Sylvie Hénon

Microscope at the Brewster angle: Direct observation of first‐order phase transitions in monolayers

A microscope employing the characteristics of the reflection at the Brewster angle has been built for the study of first‐order phase transitions in monolayers and the growth of two‐dimensional domains without adding fluorescent impurities. It takes about 2.4 s to constitute an image.

A New Determination of the Shear Modulus of the Human Erythrocyte Membrane Using Optical Tweezers

Optical tweezers are used to apply calibrated forces to human erythrocytes, via small silica beads bound to their membrane. The shear modulus mu of the membrane is inferred from measurements of the cell deformation in the small strain linear regime. We find the same result mu = 2.5 +/- 0.4 microN/m for both discotic and nearly spherical swollen cells. This value is smaller than the one deduced from micropipettes experiments. However the two methods do not operate in the same deformation regime and are not expected to lead to the same result.

Power laws in microrheology experiments on living cells: Comparative analysis and modeling.

We compare and synthesize the results of two microrheological experiments on the cytoskeleton of single cells. In the first one, the creep function J(t) of a cell stretched between two glass plates is measured after applying a constant force step. In the second one, a microbead specifically bound to transmembrane receptors is driven by an oscillating optical trap, and the viscoelastic coefficient Ge(omega) is retrieved. Both J(t) and Ge(omega) exhibit power law behaviors: J(t) = A0(t/t0)alpha and absolute value (Ge(omega)) = G0(omega/omega0)alpha, with the same exponent alpha approximately 0.2. This power law behavior is very robust; alpha is distributed over a narrow range, and shows almost no dependence on the cell type, on the nature of the protein complex which transmits the mechanical stress, nor on the typical length scale of the experiment. On the contrary, the prefactors A0 and G0 appear very sensitive to these parameters. Whereas the exponents alpha are normally distributed over the cell population, the prefactors A0 and G0 follow a log-normal repartition. These results are compared with other data published in the literature. We propose a global interpretation, based on a semiphenomenological model, which involves a broad distribution of relaxation times in the system. The model predicts the power law behavior and the statistical repartition of the mechanical parameters, as experimentally observed for the cells. Moreover, it leads to an estimate of the largest response time in the cytoskeletal network: tau(m) approximately 1000 s.

Direct measurement of the area expansion and shear moduli of the human red blood cell membrane skeleton.

The area expansion and the shear moduli of the free spectrin skeleton, freshly extracted from the membrane of a human red blood cell (RBC), are measured by using optical tweezers micromanipulation. An RBC is trapped by three silica beads bound to its membrane. After extraction, the skeleton is deformed by applying calibrated forces to the beads. The area expansion modulus K(C) and shear modulus mu(C) of the two-dimensional spectrin network are inferred from the deformations measured as functions of the applied stress. In low hypotonic buffer (25 mOsm/kg), one finds K(C) = 4.8 +/- 2.7 microN/m, mu(C) = 2.4 +/- 0.7 microN/m, and K(C)/mu(C) = 1.9 +/- 1.0. In isotonic buffer, one measures higher values for K(C), mu(C), and K(C)/mu(C), partly because the skeleton collapses in a high-ionic-strength environment. Some data concerning the time evolution of the mechanical properties of the skeleton after extraction and the influence of ATP are also reported. In the Discussion, it is shown that the measured values are consistent with estimates deduced from experiments carried out on the intact membrane and agree with theoretical and numerical predictions concerning two-dimensional networks of entropic springs.

Assessment of Mechanical Properties of Adherent Living Cells by Bead Micromanipulation: Comparison of Magnetic Twisting Cytometry vs Optical Tweezers

We compare the measurements of viscoelastic properties of adherent alveolar epithelial cells by two micromanipulation techniques: (i) magnetic twisting cytometry and (ii) optical tweezers, using microbeads of same size and similarly attached to F-actin. The values of equivalent Young modulus E, derived from linear viscoelasticity theory, become consistent when the degree of bead immersion in the cell is taken into account. E-values are smaller in (i) than in (ii): approximately 34-58 Pa vs approximately 29-258 Pa, probably because higher stress in (i) reinforces nonlinearity and cellular plasticity. Otherwise, similar relaxation time constants, around 2 s, suggest similar dissipative mechanisms.

