Dr. Céline Lichtensteiger: "Scanning probe microscopy and x-ray diffraction studies of domains and domain walls in ferroelectric thin films"
Céline Lichtensteiger, Marios Hadjimichael, Christian Weymann, Jean-Marc Triscone, Patrycja Paruch
DQMP, University of Geneva – 24 Quai Ernest Ansermet – CH – 1211 Geneva 4 - Switzerland
In ferroelectric thin films, the complex interplay between mechanical and electrostatic boundary conditions allows for the formation of a large variety of domain structures with fascinating properties. Heterostructuring and careful tuning of the epitaxial strain allow for precise control of these boundary conditions, leading to the formation of novel domain structures such as polar merons in tensile-strained PbTiO3 films and skyrmions in PbTiO3/SrTiO3 superlattices. The structural coupling across PbTiO3 layers in these systems can also lead to the formation of complex three-dimensionally ordered supercrystal structures, recently observed in tensile-strained PbTiO3/SrTiO3 and PbTiO3/SrRuO3 superlattices[4,5]. Domain structures in ferroelectric systems not only change the properties of the ferroelectric itself, but can also be used to change the properties of other materials through electrostatic and structural coupling.
Here, we will show how one can use atomic force microscopy to study ferroelectric domains and domain walls in heterostructures of PbTiO3 deposited using off-axis RF magnetron sputtering. Through an overview of our work based on samples grown on SrTiO3 substrates – imposing purely out-of plane polarisation in PbTiO3 thin films, (Pb,Sr)TiO3 solid solutions and PbTiO3/SrTiO3 superlattices, we will see how to control the presence of intrinsic ferroelectric domains as well as their size and their stability. This can be achieved by tuning different parameters such as the electrostatic boundary conditions, the layer thickness, the concentration in the solid solutions and relative layer thickness in the superlattices, or even the deposition conditions. We will then focus on more recent results obtained on DyScO3 substrates, imposing a tensile strain that favours a ferroelastic domain structure, with PbTiO3 adopting both in-plane and out-of-plane polarization orientations. Using a combination of x-ray diffraction (XRD) and atomic force microscopy (AFM), we studied the domain structure in these systems as a function of PbTiO3 layer thickness. We found that the anisotropic strain imposed by the orthorhombic substrate creates a large asymmetry in the domain configuration, with domain walls macroscopically aligned along one of the two in-plane directions. We show that above a certain critical thickness, the large structural distortions associated with the ferroelastic domains propagate through the top SrRuO3 layer, creating a modulated structure that extends beyond the ferroelectric layer thickness, with signatures observed both in XRD and AFM. Our results shine light on the complexity of ferroelastic domain structures in PbTiO3-based multilayers and their sensitivity to both electrostatic and mechanical boundary conditions.
 Catalan et al. Rev. Mod. Phys 2012
 Wang et al. Nature Materials 2020
 Das et al. Nature 2019
 Stoica et al. Nature Materials 2019
 Hadjimichael, Li, et al. Nature Materials 2021
 Zubko et al. Nature 2016
Prof. Dr. David Alsteens: "Probing Virus Binding Sites to Animal Cells Using Atomic Force Microscopy"
Nanobiophysics lab, Université catholique de Louvain, Croix du sud 4-5, bte L7.07.07, 1348 Louvain-La-Neuve, Belgium
During the last three decades, a series of key technological improvements turned atomic force microscopy (AFM) into a nanoscopic laboratory to directly observe and chemically characterize molecular and cellular biological systems under physiological conditions. I will present the key technological improvements that enable us to apply AFM as analytical laboratory to observe and quantify living biological systems at the nanoscale. I will report the use of advanced FD-based technology combined with chemically functionalized tips to probe the localization and interactions of chemical and biological sites on single native proteins and on living cells at high-resolution. I will present how an atomic force and confocal microscopy set-up allows the surface receptor landscape of cells to be imaged and the virus binding events within the first millisecond of contact with the cell to be mapped at high resolution (<50 nm). I will also highlight theoretical approaches to contour the free-energy landscape of early binding events between virus and cell surface receptors.
