Symposium CH03—Frontiers in Scanning Probe Microscopy—Beyond Imaging of Soft Materials

Several technological and natural processes are dominated by the mineral-water interactions, for example biominerelisation, corrosion etc, and hence, characterization of such interfaces is vital. Atomic Force Microscopy (AFM) has gained prominence in characterizing such surfaces.
However, the complex interplay of the tip with the hydration layers over the surface govern the AFM tip’s interaction with the hydration layers, and impede high resolution requirements needed for characterization. The theoretical molecular dynamics (MD) approaches have helped with the surface characterization. Given a hydration layer image, the search space is wide and the MD approach becomes prohibitively expensive. Here we introduce deep learning methods to reliably predict the hydration layer over a given surface.

Voltage modulation (VM) of atomic force microscopy (AFM) has been used to quantify a host of electronic, electrochemical and electromechanical functionalities across nanometer length scales. The critical importance of such information has resulted in the development of a plethora of VM-AFM techniques for exploration of either long or short-range forces. Of relevance for ferroelectrics, piezoresponse force microscopy (PFM) imaging and associated spectroscopies have effectively opened the door to the exploration of nanoscale ferroelectric properties. The rise of PFM, however, has also brought about claims of ferroelectricity in materials which were subsequently thought to be not ferroelectric,[1] even unlikely materials such as soda-lime glass. Explanations for the origins of these unexpected nanoscale phenomena have not been in short supply, including new material properties, surface-mediated polarization changes and/or spatially resolved behavior that is not present in bulk measurements. At the same time, it is well known that VM-AFM measurements are susceptible to numerous forms of crosstalk and despite efforts within the AFM community, a global approach for quantitative, crosstalk-free techniques remains elusive. In an effort to understand the true origins of the measured VM-AFM signals we demonstrate the presence of hysteretic (“false ferroelectric”) long-range interactions between the sample and cantilever body and show that these are intrinsic to traditional VM-AFM detection methods. However, we show that with interferometric displacement sensor (IDS) [2] it is possible to separate the true tip motion from the cantilever dynamics. Using the IDS we have established a rapid and simple flagging routine of false piezo and ferroelectric responses and are able to demonstrate fully quantitative and repeatable nanoelectromechanical characterization. These quantitative measurements are critical for a wide range of new devices ranging from mems actuators, memristor devices, energy storage and dynamic computer memory. While our initial work was focused on electromechanical measurements, we have extended this microscope to improved quantification of force-based mechanical measurements. Quantifying the force and displacement with this metrological AFM has enabled improved quantitative and high speed modulus mapping in a variety of environments. [3, 4]

References
[1] Vasudevan, Rama K., et al. "Ferroelectric or non-ferroelectric: Why so many materials exhibit “ferroelectricity” on the nanoscale." Applied Physics Reviews 4.2 (2017): 021302.
[2] Labuda, Aleksander, and Roger Proksch. "Quantitative measurements of electromechanical response with a combined optical beam and interferometric atomic force microscope." Applied Physics Letters 106.25 (2015): 253103.
[3] M. Kocun et al., “Fast, High Resolution, and Wide Modulus Range Nanomechanical Mapping with Bimodal Tapping Mode”, ACS Nano 11 10097 (2017).
[4] V. G. Gisbert et al., “High-Speed Nanomechanical Mapping of the Early Stages of Collagen Growth by Bimodal Force Microscopy” ACS Nano, 15 1850 (2021).

With the continued development of scanning probe microscopy techniques, manipulation of single molecules has become possible. Thereby, the manipulation path can be chosen at will and the energy landscape around the molecule can be quantified before and after manipulation. This not only allows to extract the energy barriers between potential minima along the manipulation pathway but may more generally provide unprecedented insight into the chemical interaction of individual molecules with their surroundings. To further explore the approach’s potential, we conducted controlled manipulation experiments with benzene on a Cu (100) surface as a model system using a novel manipulation protocol that despite not requiring any active feedback during execution provides the normal and lateral forces at the instance of the jump with piconewton accuracy, which leads to very robust operation. The results indicate that the molecules were either pushed, pulled, jumped to the tip, or did not move depending on the chemical surroundings of the molecules and the chemical identity of the tip. We then compared the experimentally obtained energy landscapes and barrier heights with computational results obtained by combining density functional theory with machine learning approaches. The Bayesian Optimization Structure Search (BOSS) protocol suggests that the presence of the tip lowers the energy barrier the molecule has to overcome during its motion, which evolves with the chemical identity of the tip. In addition, phenyl groups were produced from iodobenzene precursors, from which the iodine atom was split off by applying a controlled bias voltage. We then manipulated the phenyl group away from the iodine atom and measured the required energy barrier, which was found to decrease with increasing distance from the iodine atom. Calculations indicate that there are different ways the molecules can move that have different barriers, so the finding of different barrier heights may simply reflect different processes. Overall, our results indicate that a reliable methodology where surface reactions can be induced by the tip of a scanning probe microscope and the reaction pathway is chosen at will could be in reach, which would ultimately enable an individualized, site-specific understanding of single-molecule surface chemistry.

More than 10 years ago two types of Scanning Probe Microscopy (SPM) – Scanning Tunneling Microscopy (STM) [1] and non-contact Atomic Force Microscopy (nc-AFM) [2] – showed a possibility to reveal positions of atoms on small flat organic molecules in real space without any damage to the sample. These two works noticeably enhanced the field of molecular, 1 and 2D material studies in surface science (i.e. [3]). The sub-molecular resolution was achieved by usage of non-reactive and flexible tip-apices like CO-molecule [4]. Kelvin Probe Force Microscopy (KPFM)[5] is a technique proceeded with combined nc-AFM/STM instrument, which basically measures the effect of applied voltage between the microscope tip and sample on the measured force. It has possibility to measure the changes of work-function on the microscale and electric field above the sample on the nanoscale. However, the physics behind KPFM signal with flexible-tip apices showing the sub-molecular resolution, which could help with elemental recognition, remain unknown.
In this work, we will summarize up-to-date knowledge about KPFM [5,6] focusing mainly on measurements with simultaneous frequency modulation - Atomic Force Microscopy / Scanning Tunneling Microcopy instruments. Based upon this, we will present a new model for electrostatic field, which is describing the experiments with CO-metal tips [7] and metal substrates. This new electrostatic model is applied in a Density Functional Theory (DFT) calculations simulating the full tip-sample system. The results of the DFT calculations will be compared with simple mechanistic models capturing various sources of achieved signal. All these models and simulations have aim to recover the physics behind KPFM with flexible tip apices. This knowledge can help to get more information about the sample with SPM and is possibly a further step towards reliable chemical resolution in SPM.
References:
[1] R. Temirov, S. Soubatch, O. Neucheva, A. C. Lassise and F. S. Tautz, New J. Phys. 10, 053012 (2008).
[2] L, Gross, F, Mohn, N, Moll, P, Liljeroth and G. Meyer., Science 325, 1110 (2009).
[3] A. Mistry, B. Moreton, B. Schuler, F. Mohn, G. Meyer, L. Gross, A. Williams, P. Scott, G. Costantini and D. J. Fox, Chem. Eur. J. 21, 2011-2018 (2015).
[4] P. Hapala, G. Kichin, C. Wagner, F. S. Tautz, R. Temirov, and P. Jelínek, Phys. Rev. B 90, 085421 (2014).
[5] M. Nonnenmacher, M. P. O’Boyle, and H. K. Wickramasinghe, Appl. Phys. Lett. 58, 2921 (1991).
[6] L. Gross, B. Schuler, F. Mohn, N. Moll, N. Pavliček, W. Steurer, I. Scivetti, K. Kotsis, M. Persson, and G. Meye, Phys. Rev. B 90, 155455 (2014).
[7] F. Schulz, J. Ritala, O. Krejci, A. P. Seitsonen, A. S. Foster, and P. Liljeroth, ACS Nano 12, 5274−5283 (2018).

The flow of electrical current through a material can, in special circumstances, mimic the flow of a viscous fluid. This so-called ‘hydrodynamic’ regime occurs when the rate of momentum-conserving collisions between charge carriers is larger than the momentum-relaxing rate due to other scattering sources. Until very recently this condition has been difficult to satisfy in real materials, hampering experimental studies of electronic transport in this unique regime. Ultraclean graphene has emerged as an ideal platform for studying hydrodynamic flow, opening up the possibility of directly imaging fluid-like behavior such as vortex formation. In this talk, I will show scanning tunneling potentiometry (STP) measurements of quasiparticle flow around tunable electrostatic barriers in graphene on a hexagonal boron nitride substrate. Measurements are performed as carriers pass through μm-scale channels that are continuously narrowed until they are blocked entirely, forming ‘electrostatic dams’. Local STP measurements of the electrochemical potential allow for direct characterization of the evolving flow profile near the channels, which is observed to be non-Ohmic, and highly dependent on the sample temperature and the channel width. Under all flow conditions, we observe the formation of Landauer resistivity dipoles across the barriers due to scattering. However, at low temperatures (4.5 K) the channel conductance is observed to be consistent with ballistic transport, while at elevated temperatures (77 K) the conductance is observed to increase. We attribute this ‘superballistic’ transport to a viscous enhancement of the conductance originally predicted by Gurzhi, a hallmark of hydrodynamic flow. We further use these measurements to extract the electron-electron scattering length at a range of carrier densities. We compare our measurements to finite-element simulations of a Stokesian fluid with different parameters, finding good agreement at 77 K for a fluid with a kinematic viscosity of 0.7 m²/s. Our results demonstrate the incredible utility of STP in distinguishing between different electronic transport regimes, and quantifying the interaction length scales that lead to a distinct flow profiles.

