Force Spectroscopy Measurements Research Articles

E-Protein Protonation Titration-Induced Single-Particle Chemical Force Spectroscopy for Microscopic Understanding and pI Estimation of Infectious DENV.

The ionization state of amino acids on the outer surface of a virus regulates its physicochemical properties toward the sorbent surface. Serologically different strains of the dengue virus (DENV) show different extents of infectivity depending upon their interactions with a receptor on the host cell. To understand the structural dependence of E-protein protonation over its sequence dependence, we have followed E-protein titration kinetics both experimentally and theoretically for two differentially infected dengue serotypes, namely, DENV-2 and DENV-4. We have performed E-protein protonation titration-induced single-particle chemical force spectroscopy using an atomic force microscope (AFM) to measure the surface chemistry of DENV in physiological aqueous solutions not only to understand the charge distribution dynamics on the virus surface but also to estimate the isoelectric point (pI) accurately for infectious dengue viruses. Cryo-EM structure-based theoretical pI calculations of the DENV-2 surface protein were shown to be consistent with the evaluated pI value from force spectroscopy measurements. We also highlighted here the role of the microenvironment around the titrable residues (in the 3D-folded structure of the protein) in altering the pKa. This is a comprehensive study to understand how the cumulative charge distribution on the outer surface of a specific serotype of DENV regulates a prominent role of infectivity over minute changes at the genetic level.

A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force-Confocal Fluorescence Microscopy.

Integrated single-molecule force-fluorescence spectroscopy setups allow for simultaneous fluorescence imaging and mechanical force manipulation and measurements on individual molecules, providing comprehensive dynamic and spatiotemporal information. Dual-beam optical tweezers (OT) combined with a confocal scanning microscope form a force-fluorescence spectroscopy apparatus broadly used to investigate various biological processes, in particular, protein:DNA interactions. Such experiments typically involve imaging of fluorescently labeled proteins bound to DNA and force spectroscopy measurements of trapped individual DNA molecules. Here, we present a versatile state-of-the-art toolbox including the preparation of protein:DNA complex samples, design of a microfluidic flow cell incorporated with OT, automation of OT-confocal scanning measurements, and the development and implementation of a streamlined data analysis package for force and fluorescence spectroscopy data processing. Its components can be adapted to any commercialized or home-built dual-beam OT setup equipped with a confocal scanning microscope, which will facilitate single-molecule force-fluorescence spectroscopy studies on a large variety of biological systems.

Localized Plasmonic Heating for Single-Molecule DNA Rupture Measurements in Optical Tweezers.

To date, studies on the thermodynamic and kinetic processes that underlie biological function and nanomachine actuation in biological- and biology-inspired molecular constructs have primarily focused on photothermal heating of ensemble systems, highlighting the need for probes that are localized within the molecular construct and capable of resolving single-molecule response. Here we present an experimental demonstration of wavelength-selective, localized heating at the single-molecule level using the surface plasmon resonance of a 15 nm gold nanoparticle (AuNP). Our approach is compatible with force-spectroscopy measurements and can be applied to studies of the single-molecule thermodynamic properties of DNA origami nanomachines as well as biomolecular complexes. We further demonstrate wavelength selectivity and establish the temperature dependence of the reaction coordinate for base-pair disruption in the shear-rupture geometry, demonstrating the utility and flexibility of this approach for both fundamental studies of local (nanometer-scale) temperature gradients and rapid and multiplexed nanomachine actuation.

Pt-Black-Modified (Hemi)spherical AFM Sensors: In Situ Imaging of Light-Driven Hydrogen Peroxide Evolution.

In this work, we present (hemi)spherical atomic force microscopy (AFM) sensors for the detection of hydrogen peroxide. Platinum-black (Pt-B) was electrodeposited onto conductive colloidal AFM probes or directly at recessed microelectrodes located at the end of a tipless cantilever, resulting in electrocatalytically active cantilever-based sensors that have a small geometric area but, due to the porosity of the films, exhibit a large electroactive surface area. Focused ion beam-scanning electron microscopy tomography revealed the porous 3D structure of the deposited Pt-B. Given the accurate positioning capability of AFM, these probes are suitable for local in situ sensing of hydrogen peroxide and at the same time can be used for (electrochemical) force spectroscopy measurements. Detection limits for hydrogen peroxide in the nanomolar range (LOD = 68 ± 7 nM) were obtained. Stability test and first in situ proof-of-principle experiments to achieve the electrochemical imaging of hydrogen peroxide generated at a microelectrode and at photocatalytically active structured poly(heptazine imide) films are demonstrated. Force spectroscopic data of the photocatalyst films were recorded in ambient conditions, in solution, and by applying a potential, which demonstrates the versatility of these novel Pt-B-modified spherical AFM probes.