Figure. Combination of AFM and fluorescence microscopy image showing an AFM tip functionalized with a single virus while mapping virus binding sites on living mammalian cells
- M. Koehler et al., Nat Commun. (2021) Accepted
- M. Delguste et al., Nano Letters 21 (2021) 847-83
- J. Yang et al., Nat Commun 11 (2020) 4541
- M. Koehler et al. , Nat. Commun. 10 (2019) 4460
- M. Delguste et al., Sci. Adv. 4 (2018) eaat1273
- M. Eubelen et al., Science (2018) eaat1178
- R. Newton et al., Nat. Protoc. 11 (2017) 2275-2292
- D. Alsteens et al. Nat. Nanotechnol. 12 (2017) 177-183
- D. Alsteens et al. Nat. Rev. Materials 2 (2017) 17008
- D. Alsteens et al. Nat. Methods 12 (2015) 845-851
Prof. Thomas Perkins: "Probing the hidden dynamics and energetics of diverse biomolecular systems by AFM"
Single-molecule force spectroscopy (SMFS) has become a critical tool in unraveling the behavior of mechanoresponsive proteins. High-precision SMFS studies of nucleic acids and globular proteins have leveraged the stability and precision of custom-built optical traps, yielding kinetic rate constants, energetics, intermediate states, folding pathways, and finally a full 1D projection of the underlying free-energy landscape. Recently, we have developed a set of modified cantilevers for atomic force microscopy (AFM) that has enabled high-precision studies on a commercial AFM. For example, we achieved sub-pN stability over ~80 s coupled with 2-μs temporal resolution. To demonstrate broad applicability, we performed equilibrium and non-equilibrium assays on 3 classes of bio-molecules (membrane proteins, globular proteins, and structured RNA). Importantly, these studies were not limited to high forces—the traditional strength of AFM—but also characterized low-force transitions (5–30 pN). Concurrent advances in data acquisition and site-specific bio-conjugation allowed the same individual molecule to be unfolded and refolded more than 1,000 times in 15 min. The resulting increased data quantity and quality enabled free-energy landscape reconstruction from both equilibrium and non-equilibrium data for all three classes of bio-molecules. Moreover, improved constant-force assays of a protein-ligand complex yielded the height of the transition state in addition to the two traditional SMFS parameters (e.g., zero-force dissociation rate constant and the distance to the transition state). Looking forward, we anticipate that the ease-of-use of such advanced assays on a commercial AFM will accelerate the high-precision characterization of a broad range of mechano-responsive systems.
Prof. Nicholas D. Spencer: "Tailoring Hydrogel Surfaces"
Hydrogels have myriad applications ranging from contact lenses to agriculture. Their surfaces and frictional behavior can be modified by molding techniques, but the generally accepted mechanism for this has been shown to be incorrect. By extensive use of AFM nanoindentation techniques, coupled with macroscopic friction measurements, infrared spectroscopy and neutron-reflectivity studies, we have definitively pinned down the mechanism for the molding effect on hydrogels, and developed approaches that allow spatially localized frictional tailoring of hydrogels.
Dr. Thorsten Hugel: "Comprehensive biomaterial characterization by AFM and fluorescence"
Institute of Physical Chemistry and Cluster of Excellence livMatS @ FIT
University of Freiburg, Germany
The AFM is a versatile tool to investigate a large and still growing number of systems, in particular in combination with fluorescence microscopy. First, we will present an investigation of human cartilage. In a combination of AFM and fluorescence microscopy we can show that local changes in the organization of fluorescent stained cells, a marker for early osteoarthritis, lead to a significant local reduction of the elastic modulus, local thinning of the collagen fibers, and a roughening of the articular surface . This approach is currently extended towards locally measuring the frequency dependent storage and loss modulus by AFM
indentation and its correlation to fluorescence images.