We studied by non-contact AFM (nc-AFM) the formation of molecular self-assemblies on the pas-sivated surface of boron doped silicon B-Si(111)-(√3×√3)R30^○. The investigated molecules (1-(4’cyanophenyl)-2,5-bis(decyloxy)-4-(4’-iodophenyl)benzene) possesses aliphatic chains attached to a triphenyl core ended with two possible different terminations (either iodine or cyano group). The use of a passivated semiconductor substrate enables creating regular and extended structures without significant change in electronic properties of molecules [1]. Scanning tunneling microscopy and nc-AFM imaging have been performed using a low-temperature (AFM/STM (JT AFM/STM,SPECS) operated at T=4K with high stiffness Kolibri sensors (k=540 kN/m, f₀=1 MHz). The growth of a periodic molecular network is observed, formed by parallel lines made by molecule aromatic cores and interdigitated aliphatic chains placed between adjacent rows. We obtain submolecular resolution in the constant height δf images without intentional tip functionalization, but only by conditioning the tip on the silicon surface [2]. We will discuss the high-resolution AFM imaging, as well as the conformation of the molecules in the observed assemblies (e.g. changes in the interdigitated aliphatic chains ordering). Kelvin Probe Force Microscopy (KPFM) images of single molecules and molecular assemblies with sub-elementary charge sensitivity [3] and submolecular resolution will be provided and discussed, in particular with respect to the choice of the molecular terminations. They reveal the dipolar character of asymmetric molecules and are consistent with the formation of dipole-driven molecular arrays, in the case of self-assemblies. Density functional theory calculations as well as molecular dynamics simulations will be compared with experimental results.
[1] Makoudi Y,Beyer M, Lamare S, Jeannoutot J, Palmino F, Chérioux F, Nanoscale 2016, 8, 12347.
[2] Berger J, Spadafora EJ, Mutombo P, Jelínek P, Švec M, Small, 2015, 113686-3693.
[3] Turek N, Godey S., Deresmes D, Mélin T, Phys. Rev. B 2020 102, 245433.

A new solution is offered to understand an instrumental artifact which is the anomalously high atomic amplitudes on graphite surface measured by Scanning Tunneling Microscopy (STM) using the local elastic deformation theory. This is demonstrated by a series of simple experiments done to measure atomic topography of various 2D surfaces like Niobium diselenide, monolayer graphene and graphite across their monatomic step heights to show how tip-sample interaction causing the anomalous atomic amplitudes is of purely mechanical nature and not electronic. The underlying physics of Van der Waal bonding playing a major role in these interactions with ‘soft’ 2D surfaces will be uncovered.

The nanoscale structures and dynamical processes of proteins have been extensively studied by various imaging techniques such as electron microscopy and atomic force microscopy (AFM) in liquid. However, these imaging techniques can damage or perturbate the samples and do not provide chemical identification. This prevents a direct monitoring of structural and chemical evolution under physiological conditions. Herein, we demonstrate a new nondestructive platform that enables nanoscale Infrared (IR) spectroscopy for protein at graphene-aqueous solution interface by combining graphene liquid cell and Fourier Transform Infrared Nanospectroscopy (nano-FTIR). Single layer graphene separating the assembly solution and the tip minimize the sample damage and tip contamination. The S-layer protein structural evolution during and after assembling process is monitored by recording the amide I and II vibration bands, which provides unique and complimentary information to the AFM morphology in liquid. Our platform opens up broad opportunities for operando study of soft materials or nanostructures (enzyme, other membrane protein, virus, and plastic material) in their realistic condition and under external stimuli.

Investigating nucleation from solutions is challenging, because it is a consequence of unstable density fluctuations, making the structures and events that must be probed both transient in nature and small in spatial extent. Thus high-speed, molecularly resolved AFM provides a powerful and unrivalled method to investigate nucleation from solution by choosing systems that nucleate onto surfaces. Here we illustrate the deep level of fundamental insight into the pathways and energetics of nucleation made possible by this method by using it to investigate epitaxial nucleation of gibbsite (Al(OH)₃) on muscovite mica (001). AFM results combined with streaming potential measurements reveal a surface population of ions that is dominated by hydrolyzed species (Al(OH)₂⁺ and Al(OH)²⁺) even though Al³⁺ vastly dominates the bulk solution. These adsorbed ions evolve into subcritical clusters with increasing saturation state and temperature, constituting a population whose size distribution and fluctuations exhibit many similarities to the predictions of classical nucleation theory (CNT). However, severe discrepancies with CNT emerge when the values of key thermodynamic parameters are extracted from the AFM data. Moreover, the resulting gibbsite film forms simultaneously across the surface, rather than through the appearance of rare critical-size nuclei. Using kinetic Monte-Carlo (KMC) simulations, we demonstrate how this behavior can arise from electrostatic interactions between positively charged aluminum clusters and the negatively charged mica surface. Our results indicate that surface charging can dynamically modify both the chemical potential for accumulating ions on the surface, and the edge tension of the clusters. As the film grows, these interactions stabilize a nanostructured surface mesophase, which evolves gradually via a process of cluster growth and coalescence and is not limited by the classical formation of a critically sized nucleus. Our results show that high speed AFM provides a direct molecular view of nucleation at surfaces that reveals both consistencies and deviations from the simple classical picture and highlights the inherent role of surface charge in determining pathways and outcomes of nucleation.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy enables a wide array of studies, from measuring the strength of a ligand-receptor bond to elucidating the complex unfolding pathway of individual membrane proteins. Such studies and, more generally, the diverse applications of AFM across biophysics and nanotechnology are improved by enhancing the force stability, force precision, and time resolution of bioAFM. For an advanced, small-format commercial AFM, we uncovered how these three metrics were limited by the cantilever itself rather than the larger microscope structure, and then describe three increasingly sophisticated cantilever modifications using a focused ion beam that yielded enhanced data quality. For example, our most recent cantilever design, a “Warhammer,” achieved a sub-pN force precision and stability over 5 decades of time (1 ms – 100 s) coupled with 8-µs time resolution in liquid when positioned just above a surface. High-precision single-molecule studies of membrane proteins—the target for over 50% of current and future drugs—have lagged analogous studies of globular proteins due to instrumental limitations. We revisited the unfolding of the canonical membrane protein bacteriorhodopsin with a 100-fold improvement in time resolution and a 10-fold improvement in force precision. The resulting data revealed the unfolding pathway in unprecedented detail. I will conclude by discussing extensions of this capability to other diverse biomolecules, including nucleic-acid structures and globular proteins involved in hereditary heart disease. The metrological enhancements enabled by modified AFM cantilevers are not limited to single-molecule force spectroscopy but are broadly applicable across diverse AFM studies. Importantly, these modified cantilevers are robust, reusable over multiple days and, in our lab, typically modified by undergraduates. Thus, they are neither too costly nor too challenging to modify for routine use.

Understanding the chemistry and dynamics of polymers at the nanoscale and under confinement is of utmost importance for high performance and emerging materials and composites. Scanning probe microscopy (SPM) techniques promise extremely high resolution and direct, sensitive measurement of polymer chemical and mechanical properties down to 1-10 nm. However, collection and interpretation of SPM data remains complicated due to the difficulty in interpreting measured properties at such fine scales. Correlative approaches using both mechanical and chemical SPM techniques, in conjunction with modelling of tip-surface interactions, can enable discovery even at the limits of SPM resolution and performance.

Here, we demonstrate the ability to directly measure the loss tangent of poly(methyl methacrylate) (PMMA) confined to 7 nm diameter pores in an organosilicate (OCS) matrix utilizing dynamic atomic force microscopy (AFM) and modelling of the tip-sample interactions. Further, we identify a decrease in the loss tangent of PMMA when confined to an OCS matrix treated to increase silanol density on the pore surfaces. At the nanoscale, X-ray photoelectron spectroscopy as well as other optical spectroscopy techniques demonstrate that the increased silanol density leads to an increase in hydrogen bonding between the carbonyl groups in the PMMA chains and the surface silanols. The increased bonding reduces chain mobility and consequentially decreases the observed loss tangent as the surface interaction strength increases. The correlative microscopy approach enables a direct link between the observed decrease in the loss tangent of PMMA as measured by AFM and the underlying change in polymer chemistry responsible.

The highly sensitive measurements and findings described above are enabled by a technique known as gas cluster ion beam (GCIB) etching. Previously, it has been challenging to prepare composite surfaces that consist of soft polymers and hard ceramic components that are sufficiently smooth to enable high resolution mechanical property measurements. To overcome these challenges a GCIB procedure is presented that allows for damage free removal of the PMMA and OCS to produce a surface with an R_a of approximately 0.3 nm. GCIB is a promising sample preparation method that has not been extensively used in the preparation of polymer composite samples for SPM. Expanded use of GCIB should enable a broader range of samples, particularly in-situ samples, to be probed with SPM techniques.

Electrostatic force microscopy (EFM) is a modification of atomic force microscopy (AFM) which allows for a quantitative determination of localized charges and dielectric constants at the nanometer scale. In our implementation of EFM, the tip is lifted off the surface and scanned at a constant height under an applied AC and DC voltage, thereby providing sensitivity to extremely small capacitive and coulombic forces. By modeling the surface charge-cantilever force through a coulombic interaction, quantitative determinations of surface charge magnitude and location are possible down to less than 0.1 e at room temperature. In this presentation we have used EFM to study the spatial distribution and magnitude of charged regions along individual single-walled carbon nanotubes (SWCNTs). For dozens of long NTs dispersed with ionic surfactant we found surprisingly large charge density variations all along the NT with magnitudes on the order of 1 e to 15 e per 25 nm², which is roughly the EFM spatial resolution. Also, the charges had both positive and negative signs. Acquiring EFM data from before and after photoexcitation suggests that the ionic surfactant actually creates local electrostatic perturbations that act as barriers for exciton transport, which leads to distinct localized regions of charge. Additionally, we have been able to correlate single molecule photoluminescence (PL) spectroscopy and AFM microscopy images of the same individual SWCNTs. We found a highly unexpected localization of PL at sites of significant surfactant coverage. From our EFM measurements, we know these sites routinely display greater static charge accumulation, which suggests a connection between the electrostatic and optical characteristics of SWCNTs. Current efforts in our laboratory aim to provide a more detailed comparison of the charge and emissive profiles of individual NTs by correlating single molecule spectroscopy with EFM.

