Protocol for measuring mechanical properties of live cells using atomic force microscopy.

Atomic force microscope (AFM) is a powerful and versatile tool to determine the physical properties of cells. The force-distance curves obtained from AFM experiments can be used to determine the stiffness and viscoelastic properties of cells. Here, we present a protocol for the determination of viscoelasticity from live cells such as Drosophila hemocytes or mouse embryonic stem cells using AFM. This protocol has potential application in determining the physical properties of cells in healthy and diseased conditions. For complete details on the use and execution of this protocol, please refer to Mote et al. (2020),1 and Singh et al. (2023).2.

Direct and Simultaneous Measurement of the Stiffness and Internal Friction of a Single Folded Protein.

The nanomechanical response of a folded single protein, the natural nanomachine responsible for myriad biological processes, provides insight into its function. The conformational flexibility of a folded state, characterized by its viscoelasticity, allows proteins to adopt different shapes to perform their function. Despite efforts, its direct measurement has not been possible so far. We present a direct and simultaneous measurement of the stiffness and internal friction of the folded domains of the protein titin using a special interferometer based atomic force microscope. We analyzed the data by carefully separating different contributions affecting the response of the experimental probe to obtain the folded state's viscoelasticity. Above ∼95 pN of force, the individual immunoglobulins of titin transition from an elastic solid-like native state to a soft viscoelastic intermediate.

Resistive Switching in HfO2-x/La0.67Sr0.33MnO3 Heterostructures: An Intriguing Case of Low H-Field Susceptibility of an E-Field Controlled Active Interface.

High-performance nonvolatile resistive random access memories (ReRAMs) and their small stimuli control are of immense interest for high-speed computation and big-data processing in the emerging Internet of Things (IoT) arena. Here, we examine the resistive switching (RS) behavior in growth-controlled HfO2/La0.67Sr0.33MnO3 (LSMO) heterostructures and their tunability in a low magnetic field. It is demonstrated that oxygen-deficient HfO2 films show bipolar switching with a high on/off ratio, stable retention, as well as good endurance owing to the orthorhombic-rich phase constitution and charge (de)trapping-enabled Schottky-type conduction. Most importantly, we have demonstrated that RS can be tuned by a very low externally applied magnetic field (∼0-30 mT). Remarkably, application of a magnetic field of 30 mT causes RS to be fully quenched and frozen in the high resistive state (HRS) even after the removal of the magnetic field. However, the quenched state could be resurrected by applying a higher bias voltage than the one for initial switching. This is argued to be a consequence of the electronically and ionically "active" nature of the HfO2-x/LSMO interface on both sides and its susceptibility to the electric and low magnetic field effects. This result could pave the way for new designs of interface-engineered high-performance oxitronic ReRAM devices.

Validity of point-mass model in off-resonance dynamic atomic force microscopy.

The quantitative measurement of viscoelasticity of nano-scale entities is an important goal of nanotechnology research and there is considerable progress with advent of dynamic atomic force microscopy. The hydrodynamics of cantilever, the force sensor in AFM measurements, plays a pivotal role in quantitative estimates of nano-scale viscoelasticity. The point-mass (PM) model, wherein the AFM cantilever is approximated as a point-mass with mass-less spring is widely used in dynamic AFM analysis and its validity, particularly in liquid environments, is debated. It is suggested that the cantilever must be treated as a continuous rectangular beam to obtain accurate estimates of nano-scale viscoelasticity of materials it is probing. Here, we derived equations, which relate stiffness and damping coefficient of the material under investigation to measured parameters, by approximating cantilever as a point-mass and also considering the full geometric details. These equations are derived for both tip-excited as well as base-excited cantilevers. We have performed off-resonance dynamic atomic force spectroscopy on a single protein molecule to investigate the validity of widely used PM model. We performed measurements with AFMs equipped with different cantilever excitation methods as well as detection schemes to measure cantilever response. The data was analyzed using both, continuous beam model and the PM model. We found that both models yield same results when the experiments are performed in truly off-resonance regime with small amplitudes and the cantilever stiffness is much higher than the interaction stiffness. Our findings suggest that a simple PM approximation based model is adequate to describe the dynamics, provided care is taken while performing experiments so that the approximations used in these models are valid.

Mechanism of Adhesion of Natural Polymer Coatings to Chemically Modified Siloxane Polymer.

