Intracellular nanomanipulation by a photonic-force microscope with real-time acquisition of a 3D stiffness matrix.

A traditional photonic-force microscope (PFM) results in huge sets of data, which requires tedious numerical analysis. In this paper, we propose instead an analog signal processor to attain real-time capabilities while retaining the richness of the traditional PFM data. Our system is devoted to intracellular measurements and is fully interactive through the use of a haptic joystick. Using our specialized analog hardware along with a dedicated algorithm, we can extract the full 3D stiffness matrix of the optical trap in real time, including the off-diagonal cross-terms. Our system is also capable of simultaneously recording data for subsequent offline analysis. This allows us to check that a good correlation exists between the classical analysis of stiffness and our real-time measurements. We monitor the PFM beads using an optical microscope. The force-feedback mechanism of the haptic joystick helps us in interactively guiding the bead inside living cells and collecting information from its (possibly anisotropic) environment. The instantaneous stiffness measurements are also displayed in real time on a graphical user interface. The whole system has been built and is operational; here we present early results that confirm the consistency of the real-time measurements with offline computations.

Exploring the mechanical properties of single vimentin intermediate filaments by atomic force microscopy.

Intermediate filaments (IFs), together with actin filaments and microtubules, compose the cytoskeleton. Among other functions, IFs impart mechanical stability to cells when exposed to mechanical stress and act as a support when the other cytoskeletal filaments cannot keep the structural integrity of the cells. Here we present a study on the bending properties of single vimentin IFs in which we used an atomic force microscopy (AFM) tip to elastically deform single filaments hanging over a porous membrane. We obtained a value for the bending modulus of non-stabilized IFs between 300 MPa and 400 MPa. Our results together with previous ones suggest that IFs present axial sliding between their constitutive building blocks and therefore have a bending modulus that depends on the filament length. Measurements of glutaraldehyde-stabilized filaments were also performed to reduce the axial sliding between subunits and therefore provide a lower limit estimate of the Young's modulus of the filaments. The results show an increment of two to three times in the bending modulus for the stabilized IFs with respect to the non-stabilized ones, suggesting that the Young's modulus of vimentin IFs should be around 900 MPa or higher.

Nanoscale resolution in infrared imaging of protein-containing lipid membranes.

The precise imaging of biomolecular entities contributes to an understanding of the relationship between their structure and function. However, the resolution of conventional infrared microscopic imaging is diffraction limited and does not exceed a few micrometres. Atomic force microscopy, on the other hand, can detect infrared absorption down to the sub-micrometer level. In the present report, we demonstrate that for multi-bilayer lipid samples containing the plant photosynthetic pigment-protein complex LHCII, the resolution of this latter technique can be better than 20 nm. Such a high resolution is attributable to two factors: (i) the relatively high infrared absorption by the complex that is integrated perpendicular to the plane of the multilayer film, and (ii) the distinctly different mechanical properties and thermal conductivity of the lipid and protein components of the sample.

Model-based estimation of 3-D stiffness parameters in photonic-force microscopy.

We propose a system to characterize the 3-D diffusion properties of the probing bead trapped by a photonic-force microscope. We follow a model-based approach, where the model of the dynamics of the bead is given by the Langevin equation. Our procedure combines software and analog hardware to measure the corresponding stiffness matrix. We are able to estimate all its elements in real time, including off-diagonal terms. To achieve our goal, we have built a simple analog computer that performs a continuous preprocessing of the data, which can be subsequently digitized at a much lower rate than is otherwise required. We also provide an effective numerical algorithm for compensating the correlation bias introduced by a quadrant photodiode detector in the microscope. We validate our approach using simulated data and show that our bias-compensation scheme effectively improves the accuracy of the system. Moreover, we perform experiments with the real system and demonstrate real-time capabilities. Finally, we suggest a simple adjunction that would allow one to determine the mass matrix as well.

Temperature-dependent elasticity of microtubules.