Textures and phase transitions in Langmuir monolayers of fatty acids. A comparative Brewster angle microscope and polarized fluorescence microscope study

Brewster angle microscopy (BAM) and polarized fluorescence microscopy (PFM) are used to observe the distinctive textures of and the transitions between condensed phases in Langmuir monolayers of n‐alkanoic acids. BAM is sensitive to film anisotropy even when the molecules are not tilted as long as the unit cell is anisotropic. Every transition is visible with one or both of the techniques, either as a dramatic change in the degree of contrast or as a sudden alteration of the mosaic domain texture. The two techniques are generally consistent, although the presence of the fluorescent probe impurity (for PFM) causes a subtle difference in the appearance of one transition and small shifts in transition surface pressures.

Phase transitions in Gibbs films: Star textural defects in tilted mesophases

The formation of adsorbed films at the free surface of aqueous solutions of sodium hexadecanoate was observed by microscopy at the Brewster angle. A phase transition between a gas and a tilted phase occurs during these slow formations. The tilt of the molecules creates an observable optical anisotropy. We observed circular domains separated into regions of uniform tilt direction by straight defect lines, i.e., ‘‘star’’ textural defects very similar to those observed in freely suspended liquid crystal thin films and in Langmuir films of esters. The coalescence of domains leads to large regions of uniform tilt direction separated by straight or kink lines. A second transition towards a nontilted phase is observed. Quantitative information about the coefficients of the continuum elastic theory can be derived from our experiments.

Cell stiffening in response to external stress is correlated to actin recruitment.

We designed a micromanipulation device that allows the local application of a constant force on living cells, and the measurement of their stiffness. The force is applied through an Arg-Gly-Asp-coated bead adhering on the cell and trapped in optical tweezers controlled by a feedback loop. Epifluorescence observations of green fluorescent protein-actin in the cells are made during force application. We observe a stiffening of cells submitted to a constant force within a few minutes, coupled to actin recruitment both at the bead-cell contact and up to several micrometers from the stress application zone. Moreover, kinetics of stiffening and actin recruitment exhibit a strong correlation. This work presents the first quantification of the dynamics of cell mechanical reinforcement under stress, which is a novel insight into the elucidation of the more general phenomenon of cell adaptation to stress.

Spatiotemporal Analysis of Cell Response to a Rigidity Gradient: A Quantitative Study Using Multiple Optical Tweezers

We investigate the dynamic response of single cells to weak and local rigidities, applied at controlled adhesion sites. Using multiple latex beads functionalized with fibronectin, and each trapped in its own optical trap, we study the reaction in real time of single 3T3 fibroblast cells to asymmetrical tensions in the tens of pN x microm(-1) range. We show that the cell feels a rigidity gradient even at this low range of tension, and over time develops an adapted change in the force exerted on each adhesion site. The rate at which force increases is proportional to trap stiffness. Actomyosin recruitment is regulated in space and time along the rigidity gradient, resulting in a linear relationship between the amount of recruited actin and the force developed independently in trap stiffness. This time-regulated actomyosin behavior sustains a constant and rigidity-independent velocity of beads inside the traps. Our results show that the strengthening of extracellular matrix-cytoskeleton linkages along a rigidity gradient is regulated by controlling adhesion area and actomyosin recruitment, to maintain a constant deformation of the extracellular matrix.

Prestress and adhesion site dynamics control cell sensitivity to extracellular stiffness.

This study aims at improving the understanding of mechanisms responsible for cell sensitivity to extracellular environment. We explain how substrate mechanical properties can modulate the force regulation of cell sensitive elements primarily adhesion sites. We present a theoretical and experimental comparison between two radically different approaches of the force regulation of adhesion sites that depends on their either stationary or dynamic behavior. The most classical stationary model fails to predict cell sensitivity to substrate stiffness whereas the dynamic model predicts extracellular stiffness dependence. This is due to a time dependent reaction force in response to actomyosin traction force exerted on cell sensitive elements. We purposely used two cellular models, i.e., alveolar epithelial cells and alveolar macrophages exhibiting respectively stationary and dynamic adhesion sites, and compared their sensitivity to theoretical predictions. Mechanical and structural results show that alveolar epithelial cells exhibit significant prestress supported by evident stress fibers and lacks sensitivity to substrate stiffness. On the other hand, alveolar macrophages exhibit low prestress and exhibit sensitivity to substrate stiffness. Altogether, theory and experiments consistently show that adhesion site dynamics and cytoskeleton prestress control cell sensitivity to extracellular environment with an optimal sensitivity expected in the intermediate range.