Iron (Fe) is an essential metal involved in a wide spectrum of physiological functions in the body, including oxygen transport, enzymatic reactions and amino acid synthesis. Ferritin, the major iron storage protein in the mammalian body, consists of ~8 nm iron cores in the form of the iron-oxide crystal ferrihydrite. The size, distribution and oxidation state of ferritin(iron) has been shown to play an important role in health and disease. Histochemical staining, the routinely used approach to characterize iron distribution, offers limited insight into the chemical environment and composition of tissue iron. One of the properties of ferritin(iron) which has not been adequately exploited in histology is the magnetic behavior of tissue iron.
In this study we elucidate, how magnetic force microscopy (MFM), an atomic force microscopy (AFM)-based technique, can be used to map (magnetic) iron deposits in histological sections. MFM analysis was conducted on physiologically present ferritin(iron) deposits in naïve rodent spleen. To examine ferritin(iron) deposits in pathological tissues, an acutely injured rodent tissue (spinal cord) as well as tissues from chronic injury as present in brain tissue (hippocampus) from patients with Alzheimer’s disease (AD) were used. Perl’s histochemical staining was used to confirm the presence of ferric iron (Fe³⁺) and analytical transmission electron microscopy was used to verify ferritin(iron) deposits at the ultrastructural level. MFM images were analyzed to ascertain the size of magnetic deposits, the strength of the magnetic signal (phase), and fluctuations (roughness) of the MFM signal.
Our results show that while Perl’s histological staining is sensitive to the use of chemical fixatives, MFM analysis is not affected between fixed and unfixed tissues. Further, the size(s) of the magnetic deposits ascertained using MFM corresponded to that of ferritin(iron) rich lysosomal iron ascertained using electron microscopy. The size of these iron-rich deposits was significantly smaller in the diseased tissues (injured spinal cord and AD brain tissue) as compared to naïve spleen. Further naïve spleen exhibited significantly higher strength of the MFM signal when compared to injured spinal cord and brain tissue. Interestingly, the roughness of MFM signal was similar across all tissues. TEM analysis indicated that the density of lysosomal ferritin(iron) deposits, was the major factor accounting for variances in MFM phase differences in mice tissue. Thus, rich and physiologically relevant information can be obtained through MFM mapping of histological iron.
To enable a multimodal analysis of iron we further developed an indirect MFM technique where TEM, MFM and fluorescence microscopy could be performed on the very same sample. In indirect MFM the sample is physically separated from the MFM probe by an ultrathin membrane. Further studies along these directions can allow for a high throughput and multimodal analysis of the quantity and quality of iron in AD. This can be of special relevance in neurodegenerative diseases where a transition of ferrihydrite into magnetite/maghemite is reported as the two minerals differ in their magnetic properties. Finally, the high sensitivity and spatial resolution offered by MFM makes it an attractive technique for a label free mapping of iron deposits in histology.

1) Blissett, A. R., et. al. Sub-cellular In-situ Characterization of Ferritin(iron) in a Rodent Model of Spinal Cord Injury. Sci. Rep. (2018)
2) Sifford, J., et. al. Indirect magnetic force microscopy. Nanoscale Advances. (2019)
3) Walsh, K. J., et. al. Effects of fixatives on histomagnetic evaluation of iron in rodent spleen. JMMM. (2020)

Dynamic nanoscale information of cell structures and organelles plays a vital role in understanding cellular function. Nanocharacterization techniques, such as super-resolution optical microscopy, electron microscopy, and atomic force microscopy (AFM), have been applied to image the structure of cells in great detail. The major challenge is to obtain three-dimensional (3D) information at nanometer resolution without affecting the viability of the cells and avoiding interference with the process. Scanning probe microscopy methods such as AFM can generate high-resolution characterization of stiff cell surfaces, such as bacteria [1]. However, the mechanical contact between the tip and the sample deforms soft membranes, disturbing the native state of eukaryotic cells and prevents long time-lapse imaging. Here, we show a scanning ion conductance microscope (SICM) for high-speed and long-term nanoscale imaging of soft cells [2]. By implementing advances in nanopositioning, nanopore fabrication, microelectronics, and controls engineering we developed a microscopy method that can resolve spatiotemporally diverse 3D processes on the cell membrane at sub-5nm axial resolution. Moreover, we combine 3D surface data from SICM with volumetric super-resolution optical fluctuation imaging (SOFI), to generate correlative high-resolution information of live cell morphology [3]. This true non-contact technique enables a detailed look at long-period membrane events with sub-second resolution and may offer insights into cell-cell interactions for infection, immunology, and cancer research.


1: Pascal Odermatt et al. Overlapping and essential roles for molecular and mechanical mechanisms in mycobacterial cell division. Nature Physics (2019). doi:10.1038/s41567-019-0679-1
2: Samuel M. Leitao et al. Time-resolved scanning ion conductance microscopy for three-dimensional tracking of nanoscale cell surface dynamics. bioRxiv (2021). doi:10.1101/2021.05.13.444009v1
3: Vytautas Navikas, Samuel M. Leitao et al. Correlative 3D imaging of single cells using super-resolution and scanning ion-conductance microscopy. bioRxiv (2020). doi:10.1101/2020.11.09.374157

Since its invention, atomic force microscopy (AFM) has become a widely used technique in many different research disciplines from physics, through materials science to life sciences. Instruments and measurement methods alike have been developed that allow for imaging surface structures and probing the mechanical and electrical properties of samples at high spatial resolution. Along with new methods, a major aim in recent AFM development is towards improved ease of use, reliability, and integration with complimentary techniques. Our newly developed DriveAFM addresses these topics by including several technological features that improve the ease of use and extend the capabilities of the instrument. It features a fully-motorized, tip-scanning design that allows for integration with optical microscopy, flexibility regarding sample size and weight, and the possibility for automated system operation from laser adjustment to sample translation. Most importantly, we implemented photothermal cantilever excitation in this tip-scanning AFM and used this feature in several new imaging and measurement methods.

Photothermal excitation directly excites the cantilever by locally heating it at its base, and thus allows stable and clean excitation in almost any environment [1]. Here, we will show the technical difficulties and concepts implemented to include photothermal excitation in a fully motorized tip scanning AFM employing a new optomechanical design [2] as well as measurement data enabled by this implementation. The clean and unambiguous photothermal excitation of the cantilever and its textbook-like response, especially liquids, allows new kinds of measurements, e.g. time-resolved measurement of mass changes [3] or probing the nanomechanical properties of cells over a wide range of frequencies [4]. The ability to effectively drive small, fast cantilevers with photothermal excitation also below their resonance frequency allows off-resonance imaging modes being operated at higher actuation frequencies compared to typically used piezo-based actuation schemes [5].

[1] A. P. Nievergelt et al., Beilstein J. Nanotech. (2014) 5: 2459–2467
[2] J.D. Adams. U.S. Patent 10,564,181B2 (2020)
[3] D. Martinez-Martin et al., Nature (2017) 550: 500–505
[4] G. Fläschner et al., Nat. Comm. (2021) 12:2922
[5] A. P. Nievergelt et al., Nat. Nanotech. (2018) 13: 696-701

Resolution and field-of-view present a challenging tradeoff in microscopy. Atomic force microscopy (AFM), in which a cantilevered probe deflects under the influence of local forces as it scans across a substrate, is a key example of this tradeoff with high resolution imaging being largely limited to small areas. Here, we show that nanoscale motion of cantilever-free probes can be imaged using conventional optical microscopy to enable massively parallel AFM. Specifically, we realize optically reflective rigid probes on a compliant film that comprises a distributed optical lever that reports local deformation. By observing the optical contrast on each probe in an array as it is raster scanned across a sample, we demonstrate parallel imaging of surface topography with 1,088 probes with 100 nm lateral resolution and sub 10-nm vertical precision across 0.5 mm. The scalability of this approach makes it well suited for imaging applications that require high resolution over large areas such as integrated circuit quality control, optical metasurface inspection, and multi-scale characterization of biological tissue.
[1] Cao, W.; Alsharif, N.; Huang, Z.; White, A. E.; Wang, Y.; Brown, K. A., Massively parallel cantilever-free atomic force microscopy. Nat. Commun. 2021, 12 (1), 393.

In practical applications Dynamic Atomic Force Microscopy (DAFM) a (highly) non-linear oscillator, since the oscillation amplitude is (much) larger than the typical length scale of tip-sample interactions. However, it is mostly modelled as a (multifrequency) harmonic oscillator. This very simple approximation works surprisingly well, also for cases where the DAFM system is clearly operated in a large oscillation regime, and therefore non-linearity should be important. In this work, we will discuss why this apparent contradiction is in fact possible. For this, a new scheme to interpret and unify linear and nonlinear interactions is proposed. Using the classical model for the driven damped harmonic oscillator, its response is described by means of a complex number. We explicitly calculate how the time dependence of the deflection is processed by a typical Lock-In setup to obtain a point in the complex plane representing the oscillation state.

Using the Virial Theorem and work of San Paulo et al. [1] as starting point, we show how a linear DSFM system can be easily interpreted in terms of “Circles”. Essentially, as the drive frequency is tuned through the resonance frequency the outputs of a typical DSFM electronics describe to a very good approximation a “Circle” when visualized in xy-mode on an oscilloscope. Interestingly, our model shows that this “Circle” remains essentially invariant when including the non-linearity of typical tip-sample interactions. These “Circles” are distorted only by dissipation which varies with tip-sample distance. We therefore propose that this representation scheme allows a very sensitive method to separate the effect of conservative interactions (linear as well as non-linear) from the effect of dissipative interactions.

[1] A. San Paulo and R. García. PRB 66:041406 (2002).

The mechanical behavior of polymer composites is controlled by the properties of the components as well as the microstructure of the material. Because confinement effects and interphase formation can alter properties of the microphases, only measurements performed directly on the composite can provide a complete picture of this local property distribution.
In contrast, the bulk viscoelastic properties of these materials targeted during material development are usually measured using dynamic mechanical analysis, DMA (also known as dynamic mechanical spectroscopy, DMS). This measurement involves application of an oscillating stress or strain to a macroscopic sample, with the response of the sample measured as a function of oscillation frequency). When heterogeneous samples are measured using this macroscopic technique, no information about the properties of the microstructure is available – only the spatially averaged properties of the whole sample.
Atomic Force Microscopy (AFM) has the resolution and mechanical sensitivity to provide the needed local mechanical property information. However, previous AFM measurements of viscoelastic properties [1,2] have suffered from several drawbacks, making it impossible to compare directly with DMA. To address this issue, we have developed a mode combining the benefits of AFM and DMA [3]. Like bulk DMA, AFM-nDMA provides spectra of storage and loss modulus at low frequencies. Unlike bulk DMA, it can localize the measurements and generate maps with resolution of a few nanometers.
In this presentation we will demonstrate how AFM-nDMA spectra can be compared directly to bulk DMA spectra. We will review insights gained when time-temperature superposition [4] is applied to AFM-nDMA data to generate master curves, including for materials that only exist in microscopic domains within the sample. Finally, we will discuss how comparing this nanoscale viscoelastic data with that of the bulk can clarify structure-property relationships in polymer composites.
[1] B. Pittenger and D. G. Yablon, Bruker Application Note, 2017, AN149, doi: 10.13140/RG.2.2.15272.67844.
[2] M. Chyasnavichyus, S. L. Young, and V. V Tsukruk, Langmuir, 2014, 30, 10566.
[3] B. Pittenger, S. Osechinskiy, D. Yablon, and T. Mueller, JOM, 2019, 71, 3390.
[4] M. L. Williams, R. F. Landel, and J. D. Ferry, J. Am. Chem. Soc.,1955, 77, 3701.