Second, we will present fundamental insights into the formation and breaking of multivalent bonds. Since the first mechanical characterization of the silicon-carbon bond by AFM in 1999 , many other physical and chemical bonds have been characterized, usually in well defined systems. Here, we disentangle physical and
chemical bonds that form in mussel-inspired coatings. Both, the timescale of bond formation and their strength is determined. This helps to understand the interplay between adsorption (physisorption) and chemical reactions (chemisorption) in polymer coatings.
 Tschaikowsky et al., Acta Biomaterialica, in revision
 Grandbois et al., Science, 283 (5408), 1727-1730 (1999)
Dr. Bizan Balzer: "Single Molecule Friction"
Institute of Physical Chemistry and Cluster of Excellence livMatS @ FIT
University of Freiburg, Germany
Friction is defined as the resistance acting against the motion of two bodies sliding past each other. The situation becomes more complicated in soft materials, biological or biomimetic systems, which usually display complex shapes and elasticities and hence complicate the understanding of friction on a molecular scale. However, it remains still unclear how molecular friction properties affect macroscopic friction properties of those materials. The AFM is a versatile tool for imaging and force spectroscopy at the nanoscopic to mesoscopic scale which we use to understand friction and adhesion properties of molecular systems.
We use AFM-based force spectroscopy to probe single polymer adhesion and friction at interfaces in liquid environment. To that aim we have developed functionalization protocols for covalent binding of single polymers to AFM cantilever tips.  We have used vertical pulling of single polymers to probe the adhesion and mechanics of single polymers on a great variety of solid substrates. [2,3] Then, we have extended our
method towards lateral pulling of single polymers revealing different friction motifs. [4,5]
Here, we will show how to use angle dependent pulling in order to study the role of single molecular bonds and their multidirectional, velocity dependent on- and off-binding. This will enhance the understanding of the relation between single molecule friction and adhesion and will help to discover the molecular origin of friction.
Furthermore, we will present how different moieties of complex molecules such as
mucin affect the lubrication properties of surfaces.
 Kolberg et al., J. Vis. Exp., 157, e60934 (2020).
 Horinek et al., PNAS, 105(8), 2842–2847 (2008).
 Kolberg, et al., J. Am. Chem. Soc., 141 (29), 11603 (2019).
 Balzer et al., Angew. Chem. Int. Ed. 52,25, 6541 (2013).
 Balzer et al., Soft Materials 12, S106-S114 (2014).
Prof. Dr. Roland Bennewitz: "Single-molecule force spectroscopy at biomaterial surfaces"
We have investigated the mechanical properties of molecular linkers which mediate cell attachment on biomaterial surfaces by means of single-molecule force spectroscopy. Attachment and spreading of cells on poly(ethylene glycol diacrylate) (PEGDA) hydrogels depend on their mechanical properties, for examples when Young’s modulus E of a hydrogel is varied. A factor of ten in the elastic modulus E corresponded to a factor of five in the effective spring constant k of single crosslinks, indicating a transition in scaling with the mesh size ξ from the macroscopic E ∝ ξ⁻³ to the molecular k ∝ ξ⁻² . The effective stiffness of single linkers was also measured for a second polymer network based on four-arm star-PEG molecules which interpenetrated the PEGDA hydrogel.
When linkers are connected to a light-driven molecular motor, an optoregulated force can be applied to cellular receptors . The force is produced by an increasing entanglement of linkers and was quantified by flow force microscopy, where the molecular motors pull tethered beads against the flow in a microfluidic cell.
 A. Colak, B. Li, J. Blass, K. Koynov, A. del Campo, R. Bennewitz, The mechanics of single cross-links which mediate cell attachment at a hydrogel surface, Nanoscale, 11 (2019) 11596-11604.