Probing the interactions of the HIV-1 matrix protein-derived polybasic region with lipid bilayers: insights from AFM imaging and force spectroscopy.

The human immunodeficiency virus type 1 (HIV-1) matrix protein contains a highly basic region, MA-HBR, crucial for various stages of viral replication. To elucidate the interactions between the polybasic peptide MA-HBR and lipid bilayers, we employed liquid-based atomic force microscopy (AFM) imaging and force spectroscopy on lipid bilayers of differing compositions. In 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, AFM imaging revealed the formation of annulus-shaped protrusions upon exposure to the polybasic peptide, accompanied by distinctive mechanical responses characterized by enhanced bilayer puncture forces. Importantly, our AFM-based force spectroscopy measurements unveiled that MA-HBR induces interleaflet decoupling within the cohesive bilayer organization. This is evidenced by a force discontinuity observed within the bilayer's elastic deformation regime. In POPC/cholesterol bilayers, MA-HBR caused similar yet smaller annular protrusions, demonstrating an intriguing interplay with cholesterol-rich membranes. In contrast, in bilayers containing anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) lipids, MA-HBR induced unique annular protrusions, granular nanoparticles, and nanotubules, showcasing its distinctive effects in anionic lipid-enriched environments. Notably, our force spectroscopy data revealed that anionic POPS lipids weakened interleaflet adhesion within the bilayer, resulting in interleaflet decoupling, which potentially contributes to the specific bilayer perturbations induced by MA-HBR. Collectively, our findings highlight the remarkable variations in how the polybasic peptide, MA-HBR, interacts with lipid bilayers of differing compositions, shedding light on its role in host membrane restructuring during HIV-1 infection.

Nanoscale dynamical investigation of the hemoglobin complex with the bacterial protein IsdB: is their interaction stabilized by catch bonds?

Iron-regulated surface determinant B (IsdB) is a surface protein of Staphylococcus aureus that plays essential roles in host cell invasion by mediating both bacterial adhesion and hemic iron acquisition. Single-molecule experiments have recently revealed that the binding of IsdB to vitronectin and integrins is dramatically strengthened under mechanical stress conditions, promoting staphylococcal adhesion. Here we conducted atomic force spectroscopy (AFS) measurements of the interaction between IsdB and hemoglobin (Hb), in both its oxidized (metHb) and reduced forms (HbCO). While the former represents the natural substrate for IsdB, the latter is resistant to heme extraction. For the unbinding between IsdB and HbCO, we obtained a linear trend in the Bell-Evans plot, indicative of a weakening of the interaction upon mechanical stress. For the unbinding between IsdB and metHb, we found similar behavior at low loading rates. Remarkably, a non-linear trend of the complex interaction force was detected at higher force-pulling rates. Such behavior may provide some cues to the ability of IsdB to form stress-dependent bonds also with Hb, possibly enabling a more efficient heme transfer through stabilization of the transient (in vivo) IsdB-Hb complex.

Atomic force microscopy analysis of Pel polysaccharide- and type IV pili-mediated adhesion of Pseudomonas aeruginosa PA14 to an abiotic surface.

Type IV pili (TFP) contribute to the ability of microbes such as Pseudomonas aeruginosa to engage with and move across surfaces. We reported previously that P. aeruginosa TFP generate retractive forces of ∼30 pN and provided indirect evidence that TFP-mediated surface attachment was enhanced in the presence of the Pel polysaccharide. Here, we use different mutants defective in flagellar, Pel production or TFP production - alone or in combination - to decipher the relative contribution of these biofilm-promoting factors for P. aeruginosa adhesion. By means of atomic force microscopy (AFM), we show that mutating the flagellum (ΔflgK mutant) results in an increase in Pel polysaccharide production, but this increase in Pel does not result in an increase in surface adhesive properties compared to those previously described for the WT strain. By blocking Pel production in the ΔflgK mutant (ΔflgKΔpel), we directly show that TFP play a major role in the adhesion of the bacteria to hydrophobic AFM tips, but that the adhesion force is only slightly impaired by the absence of Pel. Inversely, performing single-cell force spectroscopy measurements with the mutant lacking TFP (ΔflgKΔpilA) reveals that the Pel can modulate the attachment of the bacteria to a hydrophobic substrate in a time-dependent manner. Finally, little adhesion was detected for the ΔflgKΔpilAΔpelA triple mutant, suggesting that both TFP and Pel polysaccharide make a substantial contribution to bacteria-substratum interaction events. Altogether, our data allow us to decipher the relative contribution of Pel and TFP in the early attachment by P. aeruginosa.

