s law. Therefore, the displacement of the reflected laser spot at the photodetector can be amplified. The metasurface was composed of 30 nm thick Au nano-discs with different diameters in an array of 1500 nm × 300 nm super cells. Using the fabricated metasurface mounted on a precise angle dial and a macroscale cantilever as prototypes, the concept of a metasurface-enhanced cantilever was experimentally ascertained. Results proved that the optical lever sensitivity can be easily increased. Finite element analysis showed that integration of the thin metasurface does not have a significant impact on the cantilever’s mechanical properties including stiffness and eigenfrequencies. The proposed metasurface-enhanced optical lever sensitivity may have potential applications in improving functional performances of AFM instruments and cantilever-based sensors, such as allowing much smaller imaging forces and boosting the signal-to-noise ratio.

Planar AFM macro-probes to study the biomechanical properties of large cells and 3D cell spheroids

The ability to measure mechanical response of cells under applied load is essential for developing more accurate models of cell mechanics and mechanotransduction. Living cells have been mechanically investigated by several approaches. Among them, atomic force microscopy (AFM) is widely used thanks to its high versatility and sensitivity. In the case of large cells or 3D multicellular aggregates, standard AFM probes may not be appropriate to investigate the mechanical properties of the whole biological system. Owing to their size, standard AFM probes can compress only a single somatic cell or part of it. To fill this gap, we have designed and fabricated planar AFM macro-probes compatible with commercial AFM instruments. The probes are constituted of a large flat compression plate, connected to the chip by two flexible arms, whose mechanical characteristics are tuned for specific biological applications. As proof of concept, we have used the macro-probes to measure the viscoelasticity of large spherical biological systems, which have a diameter above 100 μm: human oocytes and 3D cell spheroids. Compression experiments are combined with visual inspection, using a side-view configuration imaging, which allows us to monitor the sample morphology during the compression and to correlate it with the viscoelastic parameters. Our measurements provide a quantitative estimate of the relaxation times of such biological systems, which are discussed in relation to data present in literature. The broad applicability of the AFM macro-probes can be relevant to study the biomechanical features in any biological process involving large soft materials. Statement of SignificanceThe understanding of the role of physical factors in defining cell and tissue functions requires to develop new methods or improve the existing ones to accurately measure the biomechanical properties. AFM is a sensitive and versatile tool to measure the mechanical features from single proteins to single cells. When cells or cell aggregates exceed few tens of microns, AFM suffers from limitations. On these biological systems the control of the contact area and the application of a precise uniform compression becomes crucial. A modification of the standard cantilevers fabrication allowed us obtaining AFM macro-probes, having large planar contact area and spring constant suitable for biological investigations. They were demonstrated valuable to characterize the mechanical properties of large hierarchical biological systems.

Atomic force microscope integrated with a scanning electron microscope for correlative nanofabrication and microscopy

More than 40 years after its invention, the atomic force microscopy (AFM) can be integrated with scanning electron microscope (SEM) instruments as an increasingly capable and productive characterization tool with sub-nanometer spatial resolution. The authors have designed and developed an AFM instrument capable to be integrated into any SEM or in a combination of SEM with a focused ion-beam (FIB) tool. The combination of two or more different types of techniques like SEM, energy dispersive x-ray spectroscopy, and AFM is called correlative microscopy because analytical information from the same place of the sample can be obtained and correlated. For the first time, they introduced to the SEM/FIB tool correlative nanofabrication methods like field-emission scanning probe lithography, tip-based electron beam induced deposition, and nanomachining. The combination of all these methods provides a completely new nanotechnology instrument, which should be seen as a tool for correlative nanofabrication and microscopy. Thus, it provides for the first time the capabilities of a stand-alone instrument with the capabilities of nondestructive three-dimensional tip-based metrology and nanofabrication into the combined SEM/FIB tool. In this article, the authors describe all these methods in detail and present a brief example of the results obtained. They demonstrate that the self-sensing, self-actuating cantilevers (called active cantilevers) equipped with Diamond tip are a versatile toolkit for fast imaging and emerging nanofabrication. The AFM integrated into SEM is using active cantilevers that can characterize and generate nanostructures all in situ without the need to break-vacuum or contaminate the sample.