The nanomechanical properties of living cells, such as their surface elastic response and adhesion, have important roles in cellular processes such as morphogenesis, mechano-transduction, focal adhesion, motility, metastasis and drug delivery. Techniques based on quasi-static atomic force microscopy techniques can map these properties, but they lack the spatial and temporal resolution that is needed to observe many of the relevant details. Here, we present a dynamic atomic force microscopy method to map quantitatively the nanomechanical properties of live cells with a throughput (measured in pixels/minute) that is ∼10-1,000 times higher than that achieved with quasi-static atomic force microscopy techniques. The local properties of a cell are derived from the 0th, 1st and 2nd harmonic components of the Fourier spectrum of the AFM cantilevers interacting with the cell surface. Local stiffness, stiffness gradient and the viscoelastic dissipation of live Escherichia coli bacteria, rat fibroblasts and human red blood cells were all mapped in buffer solutions. Our method is compatible with commercial atomic force microscopes and could be used to analyse mechanical changes in tumours, cells and biofilm formation with sub-10 nm detail.

Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details. Atomic force microscopy's 7 (AFM) ability of visualizing the topography and the property of surfaces and interfaces at a molecular level 8 has enabled a rapid development in the understanding of surface phenomena. Its versatility allows the exploration of hard and soft materials in vacuum 9-12 , in air 13 , but also in complex liquids 14,15 , often allowing imaging at sub-nanometre and sometimes atomic resolution 16 . In dynamic mode 17 (vibrating cantilever), AFM has proven sensitive to the interfacial compliance of viscous liquids and provided quantitative information about the structure of liquid layers between the AFM tip and the solid surface 18 , with, in some cases, atomic resolution 15 . However, specialized instruments were used and the nature of the tip-sample interaction remains an issue of debate. Dynamic AFM has the ability to probe the solid-liquid interface [18][19][20][21] , but an interpretation of experimental results remains difficult 22,23 . Traditionally, interfaces are characterized by an interfacial energy, IE, the sum of the two surface energies in vacuum minus the work of adhesion (W SL ) necessary to separate the surfaces (Dupré Equation 24 ). The latter is de facto the energy spent to restructure the interface due to the atomistic interaction between the two materials (Fig. 1c). In practice at a solid-liquid interface this is the energy that generates density variations and structuring of the liquid close to the interface. Hereafter we will call this layer of liquid which differs from the bulk

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.

The HIV-1 genome consists of two identical RNA strands that are linked together through non-covalent interactions. A major determinant for efficient dimerization of the two RNA strands is the interaction between palindromic sequences in the dimerization initiation site. Here we use an interplay of bioinformatics, biochemistry, and atomic force microscopy to describe another conserved palindrome in the trans-activation response element (TAR) that functions as a strong dimerization site when transiently exposed to the viral nucleocapsid protein. In conjunction with the DIS interaction, the TAR dimerization induces the formation of a 65-nm higher-order circular structure in the dimeric HIV-1 RNA. Our results provide a molecular model for the role of TAR in packaging and reverse transcription of the viral genome. The unique structure of the TAR-TAR dimer renders it an intriguing therapeutic target for the treatment of HIV-1 infection.

We have used high-speed atomic force microscopy to study the dynamics of bacteriorhodopsin (bR) molecules at the free interface of the crystalline phase that occurs naturally in purple membrane.Our results reveal temporal fluctuations at the crystal edges arising from the association and dissociation of bR molecules, most predominantly pre-formed trimers. Analysis of the dissociation kinetics yields an estimate of the inter-trimer single-bond energy of -0.9 kcal/mol. Rotational motion of individual bound trimers indicates that the inter-trimer bond involves W10-W12 tryptophan residues.

Purple membranes (PM) are two-dimensional crystals formed by bacteriorhodopsin and a variety of lipids. The lipid composition and density in the cytoplasmic (CP) leaflet differ from those of the extracellular (EC) leaflet. A new way of differentiating the two sides of such asymmetric membranes using the phase signal in alternate contact atomic force microscopy is presented. This method does not require molecular resolution and is applied to study the stiffness and intertrimer lipid mobility in both leaflets of the PM independently over a broad range of pH and salt concentrations. PM stiffens with increasing salt concentration according to two different regimes. At low salt concentration, the membrane Young's normal modulus grows quickly but differentially for the EC and CP leaflets. At higher salt concentration, both leaflets behave similarly and their stiffness converges toward the native environment value. Changes in pH do not affect PM stiffness; however, the crystal assembly is less pronounced at pH > or = 10. Lipid mobility is high in the CP leaflet, especially at low salt concentration, but negligible in the EC leaflet regardless of pH or salt concentration. An independent lipid mobility study by solid-state NMR confirms and quantifies the atomic force microscopy qualitative observations.

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The physical properties of lipid bilayers comprising the cell membrane occupy the current spotlight of membrane biology. Their traditional representation as a passive 2D fluid has gradually been abandoned in favor of a more complex picture: an anisotropic time-dependent viscoelastic biphasic material, capable of transmitting or attenuating mechanical forces that regulate biological processes. In establishing new models, quantitative experiments are necessary when attempting to develop suitable techniques for dynamic measurements. Here, we map both the elastic and viscous properties of the model system 1,2-dipalmitoyl--glycero-3-phosphocholine (DPPC) lipid bilayers using multifrequency atomic force microscopy (AFM), namely amplitude modulation-frequency modulation (AM-FM) AFM imaging in an aqueous environment. Furthermore, we investigate the effect of cholesterol (Chol) on the DPPC bilayer in concentrations from 0 to 60%. The AM-AFM quantitative maps demonstrate that at low Chol concentrations, the lipid bilayer displays a distinct phase separation and is elastic, whereas at higher Chol concentration, the bilayer appears homogenous and exhibits both elastic and viscous properties. At low-Chol contents, the modulus (elastic) dominates. As the Chol insertions increases, higher energy is dissipated; and although the bilayer stiffens (increase in), the viscous component dominates (). Our results provide evidence that the lipid bilayer exhibits both elastic and viscous properties that are modulated by the presence of Chol, which may affect the propagation (elastic) or attenuation (viscous) of mechanical signals across the cell membrane.