In this work we quantitatively analyze up to eight AFM measurables that can generate materials contrast on soft matter, including (i) sliding friction force in contact mode; (ii, iii) adhesion and contact stiffness in force-distance curve mapping (both slow and fast variants, e.g. "force volume" and "peakforce" modes), (iv, v) phase imaging in net attractive- and net repulsive-regime dynamic/AC mode (aka traditional amplitude-modulation "tapping mode"); (vi, vii) contact resonance amplitude and frequency under pulsed-IR excitation (aka resonance-enhanced mode on a Bruker NanoIR3 AFM-IR system); (viii) Fourier-reconstructed conservative and dissipative force versus distance relationships analyzed and mapped using machine learning in intermodulation mode (i.e., IntermodulationProducts AB Multi Lockin Analyzer / software system).

We apply select combinations of these methods to polymer blends, nanostructured single-layer and multi-layer coatings, two-surfactant and surfactant/nanoparticle ultrathin films, and polymer/small-molecule (e.g., drug) mixtures, all being systems of interest to our industrial as well as academic collaborators. We seek improved understandings of (i) complementary information obtained (mechanical, tribological, surface energy, chemical) as well as (ii) methodological limitations and optimizations.

Material fate and degradation in situ is regulated not only by chemical interactions but also the stress and strain from coupled mechanical systems. Strain defects and breakage of nanoscale soft materials such as tissue scaffolds, biopolymers, and cells are elusive to characterize due to their subtle and slow viscoelastic response. Here, we present the application of our new technique, FT-NanoDMA, to create a multidimensional tomography of the dynamical mechanical properties of such soft materials. FT-NanoDMA stands for Fourier transformed nano dynamical mechanical analysis, which is implemented in conjunction with atomic force microscopy (AFM). It allows for simultaneous recordings at different frequencies of multiple dynamical mechanical properties of soft materials, such as the storage and loss moduli, loss tangent, etc. The FT-nanoDMA is a modality that allows for mechanical analysis of nanointerfaces and single cells with a nanoscale spatial resolution (up to 10-70 nm when recorded on fixed cells) and spans the entire biological spectroscopic range (up to 300 Hz). The images of the distribution of these parameters can be recorded at each particular depth, thereby creating a stack of images suitable to create a multidimensional tomography of single cells. We demonstrate examples, which show that the tomographic images show unique features not present in the complementary fluorescent/optical images of cells.

Poly (methyl methacrylate) (PMMA) is often used as a lightweight, shatter resistant alternative to glass, but exhibits low scratch resistance and poor ductility. It was recently shown that blending PMMA with a very low concentrations (1 wt%) of two different polyrotaxanes (PR) - unmodified PR (uPR) and modified PR (mPR) improves these properties. uPR was created by grating cyclodextrin (CD) rings onto a polycaprolactone (PCL) backbone, while the mPR had methacrylate functional groups at the end of the PCL to facilitate miscibility with PMMA. Although bulk scale dynamic mechanical analysis (DMA) and dielectric spectroscopy on these PMMA-PR blends have indicated that significant PMMA-PR interactions in these systems lead to different molecular mobility changes, which eventually contribute to the observed mechanical property changes at the bulk scale, a direct understanding of the local interfacial interactions is missing. This presentation will discuss the use of Atomic Force Microscope (AFM) based nanomechanical spectroscopic methods to directly map the local time-dependent mechanical property gradients at the interphase regions in PMMA-uPR and PMMA-mPR blends, which in turn will provide direct insights into how PMMA-PR interactions affect their mechanical behavior.

Inspired by the adhesive proteins of mussels, polydopamine (PDA) has emerged as one of the most widely employed materials for surface functionalization. Despite numerous attempts at characterization, little consensus has emerged regarding whether PDA is a covalent polymer or a noncovalent aggregate of low molecular weight species. Here, we employed single-molecule force spectroscopy (SMFS) to characterize PDA films. Retraction of a PDA-coated cantilever from an oxide surface shows the characteristic features of a polymer with contour lengths of up to 200 nm. PDA polymers are generally weakly bound to the surface through much of their contour length, with occasional “sticky” points. Our findings represent the first direct evidence for the polymeric nature of PDA and provide a foundation upon which to better understand and tailor its physicochemical properties.

Recent large interest in machine learning (ML) analysis has been extended to atomic force microscopy (AFM). In this talk, I will address three questions which typically arise when AFMers start applying ML to classify the AFM images:
1. What mode is the best to use?
2. How many images is sufficient to run machine learning classifiers and believe the results?
3. How do I know that the obtained classification is not an artifact of some fine-tuning?
I this talk, I will suggest answers to the questions. In short, the mode of operation should be robust, repeatable. This is the key. The number of images required for classification can be roughly estimated using “the rule of 10”. A more precise algorithm to identify the required number of images will be described. To be confident that one doesn’t deal with an artifact of fine-tuning or overtraining a classifier, we suggest a rather simple yet powerful method of estimating the exact amount of overtraining by using randomization of the initial classification. Examples will be given.

The possibility of non-destructively measuring living cells and their internal structures with nanoscale resolution would allow for a much greater understanding of numerous biological phenomena. To realize this goal, the use of carbon nanotube (CNT)-terminated tips in atomic force microscopy (AFM) has been shown to hold promise [1].
However, even in the very first step of the proposed “nanoendoscopic” process, consisting in the penetration of the plasma membrane, it is not always easy to give a precise interpretation of the experimental signal, which is a convolution of cell dynamics, tip shape and buffer solution.
Here, we use coarse-grained (CG) molecular dynamics simulations to investigate the effect that different widths, thicknesses and functionalization of the CNT tip can have in the penetration process. To simulate the membrane and the solvent dynamics we use the Martini 2 Force Field [2], while the CNT is simulated with a Martini-compatible force-field [3] which is more accurate in reproducing multi-layer graphene mechanical properties.
We carry out the membrane penetration by steering the CNTs downwards with constant velocity, then extracting the system's free energy using umbrella sampling, finally obtaining simulated force profiles. Additionally, we probe the possible role of the membrane skeleton mesh size [4] by emulating its effect as a position restraint on the lipids at the membrane’s edge. We directly compare our results to experimental measurements using an equivalent setup and outline the successes and limitations of the model.

[1] - Singhal, R., Orynbayeva, Z., Kalyana Sundaram, R., Niu, J., Bhattacharyya, S., & Vitol, E. et al. (2010). Multifunctional carbon-nanotube cellular endoscopes. Nature Nanotechnology, 6(1), 57-64.
[2] - Marrink, S., Risselada, H., Yefimov, S., Tieleman, D., & de Vries, A. (2007). The MARTINI Force Field - Coarse Grained Model for Biomolecular Simulations. The Journal Of Physical Chemistry B, 111(27), 7812-7824.
[3] - Ruiz, L., Xia, W., Meng, Z., & Keten, S. (2015). A coarse-grained model for the mechanical behavior of multi-layer graphene. Carbon, 82, 103-115.
[4] - Morone, N., Fujiwara, T., Murase, K., Kasai, R., Ike, H., & Yuasa, S. et al. (2006). Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography. Journal Of Cell Biology, 174(6), 851-862.

According to the World Cancer Research Fund, Breast cancer and Prostate cancer are among the most prevalent cancers in women and men, respectively. World Health Organization estimates that about 3.4 million breast and prostate cancer cases are reported each year, and about a million deaths occur worldwide due to these cancers. These cancers are almost entirely curable if detected in the early stage at the primary sites. However, most deaths occur due to metastasis when the cancer cells from the primary site migrate to a distant organ. Bone appears to be a niche for both cancers. We have developed a novel testbed using a bone mimetic nanoclay based scaffold to regenerate human bone using human mesenchymal stem cells followed by seeding prostate and breast cancer cells to generate tumors. Extensive analysis of the tumors using gene and protein expression assays and imaging confirm that the testbeds can replicate tumors during mesenchymal to epithelial transition (MET) for both cancers. Mechanical properties of cells have been used to describe the disease state for a variety of diseases. The confocal imaging of the cells from tumors shows significant changes to the cytoskeleton in terms of the quantity and organization within the cells as cancer progresses at the bone metastasis site. In the present work, for the first time, we have used mechanical response from cells from direct nanoindentation experiments on cancer cells from tumors generated on the testbeds to describe the evolution of cell properties during cancer progression at the bone metastasis sites. The nanoindentation experiments are conducted in static and dynamic modes to evaluate elastic moduli, hardness, and viscoelastic properties of cancer tumor cells. We report the evaluation of mechanical properties of cells from tumors grown on the testbed, over time, with two breast cancer cell lines (MCF-7 and MDA-MB231) at the bone metastases site using static and dynamic nanoindentation modes. A new fixture was designed and fabricated to conduct live-cell nanoindentation experiments. The force-displacement response, elastic moduli, hardness, plastic deformation, viscoelastic properties, and other mechanics characteristics were captured, along with confocal imaging of the cytoskeleton and gene and protein expressions at the same time points. Our results indicated a significant reduction in elastic modulus and increased fluid-like behavior of bone metastasized MCF-7 cells caused by depolymerization and reorganization of F-actin. On the other hand, bone metastasized MDA-MB-231 cells showed insignificant changes in elastic modulus and F-actin reorganization over time. We also measured changes to nanomechanical properties of MDA PCa2b prostate cancer cells during the MET and cancer bone metastasis progression over time. The stiffness of PCa cells decreases with metastasis; however, the mechanical plasticity increases during the same time, suggesting that PCa cells become softer on undergoing MET and softer with metastasis progression. In all cases, the imaging and gene, protein expression studies, and computational modeling point towards the depolymerization of actin and reorganization of the cytoskeleton as key factors in the evolution of cell mechanics. These studies present the use of direct nanoindentation as a potential new biomarker for metastasis progression.

Additively manufactured 316L stainless steels (AM 316L SS) using laser powder bed fusion exhibit outstanding mechanical properties such as high strength and high ductility. The rapid-solidification induced sub-grain cellular structures, a dominant microstructural feature present in the as-built material, are largely responsible. However, in term of corrosion properties, the effect of these cellular structures has yet to be confirmed. A difference in metal dissolution rate between cell walls and cell interior has been reported but the underlaying mechanisms remain unclear. To certify the use of AM 316L SS in corrosive environment, it is necessary to observe, understand, and predict the local effect of the microstructure on the resistance to corrosion, notably the initiation mechanisms. Here, we combine in-situ high-speed AFM (HS-AFM) during corrosion in HCl and NaCl solution, TEM, and SEM/EBSD to try and capture the native oxide breakdown, re-passivation, pit nucleation, and metal dissolution. We quantitively measure and map, in-situ, the metal dissolution rates in the cellular structure for different grain orientations and compare to the conventional, well annealed material.

