A Stretching Device for High-Resolution Live-Cell Imaging

Biophysical Journal, 2003

A central aspect of cellular mechanochemical signaling is a change of cytoskeletal tension upon the imposition of exogenous forces. Here we report measurements of the spatiotemporal distribution of mechanical strain in the intermediate filament cytoskeleton of endothelial cells computed from the relative displacement of endogenous green fluorescent protein (GFP)-vimentin before and after onset of shear stress. Quantitative image analysis permitted computation of the principal values and orientations of Lagrangian strain from 3-D high-resolution fluorescence intensity distributions that described intermediate filament positions. Spatially localized peaks in intermediate filament strain were repositioned after onset of shear stress. The orientation of principal strain indicated that mechanical stretching was induced across cell boundaries. This novel approach for intracellular strain mapping using an endogenous reporter demonstrates force transfer from the lumenal surface throughout the cell.

PLoS ONE, 2011

Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G9 = 0.3 kPa) to stiff (G9 = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p,0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch.

Annals of Biomedical Engineering, 2000

A stretch chamber has been developed in order to visualize the deformation of cells subjected to controlled uniaxial stretch of their substrate. A rectangular, custom-made, transparent silicone channel is used as a deformable substrate. Bovine aortic endothelial cells are plated at the bottom of the channel whose lateral deformation is controlled by two piezoelectric translators. The system is mounted on the stage of a confocal microscope where three-dimensional ͑3D͒ images of the cells can be acquired simultaneously in the three RGB channels. The first channel provides images of 216 nm fluorescent beads embedded in the cytoskeleton ͑used as internal markers͒. The second is used to image the shape of the nucleus revealed by live cell nucleic acid staining. The third one provides a transmitted light image of the cell outline. 3D images of the cell are taken before deformation, after uniaxial deformation of the substrate ͑up to 25%͒ and after relaxation. Results indicate that: ͑a͒ the cell closely follows the deformation imposed by the substrate with no measurable residual strain after relaxation, and ͑b͒ there is a clear mechanical coupling between the extracellular matrix and the nucleus, which deforms significantly under the applied substrate stretch. Suggesting that the nucleus can directly sense the mechanical environment of the cell, the latter result has potentially important implications for signal transduction. © 1998 Biomedical Engineering Society. ͓S0090-6964͑98͒01003-0͔

The mechanical micro-environment influences cellular responses such as migration, proliferation, differentiation and apoptosis. Cells are subjected to mechanical stretching in vivo, e.g., epithelial cells during embryogenesis. Current methodologies do not allow high-resolution in situ observation of cells and tissues under applied strain, which may reveal intracellular dynamics and the origin of cell mechanosensitivity. A novel polydimethylsiloxane substrate was developed, capable of applying tensile and compressive strain (up to 45%) to cells and tissues while allowing in situ observation with high-resolution optics. The strain field of the substrate was characterized experimentally using digital image correlation, and the deformation was modeled by the finite element method, using a Mooney-Rivlin hyperelastic constitutive relation. The substrate strain was found to be uniform for >95% of the substrate area. As a demonstration of the system, mechanical strain was applied to single fibroblasts transfected with GFP-actin and whole transgenic Drosophila embryos expressing GFP in all neurons during live imaging. Three observations of biological responses due to applied strain are reported: (1) dynamic rotation of intact actin stress fibers in fibroblasts; (2) lamellipodia activity and actin polymerization in fibroblasts; (3) active axonal contraction in Drosophila embryo motor neurons. The novel platform may serve as an important tool in studying the mechanoresponse of cells and tissues, including whole embryos.

PLOS ONE, 2015

Cells in the body experience various mechanical stimuli that are often essential to proper cell function. In order to study the effects of mechanical stretch on cell function, several devices have been built to deliver cyclic stretch to cells; however, they are generally not practical for live cell imaging. We introduce a novel device that allows for live cell imaging, using either an upright or inverted microscope, during the delivery of cyclic stretch, which can vary in amplitude and frequency. The device delivers equi-biaxial strain to cells seeded on an elastic membrane via indentation of the membrane. Membrane area strain was calibrated to indenter depth and the device showed repeatable and accurate delivery of strain at the scale of individual cells. At the whole cell level, changes in intracellular calcium were measured at different membrane area strains, and showed an amplitude-dependent response. At the subcellular level, the mitochondrial network was imaged at increasing membrane area strains to demonstrate that stretch can lead to mitochondrial fission in lung fibroblasts. The device is a useful tool for studying transient as well as long-term mechanotransduction as it allows for simultaneous stretching and imaging of live cells in the presence of various chemical stimuli.