Effect of temperature on organic fouling and cleaning efficiency of nanofiltration membranes for loch water treatment

We investigated the effect of solution temperature on organic fouling and cleaning of polypiperazine nanofiltration (NF) membranes. Fouling experiments were conducted in the temperature range 5 °C ≤ T ≤ 15 °C, characteristic of surface waters in northern latitudes, such as Scottish lochs. Results of laboratory-scale fouling experiments using alginate, a polysaccharide constituent of extracellular polymeric substances, showed a moderate increase in flux loss at 15 °C (41 %) compared to 5 °C (36 %). Analysis of the fouling experiments using a series-resistance model showed that the greater extent of fouling with rising temperature stems from a monotonic increase in water permeance with T. Interfacial property characterisation provided further insight into the effect of temperature on fouling determinants such as membrane hydrophobicity, nanoscale roughness, surface charge, and surface forces. No T-dependence of surface roughness was found. Similarly, we found that water contact angle is invariant within the temperature range investigated, suggesting that the observed fouling behaviour is not due to modulation of hydrophobic interactions. Conversely, colloidal-probe force spectroscopy (CPFS) measurements showed that adhesion forces become stronger with rising temperature. Further analysis showed that repulsive forces – which oppose colloidal particle deposition – become weaker with rising temperature, consistent with fouling trends. Determination of the membrane zeta potential showed that surface charge is invariant over the range of temperatures investigated, suggesting that repulsive forces have a non-electrostatic (likely steric) origin. Additionally, we assessed the effect of temperature on physical and chemical cleaning efficiency, comparing cleaning at the same temperature as the fouling experiments (5 °C ≤ T ≤ 15 °C) with cleaning at 30 °C. We observed that physical cleaning, consisting of water circulation over the fouled membrane surface, is insufficient to remove the foulant layers. On the other hand, chemical cleaning is able to restore 95 % of the original membrane permeance at 10 °C, but only 74 % at 5 °C.

Quantifying the Viscosity of Individual Submicrometer Semisolid Particles Using Atomic Force Microscopy.

Atmospheric aerosols' viscosities can vary significantly depending on their composition, mixing states, relative humidity (RH) and temperature. The diffusion time scale of atmospheric gases into an aerosol is largely governed by its viscosity, which in turn influences heterogeneous chemistry and climate-relevant aerosol effects. Quantifying the viscosity of aerosols in the semisolid phase state is particularly important as they are prevalent in the atmosphere and have a wide range of viscosities. Currently, direct viscosity measurements of submicrometer individual atmospheric aerosols are limited, largely due to the inherent size limitations of existing experimental techniques. Herein, we present a method that utilizes atomic force microscopy (AFM) to directly quantify the viscosity of substrate-deposited individual submicrometer semisolid aerosol particles as a function of RH. The method is based on AFM force spectroscopy measurements coupled with the Kelvin-Voigt viscoelastic model. Using glucose, sucrose, and raffinose as model systems, we demonstrate the accuracy of the AFM method within the viscosity range of ∼104-107 Pa s. The method is applicable to individual particles with sizes ranging from tens of nanometers to several micrometers. Furthermore, the method does not require prior knowledge on the composition of studied particles. We anticipate future measurements utilizing the AFM method on atmospheric aerosols at various RH to aid in our understanding of the range of aerosols' viscosities, the extent of particle-to-particle viscosity variability, and how these contribute to the particle diversity observable in the atmosphere.