The atomic force microscopy (AFM) combined with the force volume mode allows measurement of both indentation force curves and sample topography and is used to study cell mechanics. As was demonstrated in multiple publications, the Hertz mechanical model is not sufficient to describe cell mechanical properties in a self-consistent way. Self-consistency can be attained when using a relatively dull AFM probe and processing the data through the brush model. The brush model allows determination of both the mechanical properties of the cell body and the physical properties of the pericellular (glycocalyx) layer. Separation of the mechanical response of the pericellular layer from deformation of the cell body has demonstrated its utility in multiple studies, such as mechanics of cancerous and aging cells, and endothelial cells associated with various cardiovascular diseases. Despite its utility, the brush model has not yet been tested for its robustness with respect to the uncertainties within the model and experimental data. In other words, there is a need to demonstrate how unambiguous and precise the parameters extracted with the brush model are, when considering the uncertainty within the model and possible ambiguities of the collected force curves.
Here we address this need. We present the calculation of the elastic modulus of the cell and characteristics of the pericellular layer for a diverse set of cells: fibroblasts of zebrafish, mouse neuroblastoma cells, and human cervical epithelial cells. We perform the analysis and demonstrate that the brush model is robust. Errors in defining the modulus, which come from both model and experimental uncertainties, are within 3%, which is substantially smaller than a typical uncertainty in the spring constant of the AFM cantilever. To quantify the uncertainties in the extracted force due to the brush layer, we characterize the brush with Alexander de Gennes model, which is described by the equilibrium size of the brush layer and grafting density of the brush molecules. Although it is an obviously oversimplified approximation, we see the uncertainty in those parameters is not larger than 25%. It is worth noting that the brush parameters calculated in this way is completely independent of the calculation of the modulus.

It is widely recognized that embryogenesis is strongly associated with cell mechanical properties [1]. However, little is known about how embryonic single cells change their mechanical properties during their developing process due to a lack of technique. I will present our recent studies regarding the stiffness of cells in developing ascidian embryos with AFM [2] and the rheological measurements. The results showed that the force-distance curves measured in upper regions of embryonic cell surfaces were well fitted to the conventional Hertzian contact model, and the apparent Young’s modulus was calibrated using a modified Hertzian contact model [3] considering the tilt angle of the sample surface. The cell shapes in the height and the Young’s modulus images were almost identical, and the cell locations were in good agreement with the 3D virtual embryo model determined from confocal microscopic images, indicating that the embryonic cell stiffness measured by AFM mainly reflected the mechanical properties of cells that directly contacted the AFM probe. Furthermore, the cell stiffness exhibited a remarkable increase at the onset of cell division and then decreased after cell division, which is reminiscent of changes in an in vitro dividing single cell forming cortical actin filaments [4]. The results indicate that AFM is useful for measuring the embryonic cell stiffness in the developing process and can facilitate a better understanding of how embryonic single cells in animals are spatiotemporally regulated via mechanical cues.

References:
[1] D. Gilmour et al. Nature 541, 311 (2017). [2] Y. Fujii, et al, Commun. Biol. 4, 341 (2021). [3] Y. Fujii and T. Okajima, AIP Adv. 9, 015028 (2019). [4] R. Matzke et al. Nat.Cell. Biol. 3, 607 (2001).

In single-molecule force spectroscopy (SMFS), many studies have focused on the elasticity and conformation of polymer
chains, but little attention has been devoted to the dynamic properties of single polymer chains. In this study, we measured
the energy dissipation and elastic properties of single polystyrene (PS) and poly(N-isopropylacrylamide) (PNIPAM) chains in different solvents using a homemade piezo-control and data acquisition system, which provided more accurate information regarding the dynamic properties of the polymer chains. We quantitatively measured the chain length-dependent changes in the stiffness and viscosity of a single chain using a phenomenological model consistent with the theory of viscoelasticity for polymer chains in dilute solution. The effective viscosity of a polymer chain can be determined using the Kirkwood model, which is independent of the intrinsic viscosity of the solvent and dependent on the interaction between the polymer and solvent.

The push towards high-resolution lithography in the semiconductor industry has yielded the introduction of extreme ultraviolet (EUV) into high-volume manufacturing. However, looking forward to future technology nodes, further improvements are required in photoresist materials to keep pace with and fully utilize improvements in the EUV source and scanner. One of the critical steps is to understand the role of the dissolution process, which converts latent exposure chemistry in the photoresist into a developed pattern. This process is particularly challenging to follow due to the requirement of nanometer spatial and sub-second temporal resolution simultaneously. Most metrology of the patterning process thus ignores either the temporal component of the dissolution process by measuring just the final developed structure or the spatial component as is done in most dissolution rate monitoring techniques. In our study, a microfluidic sample stage is designed to enable the delivery of full-strength developer within fractions of a second with better concentration control and smaller dead volume for less lag and more accurate time zero determination. Moreover, our microfluidic design minimizes the mixing problem of the full-strength developer and water, which usually disrupts laser-based detection mechanism, allowing us to monitor the development process with high-speed atomic force microscopy (AFM) at the nanoscale. Thus, our system offers the ability to probe the spatially-dependent nature of the dissolution process in a fab-like environment, providing insight into exposure-dependent dissolution rate gradient, material swelling, and polymer entanglement.

Block copolymers with mechanically identical blocks having identical glass transitions such as Polystyrene-b-Poly(methyl methacrylate) (PS-b-PMMA) show poor contrast between domains, especially in vertically ordered microstructure. Addition of plasticizers that segregate into selective domains and significantly reduce the glass transition temperature of that block also increase its contrast for detection through atomic force microscopy. These additives also enhance the chemical incompatibility between blocks producing sharp interfaces that can be easily detected for the additive swollen domains. In this study, we demonstrate producing vertical domain assembly in as-cast block copolymer (BCP) thin films by controlling the casting environment using solvent mixtures and plasticizing additives. The combined interaction between the plasticizer segregated into selective domains and the solvent mixture drives the ordering front to produce through film vertical assembly in PS-b-PMMA films with cylindrical and lamellar volume fractions. The morphology is probed with atomic force microscopy to analyze the effect of additive volume fraction on domain sizes and film morphologies. Ultrafiltration membranes are an energy-efficient alternative to overcome the environmental challenges occurring from the rising amounts of mixed oil and water through oil-spills and industrial wastewater etc. Ultrafiltration membranes were fabricated from these vertically oriented structures and are promising candidates for oil-water separation applications. In addition to the uniform pore size of the membranes, the membranes have been designed to function as highly permeable as well as selective active layers on top of the polyethersulfone (PES) support membranes. This has been accomplished during the membrane preparation stage by the addition of varying amounts of plasticizing and homopolymer additives to further modify the membranes. The influence of these additives on the pore characteristics and membrane performance have accordingly been analyzed as a function of their mass ratio with respect to BCP. The created asymmetric membranes were demonstrated to be effective for oil/water separation when plasticizing additives were added in the 50%-60% mass ratio.

Macroscopic friction behaviors originate from the mechanical and tribological response of asperities at the microscopic and nanometric level. As a consequence atomic scale friction stands as a cornerstone for the field of tribology. At such small scale, materials exhibit mechanical properties reminiscent of soft materials, such as adhesion and high deformability, thus making similar experimental challenges arise. From a sliding friction perspective, the dynamics undergo different successive steps from elastic pinning to large velocity kinetic friction. Atomic force microscopy usually only probes the steady state response leaving the early sliding response unexplored. Here, we unveil a combined experimental and simulation framework based on a high-frequency oscillatory strained interface to resolve the onset of sliding aa well as the large velocity steady-state at once. A model contact of a few atoms formed between a gold tip and graphite is used to demonstrate our experimental ability to explore the different sliding regimes. We uncover peculiar behaviors, from thermolubricity for the smaller contact, to superlubricity as the contact gets larger than the unit cell of graphite. This new experimental methodology paves the way for a better understanding of sliding friction and its different dynamic stages.

To enable the forward prediction of mechanical properties of polymer nanocomposites, we have developed a novel framework that uses experimental methods and finite element analysis and we demonstrate it for a nanocomposite made of a polydimethylsiloxane (PDMS) matrix and silica particles (SiO₂). We have experimentally determined the microstructure distribution and the local mechanical properties of the extent of the interphase between the silica particles and the PDMS matrix via atomic force microscopy (AFM), and the macroscale properties of the PDMS matrix via dynamic mechanical analysis (DMA). We hypothesize that by accurately determining the local properties in the interphase we can predict the behavior of the macroscale nanocomposite as the interphase between the polymer matrix and filler has altered properties that are thought to be responsible for the enhanced properties observed in thin films and polymer nanocomposites. A key challenge in accurately determining the extent of the interphase around a nanoparticle via AFM occurs as a result of the complex contact conditions between the AFM tip and the polymer near an interphase. To address this we have developed a method similar to one used in nanoindentation experiments to eliminate the indentation depth dependence of the local property measurements and calculating depth independent properties that are not convoluted by extraneous influences. Consequently, we have performed finite element analysis (FEA) simulations to predict the macroscale properties of PDMS-SiO₂ nanocomposites and validated them against experimental data for PDMS-SiO₂ nanocomposites. This framework provides a method to achieve the forward prediction of mechanical properties of polymer nanocomposites with limited experimental work, optimizing the process to discover and develop materials with desired properties.

Electromechanical coupling underlies a broad range of diverse applications from energy harvesting to biotechnology. Voltage modulated (VM) AFM techniques including piezoresponse force microscopy (PFM) are uniquely positioned to probe electromechanical properties with spatial resolution from micrometers to nanometers. As with all AFM techniques, the goal of any PFM measurement should be towards quantification of the local material properties. Unfortunately, quantification of PFM signals remains a significant challenge. Indeed, with the growing popularity of PFM there has been a congruent rise in reports of nanoscale electromechanical functionality, including ferroelectric hysteresis, in materials that should be incapable of exhibiting piezo- or ferroelectric response. This presentation will evaluate the appropriateness of advanced modes of PFM, including interferometric and multifrequency methods, for quantification of soft and hybrid materials. A particular focus will be overcoming the susceptibility of PFM measurements to crosstalk, a pre-requisite for quantification of piezoelectric coupling coefficients. Throughout the presentation examples of quantitative measurements on soft and polymeric materials including PVDF-TrFE and hybrid organic inorganic perovskites will be described. This work was conducted at the Center for Nanophase Materials Sciences, which is a DOE Office of Science User Facility.