Central to the biological function of microtubules is their ability to modify their length which occurs by addition and removal of subunits at the ends of the polymer, both in vivo and in vitro. This dynamic behavior is strongly influenced by temperature. Here, we show that the lateral interaction between tubulin subunits forming microtubule is strongly temperature dependent. Microtubules deposited on prefabricated substrates were deformed in an atomic force microscope during imaging, in two different experimental geometries. Microtubules were modeled as anisotropic, with the Young's modulus corresponding to the resistance of protofilaments to stretching and the shear modulus describing the weak interaction between the protofilaments. Measurements involving radial compression of microtubules deposited on flat mica confirm that microtubule elasticity depends on the temperature. Bending measurements performed on microtubules deposited on lithographically fabricated substrates show that this temperature dependence is due to changing shear modulus, implying that the lateral interaction between the protofilaments is strongly determined by the temperature. These measurements are in good agreement with previously reported measurements of the disassembly rate of microtubules, demonstrating that the mechanical and dynamic properties of microtubules are closely related.

Quantitative measure of nanoscale adhesion hysteresis by ultrasonic force microscopy.

Adhesion hysteresis is the difference between the work used on separating two surfaces and the work gained on bringing them back together. Although much effort has been invested into adhesion hysteresis investigations at macroscales and microscales, its measurements at the nanolengths or even molecular lengths are still not easy. In this paper we demonstrate how to obtain quantitative measures of local adhesion hysteresis from ultrasonic force microscopy investigations. We derive analytical models fitting all the experimental cases and apply them to experimental data.

Correlations between adhesion hysteresis and friction at molecular scales.

Correlations between adhesion hysteresis and local friction are theoretically and experimentally investigated. The model is based on the classical theory of adhesional friction, contact mechanics, capillary hysteresis, and nanoscale roughness. Adhesion hysteresis was found to scale with friction through the scaling factor containing a varying ratio of adhesion energy over the reduced Young's modulus. Capillary forces can offset the relationship between adhesion hysteresis and friction. Measurements on a wide range of engineering samples with varying adhesive and elastic properties confirm the model. Adhesion hysteresis is investigated under controlled, low humidity atmosphere via ultrasonic force microscopy. Friction is measured by the friction force microscopy.

Direct observation of nondiffusive motion of a Brownian particle.

The thermal position fluctuations of a single micron-sized sphere immersed in a fluid were recorded by optical trapping interferometry with nanometer spatial and microsecond temporal resolution. We find, in accord with the theory of Brownian motion including hydrodynamic memory effects, that the transition from ballistic to diffusive motion is delayed to significantly longer times than predicted by the standard Langevin equation. This delay is a consequence of the inertia of the fluid. On the shortest time scales investigated, the sphere's inertia has a small, but measurable, effect.

Reinforcement of single-walled carbon nanotube bundles by intertube bridging.

During their production, single-walled carbon nanotubes form bundles. Owing to the weak van der Waals interaction that holds them together in the bundle, the tubes can easily slide on each other, resulting in a shear modulus comparable to that of graphite. This low shear modulus is also a major obstacle in the fabrication of macroscopic fibres composed of carbon nanotubes. Here, we have introduced stable links between neighbouring carbon nanotubes within bundles, using moderate electron-beam irradiation inside a transmission electron microscope. Concurrent measurements of the mechanical properties using an atomic force microscope show a 30-fold increase of the bending modulus, due to the formation of stable crosslinks that effectively eliminate sliding between the nanotubes. Crosslinks were modelled using first-principles calculations, showing that interstitial carbon atoms formed during irradiation in addition to carboxyl groups, can independently lead to bridge formation between neighbouring nanotubes.

Nanomechanics of microtubules.

We have determined the mechanical anisotropy of a single microtubule by simultaneously measuring the Young's and the shear moduli in vitro. This was achieved by elastically deforming the microtubule deposited on a substrate tailored by electron-beam lithography with a tip of an atomic force microscope. The shear modulus is 2 orders of magnitude lower than the Young's, giving rise to a length-dependent flexural rigidity of microtubules. The temperature dependence of the microtubule's bending stiffness in the (5-40) degrees C range shows a strong variation upon cooling coming from the increasing interaction between the protofilaments.