Combination of density-clustering and supervised classification for event identification in single-molecule force spectroscopy data

Single-molecule force spectroscopy (SMFS) measurements of the dynamics of biomolecules typically require identifying massive events and states from large data sets, such as extracting rupture forces from force-extension curves (FECs) in pulling experiments and identifying states from extension-time trajectories (ETTs) in force-clamp experiments. The former is often accomplished manually and hence is time-consuming and laborious while the latter is always impeded by the presence of baseline drift. In this study, we attempt to accurately and automatically identify the events and states from SMFS experiments with a machine learning approach, which combines clustering and classification for event identification of SMFS (ACCESS). As demonstrated by analysis of a series of data sets, ACCESS can extract the rupture forces from FECs containing multiple unfolding steps and classify the rupture forces into the corresponding conformational transitions. Moreover, ACCESS successfully identifies the unfolded and folded states even though the ETTs display severe nonmonotonic baseline drift. Besides, ACCESS is straightforward in use as it requires only three easy-to-interpret parameters. As such, we anticipate that ACCESS will be a useful, easy-to-implement and high-performance tool for event and state identification across a range of single-molecule experiments.

Characterization of Breast Cancer Aggressiveness by Cell Mechanics.

In healthy tissues, cells are in mechanical homeostasis. During cancer progression, this equilibrium is disrupted. Cancer cells alter their mechanical phenotype to a softer and more fluid-like one than that of healthy cells. This is connected to cytoskeletal remodeling, changed adhesion properties, faster cell proliferation and increased cell motility. In this work, we investigated the mechanical properties of breast cancer cells representative of different breast cancer subtypes, using MCF-7, tamoxifen-resistant MCF-7, MCF10A and MDA-MB-231 cells. We derived viscoelastic properties from atomic force microscopy force spectroscopy measurements and showed that the mechanical properties of the cells are associated with cancer cell malignancy. MCF10A are the stiffest and least fluid-like cells, while tamoxifen-resistant MCF-7 cells are the softest ones. MCF-7 and MDA-MB-231 show an intermediate mechanical phenotype. Confocal fluorescence microscopy on cytoskeletal elements shows differences in actin network organization, as well as changes in focal adhesion localization. These findings provide further evidence of distinct changes in the mechanical properties of cancer cells compared to healthy cells and add to the present understanding of the complex alterations involved in tumorigenesis.

Customization of an atomic force microscope for multidimensional measurements under environmental conditions.

Atomic force microscopy (AFM) is an analytical surface characterization tool that reveals the surface topography at a nanometer length scale while probing local chemical, mechanical, and even electronic sample properties. Both contact (performed with a constant deflection of the cantilever probe) and dynamic operation modes (enabled by demodulation of the oscillation signal under tip-sample interaction) can be employed to conduct AFM-based measurements. Although surface topography is accessible regardless of the operation mode, the resolution and the availability of the quantified surface properties depend on the mode of operation. However, advanced imaging techniques, such as frequency modulation, to achieve high resolution, quantitative surface properties are not implemented in many commercial systems. Here, we show the step-by-step customization of an atomic force microscope. The original system was capable of surface topography and basic force spectroscopy measurements while employing environmental control, such as temperature variation of the sample/tip, etc. We upgraded this original setup with additional hardware (e.g., a lock-in amplifier with phase-locked loop capacity, a high-voltage amplifier, and a new controller) and software integration while utilizing its environmental control features. We show the capabilities of the customized system with frequency modulation-based topography experiments and automated voltage and/or distance spectroscopy, time-resolved AFM, and two-dimensional force spectroscopy measurements under ambient conditions. We also illustrate the enhanced stability of the setup with active topography and frequency drift corrections. We believe that our methodology can be useful for the customization and automation of other scanning probe systems.

Oligo[2]catenane That Is Robust at Both the Microscopic and Macroscopic Scales

Polycatenanes are extremely attractive topological architectures on account of their high degrees of conformational freedom and multiple motion patterns of the mechanically interlocked macrocycles. However, exploitation of these peculiar structural and dynamic characteristics to develop robust catenane materials is still a challenging goal. Herein, we synthesize an oligo[2]catenane that showcases mechanically robust properties at both the microscopic and macroscopic scales. The key feature of the structural design is controlling the force-bearing points on the metal-coordinated core of the [2]catenane moiety that is able to maximize the energy dissipation of the oligo[2]catenane via dissociation of metal-coordination bonds and then activation of sequential intramolecular motions of circumrotation, translation, and elongation under an external force. As such, at the microscopic level, the single-molecule force spectroscopy measurement exhibits that the force to rupture dynamic bonds in the oligo[2]catenane reaches a record high of 588 ± 233 pN. At the macroscopic level, our oligo[2]catenane manifests itself as the toughest catenane material ever reported (15.2 vs 2.43 MJ/m3). These fundamental findings not only deepen the understanding of the structure-property relationship of poly[2]catenanes with a full set of dynamic features but also provide a guiding principle to fabricate high-performance mechanically interlocked catenane materials.