 Y. Zheng, M.K.L. Han, R. Zhao, J. Blass, J. Zhang, D.W. Zhou, J.-R. Colard-Itté, D. Dattler, A. Colak, M. Hoth, A.J. García, B. Qu, R. Bennewitz, N. Giuseppone, A. del Campo, Optoregulated force application to cellular receptors using molecular motors, Nature Communications, accepted (2021).
Dr. Kerstin Blank: "Mechanoresponsive proteins - from molecular mechanisms towards applications in biology and materials science"
Max Planck Institute of Colloids and Interfaces,
Potsdam Science Park, Potsdam-Golm, Germany
Proteins are essential building blocks of biogenic materials. In addition to purely protein-based materials, a wide range of different composite materials are formed, where proteins mediate specific interactions with other organic molecules (e. g. carbohydrates) or mineral surfaces. In all cases, the thermodynamic, kinetic and mechanical stability of protein interactions crucially determines material assembly, structure and function. From a mechanical point of view, function includes structural stability as well as mechanosensing, actuation and self-healing. Using single-molecule force spectroscopy, we aim to establish sequence-structure-MECHANICS relationships of protein-based material building blocks with the goal of understanding their role in Nature and to subsequently utilize these building blocks for building smart bioinspired materials. Specifically, I will focus on two examples. The first example introduces coiled coils, which are highly abundant building blocks in mammalian tissues. Using synthetic coiled coils, we have unravelled key factors that determine the stability of these structures against shear forces [1-3]. We subsequently utilized this knowledge to establish a library of mechanically calibrated coiled coils, which are now further developed as mechanosensors for cell biology applications and as mechanoresponsive hydrogel crosslinks [4-6]. The second example shows bacteria-derived proteins that control the formation of nanometre-sized magnetite crystals. Here, single-molecule force spectroscopy serves as an excellent tool to probe the kinetics and the crystal face specificity of the protein-magnetite interaction . Even though these proteins do not experience force in Nature, engineered variants with controlled binding strength can potentially serve as powerful building blocks for controlling the properties of magnetic particle-reinforced composites.
M. Goktas, C. Luo, R. M. A. Sullan, A. E. Bergues-Pupo, R. Lipowsky, A. Vila Verde, K. G. Blank (2018) Chem. Sci. 9:4610
P. López-García, M. Goktas, A. E. Bergues-Pupo, B. Koksch, D. Varón Silva, K. G. Blank (2019) Phys. Chem. Chem. Phys. 21:9145
P. López-García, A. D. de Araujo, A. E. Bergues-Pupo, I. Tunn, D. P. Fairlie, K. G. Blank (2021) Angew. Chem. Int. Ed. 60:232
I. Tunn, A. S. de Léon, K. G. Blank, M. J. Harrington (2018) Nanoscale 10:22725
I. Tunn, M. J. Harrington, K. G. Blank (2019) Biomimetics 4:25
E. M. Grad, I. Tunn, D. Voerman, A. S. de Léon, R. Hammink, K. G. Blank (2020) Front. Chem. 8:536
A. Pohl, F. Berger, R. M. A. Sullan, C. Valverde-Tercedor, K. Freindl, N. Spiridis, C. T. Lefèvre, N. Menguy, S. Klumpp, K. G. Blank, D. Faivre (2019) Nano Lett. 19: 8207
Prof. Dr. Karin Jacobs: "Quantitative bioadhesion studies by SPM and force spectroscopy"
Saarland University, Germany
A successful adhesion of proteins and/or bacteria to surfaces is a crucial step in initial biofilm formation. Using a combined experimental and computational approach, we investigated the adhesion of the pathogenic bacterium Staphylococcus aureus to smooth, hydrophilic and hydrophobic surfaces. We used atomic force microscopy-based single-cell force spectroscopy and Monte Carlo simulations to investigate the similarities and differences in adhesion to hydrophilic and hydrophobic surfaces. Our results show that binding to both types of surfaces is mediated by thermally fluctuating cell wall macromolecules that behave differently on each type of substrate . For nanorough surfaces, we find that as the size of the nanostructures increases, the adhesion forces decrease in a way that can be quantified by the area of the surface that is available for the tethering of cell wall molecules . To be able to quantitatively link surface topography and bacterial adhesion, the surface structures were characterized by a detailed morphometric analysis based on Minkowski functionals. We expect these findings to be important for understanding the adhesion behavior of many bacterial species as well as other microorganisms and even nanoparticles with soft, macromolecular coatings used for biological diagnostics, for example.