Multimodal measurements of chemical composition, electrical properties, mechanical properties, and topography by scanning probe microscopy (SPM) deliver correlations across properties at the nanoscale and provide clues to the structure-function relationship of materials. In the past, measurements with these modalities are operated separately with different operational modes of SPM. Not only do the sequential measurements require additional operation time, subject to scanner/sample drift, but also, different modalities of SPM have different spatial resolutions, which undermine correlative analysis. For example, the popular frequency-modulated Kelvin Probe Force Microscopy measures the surface potential with 30~50 nm spatial resolution under the ambient conditions, whereas the SPM measurements of chemical composition, mechanical properties, and topography can routinely achieve < 10 nm spatial resolution.

At the MRS conference, we will present our invention of an integrated SPM mode that can simultaneously provide chemical, surface potential, mechanical, and topographic imaging at < 10 nm spatial resolution under ambient conditions. First, we will present our development of pulsed force Kelvin probe force microscopy (PF-KPFM) that delivers 10 nm spatial resolution from surface potential measurement under ambient conditions. Second, we will briefly describe the peak force infrared (PFIR) microscopy on infrared nano-imaging and spectroscopy. Finally, we will introduce our invention of peak force infrared-Kelvin probe force microscopy (PFIR-KPFM) that combines PFIR and PF-KPFM in one unified SPM mode. In a single scan, the integrated PFIR-KPFM delivers simultaneous multimodal measurement at a comparable and high spatial resolution of < 10 nm. We demonstrated the PFIR-KPFM on a range of samples, from perovskite photovoltaics to protein aggregates, and revealed correlations among modalities.

Scanning probe microscopy encompasses techniques that span length scales, time scales, and operational complexity scales. Because of the existence and value of these complex measurements, it is typical for an eager, new user to require extended training together with an expert to reliably acquire the advanced, precise data they hope to collect.

In our new review in Progress in Polymer Science [1], we attempt to address the nuances of extended training by providing a resource in the form of a collection of recommendations and best practices in the nanomechanical characterization of polymers and other soft/heterogeneous materials. The review discusses and compares the variety of nanomechanical techniques available, provides details and practical considerations for real nanonomechanical experiments, and summarizes notable published works as case studies for the application of available modes for nanoscale property mapping. This presentation selects a very small subset of the content to demonstrate the utility of the review as a guide, but also to draw attention to some unanswered questions that were raised as this domain continues to mature.

Our review is concentrated on nanomechanical AFM of polymers, both because that system is one of our group's competencies, and also because it prevented the scope of the review from growing without limit. But already the narrow scope raises questions: Are these recommendations applicable to other materials, like hydrogels or metals? Can they be applied to other scanning probe microscopy techniques, like scanning tunneling microscopy or single molecule force spectroscopy? To what extent? In this presentation, we seek to lay a foundation for a greater discussion and collaboration between groups and sub-fields regarding best practices, training, and converting individual or even institutional knowledge into common knowledge.

[1] Collinson DW, Sheridan RJ, Palmeri MJ, Brinson LC (2021) Best practices and recommendations for accurate nanomechanical characterization of heterogeneous polymer systems with atomic force microscopy. Progress in Polymer Science, 101420. https://doi.org/10.1016/j.progpolymsci.2021.101420

Ferroelectric materials remain a source of physical discovery through the long history of the field. On-going development has pointed to the crucial role of domains and domain walls in ferroelectric materials. The development of scanning probe microscopy (SPM) allows visualization of domain structure and its evolution under external fields, such as electric bias applied through tip and electrodes, strain, light illumination, and heating. However, the behaviors of interest mostly are related to domain structures and concentrated in specific locations. This initializes the opportunities of automated microscopy with an automatically defined workflow for scanning sequence and data acquisition. Here, we used a Deep Kernel Learning (DKL) framework to explore the domain structure and physical properties of ferroelectric materials. We further developed a workflow based on the DKL for Piezoresponse Force Microscopy (PFM) that allows problem-specific tuning of workflow and operation in real-time, which allows exploring polarization switching and domain structure in ferroelectric materials. The structure of the DKL kernel provides insight into the physics of the process. We further build the relationship between domain wall geometry and presence of ferroelastic domain walls, as well as topography, and hysteresis loop shape. Previously in the post-experiment approach, this was accomplished via im2spec and rotational variational autoencoder (rVAE) approaches. The DKL allows to learn these relationships during the experiment. This workflow can also be adapted to other microscopy systems, such as electron microscopy and optical microscopy.

Acknowledgments: This work is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences Energy Frontier Research Centers program under Award Number DE-SC0021118. This work is conducted at the Center for Nanophase Materials Sciences, a US Department of Energy Office of Science User Facility.

HfO₂-based materials have attracted a great deal of interest because they exhibit robust ferroelectricity at extremely thin thickness and are thermodynamically compatible with conventional semiconductors, leading to a wide range of applications in ferroelectric-based devices, such as field-effect transistors, random-access memory, and tunnel junctions. However, the local uniformity of HfO₂-based materials is rarely investigated, which is important for their application. In this work, we developed an automated experiment (AE) workflow in scanning probe microscopy (SPM) to investigate the local conductivity of a Hf0.5Zr0.5O2 (HZO) thin film. This AE workflow maps a large field of view of material first and automatically locates the regions of interest (ROI); then, detailed zoom-in measurements are automatically performed in the ROIs to systematically understand the material under study. Further, to solve a universal problem—measurement drift—in microscopes, we embedded a drift correction algorithm to maintain the in-situ measurement. This drift correction method is based on a recently developed shift-invariant variational autoencoder (shift-VAE). Finally, we investigated the heterogeneous conductivity of the HZO thin films by this AE workflow, revealing the evolution of conductive hotspots around several tens of nanometers in HZO with bias voltage and time. The hotspots show complex spatiotemporal dynamics as a function of time and bias during the scanning, suggesting the presence of complex conduction mechanisms. These results underpin a wide range of HZO-based applications. We believe that the AE approach developed in this work will be universally useful for microscopy measurements.
Acknowledgments: This work is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences Energy Frontier Research Centers program under Award Number DE-SC0021118. This work is conducted at the Center for Nanophase Materials Sciences, a US Department of Energy Office of Science User Facility.

Biological processes at the base of life rely on a wide class of biomolecular machines that have characteristic nanoscale physical dimensions and whose function emerges from a correlation between their chemical and structural properties. Therefore, a fundamental objective of modern analytical methods in Physics and Chemistry is to unravel the heterogeneous chemical and physical properties of single biomolecules and relevant bio-surfaces at the nanoscale. While innovative imaging methods have been developed to characterise biomolecular processes at the nanoscale, imaging microscopies are to the most part chemically blind; thus hampering the characterisation of inhomogeneous and complex systems.
Here, we first demonstrate that Atomic Force Microscopy (AFM) provides detailed information not only on the morphological, but also on the mechanical properties of biomolecular processes and functional materials at the nanoscale [1,2]. Then, to overcome the limitations of conventional imaging microscopies, we show a real breakthrough with the development and application of Infrared Nanospectroscopy (AFM-IR) in bio- and materials science. AFM-IR combines the high spatial resolution of AFM (~1 nm) with the chemical analysis power of infrared (IR) spectroscopy to retrieve unprecedented correlative information at the nanoscale on the structural, mechanical and chemical properties of heterogeneous bio-molecular processes and functional biomaterials.[3] As most recent advances in the field, we demonstrate the achievement of single protein molecule chemical identification and structural determination.[4] Then, we show the application of this single molecule sensitivity to unravel the molecular interaction fingerprint between a small molecule and its target [3], the surface properties of artificial model membranes [6] and the structure of functional protein self-assemblies to be exploited as a novel class of biomaterials in bioscience [7-8].
Overall, the aim of our present and future research is to expand the capabilities of nanoscience for chemistry and biology to open a new window of observation on the fundamental biomolecular processes at the core of life and ageing, to shed light on the structure-activity relationship of biomolecules, and characterises functional surfaces and materials for nano- and bio- science applications.

References
[1] Ruggeri, ACS Nano, 2020.
[2] Ruggeri, PNAS, 2018.
[3] Ruggeri, Nature Comm., 2015.
[4] Ruggeri, Nature Comm., 2020.
[5] Ruggeri, Nature Comm., 2021.
[6] Marchesi, Advanced Functional Materials, 2020.
[7] Shen, Ruggeri, Nature Nanotechnology, 2020.
[8] Qamar*, Wang*, Randle*, Ruggeri*, Cell, 2018.

In the field of cell mechanics, a change in physicochemical properties of the extracellular matrix (ECM) can cause profound functional modifications, such as differentiation, replication, migration, and apoptosis. Thus the physicochemical properties of culture substrates were modulated in order to mimic in vitro the complexity of physiological environments. In this work, polydimethylsiloxane and hydroxy-polyacrylamide hydrogel (hydroxy-PAAm) were used to study the impact of matrix stiffness on endothelial cell mechanics. Firstly, the viscoelastic properties were estimated in physiological conditions by nano Dynamic Mechanical Analysis, based on single force curves, while the probe is indenting the surface with nano-Newton forces. Our findings show a linear stiffness gradient while the bis-acrylamide/acrylamide ratio increases. In addition, we show that the adhesion from PeakForce Quantitative NanoMechanics (PF QNM) is higher significantly than the value obtained in Force Volume. Then we quantitatively mapped the nanomechanical properties of human umbilical vein endothelial cells (HUVECs) plated on polydimethylsiloxane and hydrogels. We found that the elastic modulus of the cell body is about 12-18 kPa which is higher than the cell lamellipodia (5-10 kPa) with Tan δ approximately 0.5 for cell body.