Deep learning for precise axial localization of trapped microspheres in reflective optical systems.

High-precision axial localization measurement is an important part of micro-nanometer optical measurement, but there have been issues such as low calibration efficiency, poor accuracy, and cumbersome measurement, especially in reflected light illumination systems, where the lack of clarity of imaging details leads to the low accuracy of commonly used methods. Herein, we develop a trained residual neural network coupled with a convenient data acquisition strategy to address this challenge. Our method improves the axial localization precision of microspheres in both reflective illumination systems and transmission illumination systems. Using this new localization method, the reference position of the trapped microsphere can be extracted from the identification results, namely the "positioning point" among the experimental groups. This point relies on the unique signal characteristics of each sample measurement, eliminates systematic repeatability errors when performing identification across samples, and improves the localization precision of different samples. This method has been verified on both transmission and reflected illumination optical tweezers platforms. We will bring greater convenience to measurements in solution environments and will provide higher-order guarantees for force spectroscopy measurements in scenarios such as microsphere-based super-resolution microscopy and the surface mechanical properties of adherent flexible materials and cells.

Influence of Surface Chemistry of Fiber and Lignocellulosic Materials on Adhesion Properties with Polybutylene Succinate at Nanoscale.

The production of bio-based composites with enhanced characteristics constitutes a strategic action to minimize the use of fossil fuel resources. The mechanical performances of these materials are related to the specific properties of their components, as well as to the quality of the interface between the matrix and the fibers. In a previous research study, it was shown that the polarity of the matrix played a key role in the mechanisms of fiber breakage during processing, as well as on the final properties of the composite. However, some key questions remained unanswered, and new investigations were necessary to improve the knowledge of the interactions between a lignocellulosic material and a polar matrix. In this work, for the first time, atomic force microscopy based on force spectroscopy measurements was carried out using functionalized tips to characterize the intermolecular interactions at the single molecule level, taking place between poly(butylene succinate) and four different plant fibers. The efficiency of the tip functionalization was checked out by scanning electron microscopy and energy-dispersive X-ray spectroscopy, whereas the fibers chemistry was characterized by Fourier-transform infrared spectroscopy. Larger interactions at the nanoscale level were found between the matrix and hypolignified fibers compared to lignified ones, as in control experiments on single lignocellulosic polymer films. These results could significantly aid in the design of the most appropriate composite composition depending on its final use.

Single-Molecule Force Spectroscopy Reveals Cation-π Interactions in Aqueous Media Are Highly Affected by Cation Dehydration.

Cation-π interactions underlie many important processes in biology and materials science. However, experimental investigations of cation-π interactions in aqueous media remain challenging. Here, we studied the cation-π binding strength and mechanism by pulling two hydrophobic polymers with distinct cation binding properties, i.e., poly-pentafluorostyrene and polystyrene, in aqueous media using single-molecule force spectroscopy and nuclear magnetic resonance measurement. We found that the interaction strengths linearly depend on the cation concentrations, following the order of Li^{+}<NH_{4}^{+}<Na^{+}<K^{+}. The binding energies are 0.03-0.23 kJ mol^{-1} M^{-1}. This order is distinct from the strength of cation-π interactions in gas phase and may be caused by the different dehydration ability of the cations. Taken together, our method provides a unique perspective to investigate cation-π interactions under physiologically relevant conditions.