 E. Maikranz et al., Nanoscale 12 (2020) 19267 DOI: 10.1039/d0nr03134h
 C. Spengler et al., Nanoscale 11 (2019) 19713 DOI: 10.1039/C9NR04375F
Dr. Alice Pyne: "Quantitative bioadhesion studies by SPM and force spectroscopy"
University of Sheffield, Sir Robert Hadfield Building, Sheffield, S1 3JD, UK
Understanding how DNA behaves in its cellular environment is a challenge of complexity, which can be enhanced by a better understanding of the fundamental properties of DNA. In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. We overcome these limitations by combining high-resolution AFM¹ and atomistic MD simulations to resolve the structure, conformation and dynamics of supercoiled DNA to the base-pair level (Figure 1)².
We use DNA minicircles, only twice the persistence length of DNA, to probe the structure and function of negatively-supercoiled DNA. These minicircles are small enough to be simulated at the atomistic level by MD and to be visualized at high (double-helix) resolution by AFM experiments in solution We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility³. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.
Figure 1. Structural diversity in supercoiled DNA minicircles. Synergistic high-resolution AFM images (a-d) and MD snapshots (e) of natively supercoiled DNA minicircles show striking structural diversity in natively supercoiled DNA minicircles. Scale bars: 10 nm. Height scale (inset): 2.5 nm for all images
1. Pyne, A., Thompson, R., Leung, C., Roy, D. & Hoogenboom, B. W. Single-Molecule Reconstruction of Oligonucleotide Secondary Structure by Atomic Force Microscopy. Small 10, 3257–3261 (2014).
2. Pyne, A. L. B. et al. Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides. Nature Communications 12, 1053 (2021).
3. Beton, J. G. et al. TopoStats – A program for automated tracing of biomolecules from AFM images. Methods (2021) doi:10.1016/j.ymeth.2021.01.008.
Prof. Dr. Michael Nash: "Engineering biophysical properties of molecular therapeutics"
University of Basel & ETH Zurich, Switzerland
Mechanical anisotropy is a general property of biomolecules that manifests when mechanical forces are applied from different directions. Due to its vectorial nature, mechanical force always has not only a magnitude but also an associated direction, and molecular deformation is significantly modulated by the anchoring positions through which forces are applied. This talk will focus on recent work in the Nash Lab optimizing molecular pulling geometries of biotherapeutics. We report a new AFM measurement setup and bioorthogonal protein engineering workflow that allows us to apply tension to non-antibody therapeutic binding scaffolds through controlled anchor points at the N-terminus, C-terminus or at internal sequence positions. This method has revealed that dissociating complexes by pulling from internal sequence positions significantly increases the unbinding forces. Anchor point optimization can be considered a new paradigm for improving binding strength of therapeutic scaffolds that does not require mutagenesis of binding paratopes, with implications for the design of drug delivery systems optimized to work under shear flow.
 Liu, Z.; Moreira, R. A.; Dujmović, A.; Liu, H.; Yang, B.; Poma, A. B.; Nash, M. A. Optimizing Mechanostable Anchor Points of Engineered Lipocalin in Complex with CTLA-4. Bioarxiv, 2021, 2021.03.09.434559. https://doi.org/10.1101/2021.03.09.434559.