The bladder is a highly variable mechanical microenvironment expanding from a few to hundreds of kPa due to functional reasons. Interestingly, such a large range of functional stiffness seems not to influence the mechanical properties of urothelial cancer cells as they become more deformable already at the early stages of cancer progression as measured by atomic force microscopy (AFM). Several structural components contribute to the mechanical properties of bladder cancer cells. Primarily, they are related to the organization of polymerized actin form (actin filaments) and the overall actin content. Cell mechanics alter dynamically in response to external or internal stimuli, including the organization of cells (single cells, monolayers or more complex structures) and the interplay of cellular deformability with the surrounding microenvironment (i.e., extracellular matrix, ECM). All steps of cancer progression involve specific interactions between cell surface receptors and multiple components of the ECM, including basement membranes (BMs), mainly composed of laminins or type IV collagen. This structural scaffold, making a physical barrier, contributes to such phenomena as cell adhesion, proliferation, and migration. Laminin binding to receptors present on a cell surface initiates cell invasion through BMs. The actin cytoskeleton is linked with the focal adhesion molecules, thus altered expressions of related molecules will affect cell deformability. This leads to glycocalyx as the other component contributing to bladder cancer mechanics at the cellular level. By applying the brush model, the mechanical properties of the cells studied under specific cleavage conditions were correlated with the presence of the particular type of the pericellular glycocalyx layer. Distinct contributions of cell structural elements to cell biomechanics form a question of the leading cause of the alterations in mechanical properties of cells. The working hypothesis can be summarized as follows: mechanical properties of local cellular microenvironment define the invasive and migratory phenotype of cancerous cells through irreversible changes in composition and structure of both proteoglycan’s component of the glycocalyx and expression of the adhesive molecules leading to cytoskeleton reorganization. Cells leaving the primary tumour site carry this information and use it to form a secondary, metastatic site.

Atomic force microscopy (AFM) is uniquely capable of characterizing the 3-dimensional modulus variations that arise in vat photopolymerization additive manufacturing processes such as stereolithography, digital light processing and inkjet. 3D control of part shape and properties is essential to realize additive manufacturing opportunities in critical applications such as organ printing and dentistry. We will discuss how AFM can be used both ex-situ and in-situ to the printing process to inform where heterogeneity and shape deviation arise in printed parts. We will then employ and verify various models to correct and control for such variations, yielding geometrically and mechanically precise parts. Ex site characterization of as-printed parts, both pristine and cryo-ultramicrotomed reveals true process control of properties based on light exposure and feature size. In-situ characterization complements ex-situ by revealing differences between resin, green-state and final part properties. Overall, the interplay between characterization and control enables new freedoms in 3D printed part design, particularly at the smallest length scales.

Atomic Force Microscopy (AFM) is a powerful technique in biophysics and nanomedicine, since it allows imaging and manipulating nanostructures in physiological conditions on a single molecule level [1]. Magnetic Force Microscopy (MFM) is an AFM-based technique where a nanometric magnetic probe is scanned in close proximity to a surface detecting the local magnetic fields gradients near it. MFM has been applied to the study of a variety of magnetic systems, including magnetic nanoparticles. Despite the importance of studying magnetic nanostructures with biological applications in physiological conditions, the applicability of MFM to these systems was limited up to now because of the difficulty in developing MFM for detecting magnetic interactions in liquids. This is a consequence of the higher damping forces acting on the cantilever when working in liquid environment, as compared to air, which results in a significant loss of sensitivity of the MFM signal. In the work presented here, we start by introducing the necessary development for MFM imaging in liquid media [2] and discussing the influence of the chosen cantilever on the obtained MFM signal-to-noise ratio. Then, we present the development of new magnetic probes fabricated by Focused Electron Beam Induced Deposition (FEBID) using specially designed cantilevers for liquid medium and demonstrating that further improvement on the performance can be gained [3].
[1] Y. F. Dufrêne et al. Nature Nanotechnology 12, 295–307 (2017).
[2] P. Ares et al. Small 11, 36, 4731–4736 (2015).
[3] M. Jaafar et al, Nanoscale, 12, 10090–10097 (2020).

Many of the processes occurring at electrolyte-solid interfaces, such as corrosion, catalysis and heterogeneous nucleation, as well as the interactions and response dynamics of colloidal systems, are mediated by the solution near the interface. However, our knowledge of interfacial solution structure (ISS) has been limited by a dearth of experimental techniques that can visualize ISS at the atomic scale. AFM-based three-dimensional fast force mapping (3D FFM) is a recently developed method that provides this ability. During FFM, changes in phase and amplitude (or frequency) of an oscillating AFM tip due to interactions with its surroundings is recorded as the tip approaches and retreats from the surface. By collecting these 1D force curves over a 2D area, a 3D map of solution structure is created. As part of an effort to understand the interactions and dynamics leading to growth of nanomaterials through the process of oriented attachment (OA), we are using 3D FFM to visualize solution structure at a variety of crystal interfaces. Here we report on studies of the Zn (0001) interface in which we pursue three novel directions. First, we attempt to understand how the ISS depends on the nature of the solvent by examining both water and ethanol. Second, we utilize the polar nature of ZnO to investigate how differences in surface termination — O vs Zn — impact the ISS. Third, we introduce custom-made nanocrystal AFM probes fabricated by depositing ZnO onto commercial silicon probes to understand the structure seen by ZnO particles as they approach one another during OA. Our results to date demonstrate distinct differences between 3D FFM data collected in water and ethanol. In particular, a strong attractive force at a tip-sample separation of ~1nm is measured in water, yet absent in ethanol. Measurements on Zn- and O-terminated surfaces reveal differences in both the surface morphology and the interfacial structure. We successfully used the ZnO tips to execute FFM; the initial data show that the tip chemistry strongly influences the solution structure confined between the two surfaces. In summary, our findings reveal a complex dependence of ISS on surface termination, solvent composition and tip chemistry. By resolving the key limitation of conventional 3D FFM – namely the discrepancy between the tip and sample materials – we provide a more straightforward basis for predicting long-range interactions between ZnO particles, delineating the solution structure, and advancing the quantitative interpretation of 3D FFM data.

Charge transport in π-stacked aromatic rings is central to biological processes and organic electronics. However, direct experimental investigation of intermolecular charge transport between aromatics at molecular level is challenging. In this work, we use a supramolecular self-assembly approach to control the stacking geometry of two pyridinium molecules, thereby providing a promising model system to investigate intermolecular charge transport behaviors in discrete pyridinium dimers. The charge transport properties of free and dimerized pyridiniums were characterized using a scanning tunneling microscope-break junction (STM-BJ) technique, thereby enabling direct measurement of single-molecule conductance. Our results show that π-stacked pyridinium dimers exhibit up to 10-fold enhanced molecular conductance compared to single pyridinium. The experimental results were further analyzed with insights from density function theory (DFT) calculations. From a broad perspective, we anticipate that merging supramolecular chemistry and single-molecule conductance experiments will afford new functional molecular electronics.

About ten years after invention of Scanning Tunneling Microscope (STM), scientists found that STM can be used to modify sample surfaces at atomic scale. This led to the invention of STM-based lithography in which the STM tip is used as a source of a very fine and low energy electron beam to pattern a monolayer resist adsorbed on the sample surface with ultimate atomic resolution and precision. As a result, STM has been proposed as a viable alternative to conventional e-beam lithography whose resolution and precision is fundamentally limited to a few nanometers.
Despite this superiority in resolution, slow speed and low throughput have been major downsides of the STM which hinder its widespread use. A key contributor to these limitations is its relatively large nanopositioner, i.e. the piezotube. For this drawback, miniaturization stands out as a practical solution for the bulkiness of the STM piezotube. A Microelectromechanical-System (MEMS) STM nanopositioner can offer higher bandwidth and an opportunity for parallel operation of a multi-tip STM due to its small footprint.
In our previous work [1], we presented a MEMS-based STM system. There, we integrated a one-degree-of-freedom MEMS STM nanopositioner into a commercial Ultrahigh-Vacuum (UHV) STM system to replace the Z axis of its piezotube. As a result, a hybrid system is achieved in which the MEMS device provides the Z-axis motion which requires higher sensitivity, while the XY-plane motions are still delivered by the piezotube during the scan. Compared to the Z-axis of the piezotube, the MEMS device features tenfold higher bandwidth (i.e. 10 kHz) with the same range of motion (1-2 µm).
In this work, we further examine the practical benefits of this hybrid system over a conventional piezo-based STM. Thanks to the MEMS nanopositioner, higher scan speeds are achieved during both imaging and lithography compared to the original UHV STM system. Dimer row resolution images of Si(001):H were obtained at scan speeds over 2 µm/s with the hybrid STM in conjunction with the improved control loop [2].
STM-based lithography was also tested with the proposed hybrid system. With the greater sensitivity of the MEMS Z-actuator and the improved control loop, it is expected that lithography can operate at much higher tunnel current setpoints, allowing for much faster lithography. In our experiments, the sample used was a H-terminated Si(100)-2×1 surface. First, the sample surface was imaged, and its lattice was detected based on the image. Then, H atoms were selectively desorbed by moving the tip along a predefined trajectory over the sample surface using an appropriate bias voltage (+4 V) and tunnel currents up to 40 nA, providing write speeds up to 100 nm/s. The results obtained in this work demonstrate that the proposed MEMS-based STM system is conducive to high-speed atomic-scale lithography.

Acknowledgements
This work is supported by the U.S. Department of Energy’s Office of Energy Efficiency and Renewable Energy under the Advanced Manufacturing Office Award No. DEEE0008322. The authors wish to thank Zyvex Labs members, William R. Owen and Robin Santini for their assistance in setting up the experimental testbeds.

References
[1] Alipour, Afshin, et al. “Atomic-precision imaging with an on-chip scanning tunneling microscope integrated into a commercial ultrahigh vacuum STM system.” Journal of Vacuum Science & Technology B (2021) (Accepted for publication).
[2] Tajaddodianfar, Farid, et al. "Scanning tunneling microscope control: a self-tuning PI controller based on online local barrier height estimation." IEEE Transactions on Control Systems Technology 27.5 (2018): 2004-2015.

Domain structures and topological defects in ferroelectric materials underpin a broad range of applications ranging from materials with giant electromechanical responses to domain wall electronics. Correspondingly, exploring the functionalities of domain walls and controlled modifications of domain structures is of interest for a broad spectrum of applications. However, the dynamic nature of these objects severely constrains piezoresponse force microscopy approaches to explore their functionality. Here, to overcome this challenge, a real space image-based feedback approach is utilized to control the atomic force microscope tip bias during ferroelectric switching allowing for modification routes triggered by domain states under the tip. Specifically, the complex trigger system (i.e., “FerroBot”) is to used study frustrated and metastable domain and domain-wall dynamics in a Pb_xSr_(1-x)TiO₃ system. We have demonstrated that microscopic mechanisms of the domain-wall dynamics can be identified, i.e., domain-wall bending can be separated from irreversible domain reconfiguration regimes. In conjunction, phase-field modeling was used to corroborate the observed mechanisms. This study highlights a new pathway toward discovery and control of metastable states in ferroelectrics, and more generally paves way for automated systems for controlled modification of domain walls and defects to improve material properties. This work was supported by the U.S. Department of Energy, Office of Science, Basic Energy Sciences, Materials Sciences and Engineering Division at Oak Ridge National Laboratory’s Center for Nanophase Materials Science, which is a U.S. DOE Office of Science User Facility.

