Probing the Effect of Molecular Nonuniformity in Directed Self-Assembly of Diblock Copolymers in Nanoconfined Space.

Various complex self-assembled morphologies of lamellar- and cylinder-forming block copolymers comprising poly(dimethylsiloxane)-b-polylactide (PDMS-b-PLA) confined in cylindrical channels were generated. Combining top-down lithography with bottom-up block copolymer self-assembly grants access to morphologies that are otherwise inaccessible with the bulk materials. Channel diameter (D) was systematically varied with four diblock copolymers having different compositions and bulk domain spacing (L0), corresponding to a range of frustration ratios (D/L0 from 2 to 4). Excessive packing frustration imposed by the channels leads to contorted domains. The resulting morphologies depend strongly on both D/L0 and copolymer composition. Under several circumstances, mixtures of complex morphologies were observed, which hypothetically arise from the severe sensitivity to D/L0 combined with the inherent compositional/molar mass dispersities associated with the nonuniform synthetic materials and silicon templates. Stochastic calculations offer compelling support for the hypothesis, and tractable pathways toward solving this apparent conundrum are proposed. The materials hold great promise for next-generation nanofabrication to address several emerging technologies, offering significantly enhanced versatility to basic diblock copolymers as templates for fabricating complex nanoscale objects.

Edge transfer lithography using alkanethiol inks.

Edge lithographic patterning techniques are based on the utilization of the edges of micrometer-sized template features for the reproduction of submicrometer structures. Edge transfer lithography (ETL) permits local surface modification in a single step by depositing self-assembled monolayers onto a metal substrate selectively along the feature edges of an elastomeric stamp. In this report two stamp designs are described that now allow for the use of alkanethiol inks in ETL and their use as etch resists to reproduce submicrometer structures in gold. Anisotropically modified stamps are shown to combine the potential for very high-resolution patterning with the versatility and simplicity of microcontact printing.

Viscoelastic properties of single attached cells under compression.

The viscoelastic properties of single, attached C2C12 myoblasts were measured using a recently developed cell loading device. The device allows global compression of an attached cell, while simultaneously measuring the associated forces. The viscoelastic properties were examined by performing a series of dynamic experiments over two frequency decades (0.1-10 Hz) and at a range of axial strains (approximately 10-40%). Confocal laser scanning microscopy was used to visualize the cell during these experiments. To analyze the experimentally obtained force-deformation curves, a nonlinear viscoelastic model was developed. The nonlinear viscoelastic model was able to describe the complete series of dynamic experiments using only a single set of parameters, yielding an elastic modulus of 2120 +/- 900 Pa for the elastic spring, an elastic modulus of 1960 +/- 1350 for the nonlinear spring, and a relaxation time constant of 0.3 +/- 0.12 s. To our knowledge, it is the first time that the global viscoelastic properties of attached cells have been quantified over such a wide range of strains. Furthermore, the experiments were performed under optimal environmental conditions and the results are, therefore, believed to reflect the viscoelastic mechanical behavior of cells, such as would be present in vivo.

Anisotropic, three-dimensional deformation of single attached cells under compression.

Quantifying three-dimensional deformation of cells under mechanical load is relevant when studying cell deformation in relation to cellular functioning. Because most cells are anchorage dependent for normal functioning, it is desired to study cells in their attached configuration. This study reports new three-dimensional morphometric measurements of cell deformation during stepwise compression experiments with a recently developed cell loading device. The device allows global, unconfined compression of individual, attached cells under optimal environmental conditions. Three-dimensional images of fluorescently stained myoblasts were recorded with confocal microscopy and analyzed with image restoration and three-dimensional image reconstruction software to quantify cell deformation. In response to compression, cell width, cross-sectional area, and surface area increased significantly with applied strain, whereas cell volume remained constant. Interestingly, the cell and the nucleus deformed perpendicular to the direction of actin filaments present along the long axis of the cell. This strongly suggests that this anisotropic deformation can be attributed to the preferred orientation of actin filaments. A shape factor was introduced to quantify the global shape of attached cells. The increase of this factor during compression reflected the anisotropic deformation of the cell.

