Spatially Multiplexed Micro-Spectrophotometry in Bright Field Mode for Thin Film Characterization.

Thickness characterization of thin films is of primary importance in a variety of nanotechnology applications, either in the semiconductor industry, quality control in nanofabrication processes or engineering of nanoelectromechanical systems (NEMS) because small thickness variability can strongly compromise the device performance. Here, we present an alternative optical method in bright field mode called Spatially Multiplexed Micro-Spectrophotometry that allows rapid and non-destructive characterization of thin films over areas of mm² and with 1 µm of lateral resolution. We demonstrate an accuracy of 0.1% in the thickness characterization through measurements performed on four microcantilevers that expand an area of 1.8 mm² in one minute of analysis time. The measured thickness variation in the range of few tens of nm translates into a mechanical variability that produces an error of up to 2% in the response of the studied devices when they are used to measure surface stress variations.

Fabrication and mechanical characterization of semi-free-standing (conjugated) polymer thin films.

Polymers undergo severe low-dimensionality effects when they are confined to ultrathin films since most of the structural and dynamical processes involving polymer molecules are correlated to length scales of the order of nanometers. However, the real influence of the size limitation over such processes is often hard to identify as it is masked by interfacial effects. We present the fabrication of a new type of nanostructure consisting of poly[[9-(1-octylnonyl)-9H-carbazole-2,7-diyl]-2,5-thiophenediyl-2,1,3-benzothiadiazole-4,7-diyl-2,5-thiophenediyl] (PCDTBT) thin film that is held up exclusively over tips of poly(ether-ether-ketone) (PEEK) nanopillars. The fabrication method exploits the nonwetting behavior of PCDTBT onto an ordered PEEK nanopillar array when the mobility of the PCDTBT molecules is enhanced by a solvent annealing process. We use this new configuration to characterize the mechanical behavior of free-standing thin film regions, thus in the absence of underlaying substrate, by means of an atomic force microscope (AFM) setup. First, we study how the finite thickness and/or the presence of the underlying substrate influences the mechanical modulus of the material in the linear elastic regime. Moreover, we analyze deep indentations up to the rupture of the thin film, which allow for the measurement of important mechanical features of the nanoconfined polymer, such as its yield strain, the rupture strain, the bending rigidity, etc., which are impossible to investigate in thin films deposited on substrates.

Torsion and curvature of FtsZ filaments.

FtsZ filaments participate in bacterial cell division, but it is still not clear how their dynamic polymerization and shape exert force on the underlying membrane. We present a theoretical description of individual filaments that incorporates information from molecular dynamic simulations. The structure of the crystallized Methanococcus jannaschii FtsZ dimer was used to model a FtsZ pentamer that showed a curvature and a twist. The estimated bending and torsion angles between monomers and their fluctuations were included in the theoretical description. The MD data also permitted positioning the curvature with respect to the protein coordinates and allowed us to explore the effect of the relative orientation of the preferred curvature with respect to the surface plane. We find that maximum tension is attained when filaments are firmly attached and oriented with their curvature perpendicular to the surface and that the twist serves as a valve to release or to tighten the tension exerted by the curved filaments on the membrane. The theoretical model also shows that the presence of torsion can explain the shape distribution of short filaments observed by Atomic Force Microscopy in previously published experiments. New experiments with FtsZ covalently attached to lipid membranes show that the filament on-plane curvature depends on lipid head charge, confirming the predicted monomer orientation effects. This new model underlines the fact that the combination of the three elements, filament curvature, twist and the strength and orientation of its surface attachment, can modulate the force exerted on the membrane during cell division.

Polymorphism of FtsZ filaments on lipid surfaces: role of monomer orientation.

FtsZ is a bacterial cytoskeletal protein involved in cell division. It forms a ringlike structure that attaches to the membrane to complete bacterial division. It binds and hydrolyzes GTP, assembling into polymers in a GTP-dependent manner. To test how the orientation of the monomers affects the curvature of the filaments on a surface, we performed site-directed mutagenesis on the E. coli FtsZ protein to insert cysteine residues at lateral locations to orient FtsZ on planar lipid bilayers. The E93C and S255C mutants were overproduced, purified, and found to be functionally active in solution, as well as being capable of sustaining cell division in vivo in complementation assays. Atomic force microscopy was used to observe the shape of the filament fibers formed on the surface. The FtsZ mutants were covalently linked to the lipids and could be polymerized on the bilayer surface in the presence of GTP. Unexpectedly, both mutants assembled into straight structures. E93C formed a well-defined lattice with monomers interacting at 60° and 120° angles, whereas S255C formed a more open array of straight thicker filament aggregates. These results indicate that filament curvature and bending are not fixed and that they can be modulated by the orientation of the monomers with respect to the membrane surface. As filament curvature has been associated with the force generation mechanism, these results point to a possible role of filament membrane attachment in lateral association and curvature, elements currently identified as relevant for force generation.

Nanomechanical properties of composite protein networks of erythroid membranes at lipid surfaces.

Erythrocyte membranes have been particularly useful as a model for studies of membrane structure and mechanics. Native erythroid membranes can be electroformed as giant unilamellar vesicles (eGUVs). In the presence of ATP, the erythroid membrane proteins of eGUVs rearrange into protein networks at the microscale. Here, we present a detailed nanomechanical study of individual protein microfilaments forming the protein networks of eGUVs when spread on supporting surfaces. Using Peak Force tapping Atomic Force Microscopy (PF-AFM) in liquid environment we have obtained the mechanical maps of the composite lipid-protein networks supported on solid surface. In the absence of ATP, the protein pool was characterized by a Young's Modulus Epool≈5-15MPa whereas the complex filaments were found softer after protein supramolecular rearrangement; Efil≈0.4MPa. The observed protein softening and reassembling could be relevant for understanding the mechanisms of cytoskeleton reorganization found in pathological erythrocytes or erythrocytes that are affected by biological agents.