Surface coatings play an important role in improving the performance of biomedical implants. Polydimethylsiloxane (PDMS) is a commonly used material for biomedical implants, and surface-coated PDMS implants frequently face problems such as delamination or cracking of the coating. In this work, we have measured the performance of nano-coatings of the biocompatible protein polymer silk fibroin (SF) on pristine as well as modified PDMS surfaces. The PDMS surfaces have been modified using oxygen plasma treatment and 3-amino-propyl-triethoxy-silane (APTES) treatment. Although these techniques of PDMS modification have been known, their effects on adhesion of SF nano-coatings have not been studied. Interestingly, testing of the coated samples using a bulk technique such as tensile and bending deformation showed that the SF nano-coating exhibits improved crack resistance when the PDMS surface has been modified using APTES treatment as compared to an oxygen plasma treatment. These results were validated at the microscopic and mesoscopic length scales through nano-scratch and nano-indentation measurements. Further, we developed a unique method using modified atomic force microscopy to measure the adhesive energy between treated PDMS surfaces and SF molecules. These measurements indicated that the adhesive strength of PDMS-APTES-SF is 10 times more compared to PDMS-O2-SF due to the higher number of molecular linkages formed in this nanoscale contact. This lower number of molecular linkages in the PDMS-O2 indicates that only fewer numbers of surface hydroxyl groups interact with the SF protein through secondary interactions such as hydrogen bonding. On the other hand, a larger number of amine groups present on PDMS-APTES surface hydrogen bond with the polar amino acids present on the silk fibroin protein chain, resulting in better adhesion. Thus, APTES modification to the PDMS substrate results in improved adhesion of nano-coating to the substrate and enhances the delamination and crack resistance of the nano-coatings.

The nano-scale viscoelasticity using atomic force microscopy in liquid environment.

We measured viscoelasticity of two nanoscale systems, single protein molecules and molecular layers of water confined between solid walls. In order to quantify the viscoelastic response of these nanoscale systems in liquid environment, the measurements are performed using two types of atomic force microscopes (AFMs), which employ different detection schemes to measure the cantilever response. We used a deflection detection scheme, available in commercial AFMs, that measures cantilever bending and a fibre-interferometer based detection which measures cantilever displacement. The hydrodynamics of the cantilever is modelled using Euler-Bernoulli equation with appropriate boundary conditions which accommodate both detection schemes. In a direct contradiction with many reports in the literature, the dissipation coefficient of a single octomer of titin I278 is found to be immeasurably low. The upper bound on the dissipation coefficient is 5 × 10-7 kg s-1, which is much lower than the reported values. The entropic stiffness of single unfolded domains of protein measured using both methods is in the range of 10 mN m-1. We show that in a conventional deflection detection measurement, the phase of the bending signal can be a primary source of artefacts in the dissipation estimates. It is recognized that the measurement of cantilever displacement, which has negligibly small phase lag due to hydrodynamics of the cantilever at low excitation frequencies, is better suited for ensuring artefact-free measurement of viscoelasticity compared to the measurement of the cantilever bending. Further, it was possible to measure dissipation in molecular layers of water confined between the tip and the substrate using fibre interferometer based AFM with similar experimental parameters. It confirms that the dissipation coefficient of a single I278 is below the detection limit of AFM. The results shed light on the discrepancy observed in the measured diffusional dynamics of protein collapse measured using Force spectroscopic techniques and single-molecule optical techniques.

Differential tissue stiffness of body column facilitates locomotion of Hydra on solid substrates.

The bell-shaped members of the Cnidaria typically move around by swimming, whereas the Hydra polyp can perform locomotion on solid substrates in an aquatic environment. To address the biomechanics of locomotion on rigid substrates, we studied the 'somersaulting' locomotion in Hydra We applied atomic force microscopy to measure the local mechanical properties of Hydra's body column and identified the existence of differential Young's modulus between the shoulder region versus rest of the body column at 3:1 ratio. We show that somersaulting primarily depends on differential tissue stiffness of the body column and is explained by computational models that accurately recapitulate the mechanics involved in this process. We demonstrate that perturbation of the observed stiffness variation in the body column by modulating the extracellular matrix polymerization impairs the 'somersault' movement. These results provide a mechanistic basis for the evolutionary significance of differential extracellular matrix properties and tissue stiffness.