Prof. Dr. Georg Papastavrou "New degrees of freedom in AFM: Combining AFM with Nanofluidics"
University of Bayreuth, Germany
Atomic force microscopy (AFM) has developed in the last decades to a ‘classical’ surface analytical technique. Based on the simple idea of ‘scratching’ with a sharp tip over a surface, AFM became the origin of a plethora of imaging modes and combinations with other techniques. These AFM-based techniques impressively demonstrate the potential of and the need for further analytical techniques with high lateral resolution.
Here, some applications of the combination of AFM with nanofluidic techniques, also known as Fluid Force Microscopy (FluidFM), is presented. The basic idea is to fabricate AFM-cantilevers with an internal channel and an aperture in the micro- to nanometer range at the end. Originally this technique was intended to be used in the biomedical field, for example to manipulate single cells and bacteria. However, the possibility to eject and aspirate fluids, respectively, is much more universal and can be applied also in material or colloid science.
The colloidal probe technique, which is based on AFM, revolutionized direct force measurements. Unfortunately, for many applications, the surface chemistry and the minimum dimensions for the probe particles restricted the general use of this technique. The FluidFM-approach allowed for the first time to overcome these limitations. We demonstrated the use of 300 nm sized carboxylate-modified latex particles as well as sub-micron core-shell particles with a soft exterior layer as probes. Moreover, by utilizing electrokinetic effects in the interior channel, it became possible to detect the aspiration of small colloidal particles to the cantilever’s aperture in order to automatize the process.
Furthermore, the FluidFM-technique allows for structing or deposition of materials on the micrometer-level. We demonstrate a proof-of-principle for the etching of patterns in very soft hydrogel films. The resulting ‘chemical writing’ process has been studied in detail and the influence of various parameters, such as applied pressure and time, has been validated.
 Meister, A. et al. FluidFM: Combining Atomic Force Microscopy and Nanofluidics in a Universal Liquid Delivery System for Single Cell Applications and Beyond. Nano Letters 9, 2501-2507 (2009).
 Mark, A. et al. The Next Generation of Colloidal Probes: A Universal Approach for Soft and Ultra‐Small Particles. Small 15, 1902976 (2019).
 Mark, A. et al. Electrokinetics in Micro-channeled Cantilevers: Extending the Toolbox for Reversible Colloidal Probes and AFM-Based Nanofluidics. Scientific Reports 9, 20294 (2019).
 Helfricht, N. et al. Writing with Fluid: Structuring Hydrogels with Micrometer Precision by AFM in Combination with Nanofluidics. Small 13, 1700962 (2017).
Prof. Dr. Lukas Eng "Applying Sample Strain in-situ –
A Multimodal Nanoscale Analysis including scanning probe microscopy"
Ekta Singh ¹ and Lukas M. Eng ¹,²
¹ Institute of Applied Physics, TU Dresden, Nöthnitzerstr. 61, 01187 Dresden, Germany
² ct.qmat: Würzburg-Dresden Cluster of Excellence - EXC 2147, TU Dresden, Germany
Probing intriguing sample properties by applying external stimuli in-situ is of uttermost benefit to both fundamental research and applied sciences. Magnetic and electrical fields certainly count to the two most-spread vector fields that have been widely used into this, for probing material’s phase transitions or hysteretic behavior. What is less know is the application of mechanical stress fields, i.e. in-situ straining the sample both tensile and compressively.
Here, we report on the combination of an uniaxial stress cell within the NX-10 scanning-force-microscope from ParkSystems, with the goal to inspect the nanoscale properties of 2-dimensional materials (2DMs) or ferroelectric domains and domain walls (DWs) under uniaxial mechanical stimulus. We will show how to integrate that system into a COVID-19-friendly, hence full remotely-controlled user platform. Moreover, examples of the strain impact on a variety of samples will be presented, such as DW conductivity in bulk LiNbO3 as measured by cAFM, for BiFeO3 ferroelectric thin-films investigated by PFM, and properties of MoS2 as the prototype 2DM. All these experiments are backed up with complementary techniques such as µ-Raman spectroscopy or macroscopic transport measurements.