Recently, semiconductor nanowires (NW) have gained more and more attention, as they are the building blocks of various future functional devices at the nanoscale. The measurement and quantitative determination of doping and doping profile within semiconductor nanowires (NW) are crucial in order to develop the technology using them. Specific methods and techniques are in high demand to understand the incorporation of doping into such one-dimensional structures. In the last two decades, scanning capacitance microscopy (SCM) and scanning spreading resistance microscopy (SSRM) based on atomic force microscopy, has emerged as promising tools for two-dimensional high resolution carrier/dopant profiling. In SCM, the capacitance change providing by an alternating bias applied between the tip/sample system under a DC bias to alternately accumulate and deplete carriers within the semiconductor underneath the local tip is dependent on the local carrier concentration of the semiconductor. In SSRM, the local resistivity, allowing the determination of the doping concentration, is determined via the spreading resistance measurement at the tip/sample system, applying an additional DC bias on the sample and using a logarithmic current amplifier. As multi-parameter experiments, these two techniques need of an accurate calibration method for a quantitative doping analysis.
In this study we present first a doping calibration method based on cross-sectional scanning of multilayers samples ,with , for pedagogic demonstration, different Ga doping concentration in n-type ZnO allowing the quantitative measurement by SCM and SSRM. Then, to study NWs, we have developed a methodology of sample preparation, based on dip-coating filling of NWs field. The dip-coating parameters as coating solution, removal rate and NW field morphology have been controlled by SEM, ellipsometry and atomic force microscopy topography in order to optimize the filling and polishing process.
As examples, some results are presented on ZnO and GaN NW: One important results has been to be able to measure using SCM and SSRM, the non-intentionally n-type doping (nid) of ZnO nanowires, well estimated at 210¹⁸cm^-3, explaining the difficulty to turn these NWs into p-type during p-type doping experiments, a crucial problematic in ZnO. Using antimony (Sb) doping in nid ZnO core/ Sb ZnO shell NW structures, we have successfully determined the decrease of carrier concentration with respect to the nid ZnO core, which can be ascribed to the formation of Sb-related acceptors compensating the native donors. The understanding of this electric compensation mechanism is the clear signature of a p-type Sb doping feasibility in ZnO NW. This important result opens the way to succeed in the p-type doping in ZnO.
The generalization of this doping profiling methodology to other NW semiconductors is also pointed out for instance to study the non homogeneity of Si and Mg doping in GaN NWs.

Since the interface between ionic liquids (ILs) and solids always plays a critical role in important applications such as coating, lubrication, energy storage, and catalysis, it is essential to uncover the molecular structure and dynamics of ILs confined to solid surfaces. Here, we present our direct observation of a unique double-layering quantized growth of three IL (i.e., [Emim][FAP], [Bmim][FAP] and [Hmim][FAP]) nanofilms on mica. AFM results show that the IL nanofilms initially grow only by covering more surface areas at the constant film thickness of 2 monolayers (ML) until a quantized increase in the film thickness by another 2 ML. Based on the AFM results, we propose a double-layering model describing the molecular structure of IL cations and anions on the mica surface. The interesting double-layering structure can be explained as the result of several competing interactions at the IL-mica interface. Meanwhile, the time-dependent AFM results indicate that the topography of IL nanofilms could change with time, and the mobility of the nanofilm is lower for ILs with longer alkyl chains, which can be attributed to the more ordered packing between longer alkyl chains. Our findings have important implications on the molecular structure and dynamics of ILs confined to solid surfaces.

The dynamic shapes of biologial tissues emerge from a complex interplay of physics, chemistry and genetics, which determines--at each temporal and spatial scale--the mechanical properties that eventually form the adaptive structures of living organisms. Shape and mechanical stability of living organisms rely on precise control in time and space of growth, which is achieved by dynamically tuning the mechanical (viscous and elastic) properties of their hierarchically built structures from the nanometer up. It is now well-established that cellular behaviour (including stem cell differentiation) crucially depends on the mechanical properties of the cells’ environment. Attention has been directed towards the importance of the stiffness of the natural (extracellular matrix, ECM) or artificial matrices where cells grow, with the purpose of either understanding mechanotransduction, or controlling the behaviour of cells in tissue engineering. While stiffness (i.e. the capacity of a material to elastically store mechanical energy) has been the focus of most experimental research, neither cells or matrices are elastic. Biological systems dissipate energy (i.e. they are viscous) and hence they do not respond to mechanical deformations instantaneously (like an ideal Hookean spring), but present different time responses at different spatial scales that characterise their responses to external stimuli. Measuring viscoelasticity (especially at the nanoscale) has remained experimentally challenging [1,2]. I will present atomic force microscopy (AFM)- based techniques developed in my lab to measure and map the nano-viscoelasticity of living organisms, cells, membranes, collagen, ECMs, and tissue engineering matrices across the spatial and temporal (from Hz to 100s of kHz) scales, and chirp-based spectroscopic techniques to assess viscoelasticity from Hz to 100s kHz at the nano and micro scale developed in my lab. I will also present tests for assessing which viscoelasic model better fits the experimental AFM results. Our results have uncovered that extracellular matrices of both plants [3] and tumours present an almost perfect linear viscoelastic behaviour.
References:
[1] “Multifrequency AFM reveals lipid membrane mechanical properties and the effect of cholesterol in modulating viscoelasticity” 2019. Z Al-Rekabi, S Contera; Proceedings of the National Academy of Sciences 115 (11), 2658-2663.
[2] “Mapping nanomechanical properties of live cells using multi-harmonic atomic force microscopy” 2011 A Raman, S Trigueros, A Cartagena, APZ Stevenson, M Susilo, E Nauman, S Contera. Nature Nanotechnology 6 (12), 809.
[3] “Mapping cellular nanoscale viscoelasticity and relaxation times relevant to growth of living Arabidopsis thaliana plants using multifrequency AFM” 2021 J Seifert, C Kirchhelle, I Moore, S Contera. Acta Biomaterialia 121, 371-382

Nanoimaging and probing of physical-chemical characteristics is central to the development of new nanomaterials for energy and environment, communication, computing and security as well as for health and medicine. While all these areas are of significant importance, health and medical applications remain the most challenging because they involve interactions with living organisms. Inherently, such interactions are less predictable and directly linked to human health, which highlights their societal impact. Therefore, resolving the principles that govern biological behaviours and developing modern technologies that exploit new capabilities offered by nanomaterials to control multicellular environment is likely to result in significant advances in a quest for innovative therapies for major health threats including multi drug resistance, cancer and diabetes.
We know that biological responses are not controlled by a single material parameter. It is rather an interplay of many parameters such as size, shape, surface charge, chemistry and mechanical properties that defines the biological outcome. Therefore, correlative characterisation of structural, physical and biological interactions at the nanoscale is paramount in order to correlate nanomaterials to their biological modulatory capabilities. Precise knowledge of key characteristics of nanomaterials will ultimately allow us to predict their safety and fate in biological systems and in the environment. It will unlock new opportunities to create safe-by-design systems for various biomedical applications.
To overcome limitations of current methods we proposed a correlative approach that uses nanoscale infrared spectroscopy, Lorentz contact resonance spectroscopy and Molecular force probe spectroscopy to interrogate nanoscale mechanical properties and map chemical structure of nanoparticles. To demonstrate strength of the nanocorrescopy in nanomaterials characterisation we used a copolymer composite mPEG-PLGA (methoxy-poly (ethylene glycol)-b-poly (lactic-co-glycolic) acid) nanorods. We demonstrated that the nanocorresocpy is capable of characterising precisely distribution of viscoelastic and chemical properties with the spatial resolution of <150 nm and <20 nm respectively. We further showed that this methodology enable characterisation of the corona formation on nanoparticles and for the first time assess its evolution in real time. We believe that the nanocorrescopy provides new opportunities to characterise variety of biomaterials used in biomedicine to ensure their safety, reliability, and sustainability.

In-situ AFM typically possesses the single-molecule detection sensitivity of the antibody-antigen binding, however, yielding inevitably great limitations due to operational issues from a practical application viewpoint. Here, we propose a method of an ex-situ AFM-molecule chip technique with the feature of graphically superimposed alignment that enables ex-situ AFM analysis of an immobilized antibody at the same location on a semiconductor chip surface before and after incubation with its antigen, which makes all required chemical/biological treatments feasibly executed in the standard laboratory manner, allowing the single-molecule ex-situ AFM detection in a way with more practicality, flexibility, and versatility. Accordingly, by taking the hepatitis B virus X protein (HBx) for instance, the individual information of the topographical characteristics for the immobilized IgG antibodies/aggregates on the chip surface can be extracted using this ex-situ AFM analysis method, and further analyzed statistically. Experimental observations revealed that individual IgG antibodies covalently immobilized on the aldehyde-terminated chip surface, presumably featuring a predominately head-on orientation, possessed an AFM height of approximately 5.416 nm in statistical analysis. Furthermore, information regarding the AFM height change of individual IgG antibody/aggregate arising from the conformational change in the IgG-HBx complex formation was investigated in a statistical manner as well, leading to an increased height of approximately 1 nm. To broaden the application spectrum of the ex-situ AFM-molecule chip technique, we attempted to apply this technique to identify the locations and spatial distributions of the immobilized molecules on the bioFET surfaces. The surface topographical measurements at the same location on the nanowire bioFET surface were recorded after the chip cleaning, antibody modification, and antigen incubation steps, respectively. As a result, information regarding the correlation between the spatial distributions of the biological molecules on the bioFET surface and the corresponding electrical characteristics of the nanowire bioFETs can be obtained, highlighting that the single-molecule ex-situ analysis method can be beneficial to the development of bioFET applications. More importantly, other analysis modules in the SPM family can also be employed to simultaneously record various fundamental characteristics of the molecules on the chip surface at the single-molecule level while recording the topographical information of the chip surface using an AFM module, significantly revealing versatile applications of the ex-situ SPM-molecule chip technique (here using the SPM to replace the AFM for highlighting the potentials of multi-functional applications) in multidisciplinary areas across materials, chemistry, physics, and biology.