Decreased mechanical stiffness in LMNA-/- cells is caused by defective nucleo-cytoskeletal integrity: implications for the development of laminopathies.

Laminopathies comprise a group of inherited diseases with variable clinical phenotypes, caused by mutations in the lamin A/C gene (LMNA). A prominent feature in several of these diseases is muscle wasting, as seen in Emery-Dreifuss muscle dystrophy, dilated cardiomyopathy and limb-girdle muscular dystrophy. Although the mechanisms underlying this phenotype remain largely obscure, two major working hypotheses are currently being investigated, namely, defects in gene regulation and/or abnormalities in nuclear architecture causing cellular fragility. In this study, using a newly developed cell compression device we have tested the latter hypothesis. The device allows controlled application of mechanical load onto single living cells, with simultaneous visualization of cellular deformation and quantitation of resistance. With the device, we have compared wild-type (MEF+/+) and LMNA knockout (MEF-/-) mouse embryonic fibroblasts (MEFs), and found that MEF-/- cells show a significantly decreased mechanical stiffness and a significantly lower bursting force. Partial rescue of the phenotype by transfection with either lamin A or lamin C prevented gross nuclear disruption, as seen in MEF-/- cells, but was unable to fully restore mechanical stiffness in these cells. Our studies show a direct correlation between absence of LMNA proteins and nuclear fragility in living cells. Simultaneous recordings by confocal microscopy revealed that the nuclei in MEF-/- cells, in contrast to MEF+/+ cells, exhibited an isotropic deformation upon indentation, despite an anisotropic deformation of the cell as a whole. This nuclear behaviour is indicative for a loss of interaction of the disturbed nucleus with the surrounding cytoskeleton. In addition, careful investigation of the three-dimensional organization of actin-, vimentin- and tubulin-based filaments showed a disturbed interaction of these structures in MEF-/- cells. Therefore, we suggest that in addition to the loss of nuclear stiffness, the loss of a physical interaction between nuclear structures (i.e. lamins) and the cytoskeleton is causing more general cellular weakness and emphasizes a potential key function for lamins in maintaining cellular tensegrity.

In vitro models to study compressive strain-induced muscle cell damage.

Skeletal muscle tissue is highly susceptible to sustained compressive straining, eventually leading to tissue breakdown in the form of pressure sores. This breakdown begins at the cellular level and is believed to be triggered by sustained cell deformation. To study the relationship between compressive strain-induced muscle cell deformation and damage, and to investigate the role of cell-cell interactions, cell-matrix interactions and tissue geometry in this process, in vitro models of single cells, monolayers and 3D tissue analogs under compression are being developed. Compression is induced using specially designed loading devices, while cell deformation is visualised with confocal microscopy. Cell damage is assessed from viability tests, vital microscopy and histological or biochemical analyses. Preliminary results from a 3D cell seeded agarose model indicate that cell deformation is indeed an important trigger for cell damage; sustained compression of the model at 20% strain results in a significant increase in cell damage with time of compression, whereas damage in unstrained controls remains constant over time.

Photoinduced electron transfer in a mesogenic donor-acceptor-donor system.

A novel donor-acceptor-donor molecule consisting of two oligo(p-phenylene vinylene) (OPV4) units attached to a central perylene bisimide (PERY) core is described. This OPV4-PERY-OPV4 is the first mesogenic molecule that incorporates both p- and n-type semiconducting properties and possesses a liquid-crystalline mesophase, in which donor and acceptor functionalities self-assemble into an ordered material. Upon photoexcitation of the donor, a subpicosecond electron-transfer reaction occurs in OPV4-PERY-OPV4, both in solution and in (ordered) thin solid films. The lifetime of the charge-separated state is significantly longer in (ordered) thin films than in solution as a result of a reduction of geminate recombination by migration and spatial separation of charges in the film.