Effect of Actin Organization on the Stiffness of Living Breast Cancer Cells Revealed by Peak-Force Modulation Atomic Force Microscopy.

We study the correlation between cytoskeleton organization and stiffness of three epithelial breast cancer cells lines with different degrees of malignancy: MCF-10A (healthy), MCF-7 (tumorigenic/noninvasive), and MDA-MB-231 (tumorigenic/invasive). Peak-force modulation atomic force microscopy is used for high-resolution topography and stiffness imaging of actin filaments within living cells. In healthy cells, local stiffness is maximum where filamentous actin is organized as well-aligned stress fibers, resulting in apparent Young's modulus values up to 1 order of magnitude larger than those in regions where these structures are not observed, but these organized actin fibers are barely observed in tumorigenic cells. We further investigate cytoskeleton conformation in the three cell lines by immunofluorescence confocal microscopy. The combination of both techniques determines that actin stress fibers are present at apical regions of healthy cells, while in tumorigenic cells they appear only at basal regions, where they cannot contribute to stiffness as probed by atomic force microscopy. These results substantiate that actin stress fibers provide a dominant contribution to stiffness in healthy cells, while the elasticity of tumorigenic cells appears not predominantly determined by these structures. We also discuss the effects of the high-frequency indentations inherent to peak-force atomic force microscopy for the identification of mechanical cancer biomarkers. Whereas conventional low loading rate indentations (1 Hz) result in slightly differentiated average stiffness for each cell line, in high-frequency measurements (250 Hz) healthy cells are clearly discernible from both tumorigenic cells with an enhanced stiffness ratio; however, the two cancerous cell lines produced indistinguishable results.

Surface light-scattering at the air-liquid interface: from newtonian to viscoelastic polymer solutions.

The dynamics of the liquid-air interface of aqueous solutions of a tensioactive triblock copolymer (Pluronic F-68) has been studied using surface quasielastic light scattering over a broad range of concentrations and temperatures. Ancillary surface tension and bulk rheometry data have been obtained for the same system. The results show that the classical theoretical spectrum for monolayers on a Newtonian fluid can be applied only for concentrations below 4.10(-2) mM. For concentrations above c = 14 mM a clear peak centered at zero frequency appears in the spectrum. This feature is incompatible with the classical theoretical spectrum. The SQELS spectra have been described in terms of the theory of Wang and Huang [Wang, C. H.; Huang, Q. R. J. Chem. Phys. 1997, 107, 5898] considering that the loss modulus of the concentrated solutions shows the existence of two relaxation modes even at low frequencies. The theory is able to explain the existence of a peak centered at zero frequency in the spectra, and the theoretical spectra point out the existence of an elastic peak together with the capillary one. There is a reasonable agreement between the relaxation times and the product Gtau obtained from the fits of the SQELS spectra to the theory of Wang and Huang and those obtained from bulk rheology.

Modeling the interplay between protein and lipid aggregation in supported membranes.

We present a theoretical model that deals with the complex interplay between lipid segregation and the self-aggregation of lipid-attached proteins. The model, in contrast to previous ones that consider proteins only as passive elements affecting the lipid distribution, describes the system including three terms: the dynamic interactions between protein monomers, the interactions between lipid components, and a mixed term considering protein-lipid interactions. It is used to explain experimental results performed on a well-defined system in which a self-aggregating soluble bacterial cytoskeletal protein polymerizes on a lipid bilayer containing two lipid components. All the elements considered in a previously described protein model, including torsion of the monomers within the filament, are needed to account for the observed filament shapes. The model also points out that lipid segregation can affect the length and curvature of the filaments and that the dynamic behavior of the lipids and proteins can have different time scales, giving rise to memory effects. This simple model that considers a dynamic protein assembly on a fluid and active lipid surface can be easily extended to other biologically relevant situations in which the interplay between protein and lipid aggregation is needed to fully describe the system.

X-ray diffraction, calorimetric, and dielectric relaxation study of the amorphous and smectic states of a main chain liquid crystalline polymer.

Liquid crystalline polymers (LCPs) are complex systems that include features of both orientationally ordered mesophases and amorphous polymers. Frequently, the isotropic amorphous state cannot be studied due to the rapid mesophase formation. Here, a new main chain LCP, poly(triethyleneglycol methyl p,p'-bibenzoate), PTEMeB, has been synthesized. It shows a rather slow mesophase formation making possible to study independently both the amorphous and the liquid crystalline states. The structure and phase transitions of PTEMeB have been investigated by calorimetry, variable-temperature MAXS/WAXS employing synchrotron radiation, and X-ray diffraction in oriented fibers. These experiments have pointed out the presence of two glass transitions, related to the amorphous or to the liquid crystal phases. Additionally, the mesophase seems to be a coexistence of orthogonal and tilted smectic phases. A dielectric relaxation study of PTEMeB over broad ranges of temperature and pressure has been performed. The dynamic glass transition turns out to be slower for the amorphous state than for the liquid crystal. Monitoring of the α relaxation has allowed us to follow the isothermal mesophase formation at atmospheric pressure. Additionally, the dynamical behavior at high pressures has pointed out the fast formation of the mesophase induced by sudden pressure changes.