Stability of supported hybrid lipid bilayers on chemically and topographically-modified surfaces

Hybrid lipid bilayers are a particular case of supported lipid bilayers with the two monolayer leaflets composed by different types of molecules. These nanostructures can be produced in a well-controlled array fashion and are suitable for the study of biomembrane-related phenomena via electrochemical or plasmonic sensing. Understanding how the underlying solid surface affects the supported membrane formation and organization is necessary for the potential use of these hybrid platforms in applications for which surfaces are not flat and topographically complex. Here we assess the role of lipid phase, substrate surface energy and topography on the formation and stability of hybrid supported membranes from vesicle precursors using complementary surface-sensitive techniques, namely quartz crystal microbalance with dissipation and atomic force microscopy. The stability of hybrid bilayers against thermal and osmotic changes is evaluated and compared to standard supported lipid bilayers formed onto hydrophilic SiO2. Force spectroscopy measurements reveal an overall weaker lateral organization of hybrid membranes as a result of the underlying self-assembled monolayer being not optimally organized. Hybrid bilayers display a decoupled behavior between the two leaflets when vertically compressed at constant speed. On microcontact printed Au surfaces, hybrid bilayers were formed over printed patches, while surprisingly, supported lipid bilayers were observed on non-patterned Au regions suggesting a non-trivial self-assembled monolayer reorganization when in aqueous environment.

Measuring DNA extension during unfolding in a centrifugal force microscope

DNA in sperm cells can form compact toroids when bound with proteins from the protamine family. To study DNA folding and toroid formation, we have built a centrifugal force microscope (CFM) to unfold DNA and observe the intermediate folding states. In a CFM, micron-sized beads tethered to the sample by a DNA strand are placed into a microscope within a desktop centrifuge. The centrifuge applies a centrifugal force (a pseudoforce caused by inertia) to our sample, unfolding the DNA. Recording and analyzing the bead motion can yield information on the extension of the DNA over time and the intermediate stages of DNA condensation. Our CFM is operational at low centrifuge speeds, but we did not have a method for measuring the extension of the DNA. Here, we will discuss our progress in creating a program that gathers and analyzes video microscopy data from the CFM to measure extension in real time. This program will be useful in collecting data on the pathway of DNA condensation and for other force spectroscopy measurements with the CFM.

Cytoskeletal anchorage of different Dsg3 pools revealed by combination of hybrid STED/SMFS-AFM

Desmoglein 3 (Dsg3) is a desmosomal cadherin mediating cell adhesion within desmosomes and is the antigen of the autoimmune blistering skin disease pemphigus vulgaris. Therefore, understanding of the complex desmosome turnover process is of high biomedical relevance. Recently, super resolution microscopy was used to characterize desmosome composition and turnover. However, studies were limited because adhesion measurements on living cells were not possible in parallel. Before desmosomal cadherins are incorporated into nascent desmosomes, they are not bound to intermediate filaments but were suggested to be associated with the actin cytoskeleton. However, direct proof that adhesion of a pool of desmosomal cadherins is dependent on actin is missing. Here, we applied single-molecule force spectroscopy measurements with the novel single molecule hybrid-technique STED/SMFS-AFM to investigate the cytoskeletal anchorage of Dsg3 on living keratinocytes for the first time. By application of pharmacological agents we discriminated two different Dsg3 pools, only one of which is anchored to actin filaments. We applied the actin polymerization inhibitor Latrunculin B to modify the actin cytoskeleton and the PKCα activator PMA to modulate intermediate filament anchorage. On the cellular surface Dsg3 adhesion was actin-dependent. In contrast, at cell–cell contacts, Dsg3 adhesion was independent from actin but rather is regulated by PKC which is well established to control desmosome turn-over via intermediate filament anchorage. Taken together, using the novel STED/SMFS-AFM technique, we demonstrated the existence of two Dsg3 pools with different cytoskeletal anchorage mechanisms.

Condensed tannins act as anthelmintics by increasing the rigidity of the nematode cuticle

Tannins and tanniferous plant extracts have been discussed as sustainable means for helminth control in the past two decades in response to a dramatic increase of resistances towards standard anthelmintics. While their bioactivities have been broadly investigated in vitro and in vivo, less is known about their mode of action in nematodes, apart from their protein binding properties. In the current study we therefore investigated the impact of a phytochemically well characterized plant extract from Combretum mucronatum, known to contain procyanidins as the active compounds, on the model organism Caenorhabditis elegans. By different microscopic techniques, the cuticle was identified as the main binding site for tannins, whereas underlying tissues did not seem to be affected. In addition to disruptions of the cuticle structure, molting defects occurred at all larval stages. Finally, an increased rigidity of the nematodes’ cuticle due to binding of tannins was confirmed by force spectroscopic measurements. This could be a key finding to explain several anthelmintic activities reported for tannins, especially impairment of molting or exsheathment as well as locomotion.