The Analyst, 2012

Mechanical forces affect biological systems in their natural environment in a widespread manner. Mechanical stress may either stimulate cells or even induce pathological processes. Cells sensing mechanical stress usually respond to such stressors with proliferation or differentiation. Hence, for in vitro studies, the ability to impose a controlled mechanical stress on cells combined with appropriate analytical tools providing an immediate answer is essential to understand such fundamental processes. Here, we present a novel uniaxial motorized cell stretching device that has been integrated into a combined fluorescence microscope (FM)-atomic force microscope (AFM) system, thereby enabling high-resolution topographic and fluorescent live cell imaging. This unique tool allows the investigation of mechanotransduction processes, as the cells may be exposed to deliberately controlled mechanical stress while simultaneously facilitating fluorescence imaging and AFM studies. The developed stretching device allows applying reproducible uniaxial strain from physiologically relevant to hyperphysiological levels to cultured cells grown on elastic polydimethylsiloxane (PDMS) membranes. Exemplarily, stretching experiments are shown for transfected squamous cell carcinoma cells (SCC-25) expressing fluorescent labeled cytokeratin, whereby fluorescence imaging and simultaneously performed AFM measurements reveal the cytokeratin (CSK) network. Topographical changes and mechanical characteristics such as elasticity changes were determined via AFM while the cells were exposed to mechanical stress. By applying a cell deformation of approx. 20%, changes in the Young's modulus of the cytoskeletal network due to stretching of the cells were observed. Consequently, integrating a stretching device into the combined atomic force-fluorescence microscope provides a unique tool for dynamically analyzing structural remodeling and mechanical properties in mechanically stressed cells.

Biophysical Journal, 2008

The mechanical properties of the living cell are intimately related to cell signaling biology through cytoskeletal tension. The tension borne by the cytoskeleton (CSK) is in part generated internally by the actomyosin machinery and externally by stretch. Here we studied how cytoskeletal tension is modified during stretch and the tensional changes undergone by the sites of cell-matrix interaction. To this end we developed a novel technique to map cell-matrix stresses during application of stretch. We found that cell-matrix stresses increased with imposition of stretch but dropped below baseline levels on stretch release. Inhibition of the actomyosin machinery resulted in a larger relative increase in CSK tension with stretch and in a smaller drop in tension after stretch release. Cell-matrix stress maps showed that the loci of cell adhesion initially bearing greater stress also exhibited larger drops in traction forces after stretch removal. Our results suggest that stretch partially disrupts the actinmyosin apparatus and the cytoskeletal structures that support the largest CSK tension. These findings indicate that cells use the mechanical energy injected by stretch to rapidly reorganize their structure and redistribute tension.

Journal of Biomechanical Engineering, 2011

Mechanical forces are key regulators of cell function with varying loads capable of modulating behaviors such as alignment, migration, phenotype modulation, and others. Historically, cell-stretching experiments have employed mechanically simple environments (e.g., uniform uniaxial or equibiaxial stretches). However, stretch distributions in vivo can be highly non-uniform, particularly in cases of disease or subsequent to interventional treatments. Herein, we present a cell-stretching device capable of subjecting cells to controllable gradients in biaxial stretch via radial deformation of circular elastomeric membranes. By including either a defect or a rigid fixation at the center of the membrane, various gradients are generated. Capabilities of the device were quantified by tracking marked positions of the membrane while applying various loads, and experimental feasibility was assessed by conducting preliminary experiments with 3T3 fibroblasts and 10T1/2 cells subjected to 24 h of ...

ObjectiveAdherent cell behavior is influenced by a complex interplay of factors, including chemical and mechanical signals.In vitroexperiments that mimic the mechanical environment experienced by cellsin vivoare crucial for understanding cellular behavior and the progression of disease. In this study, we developed and validated a low-cost pneumatically-controlled cell stretcher with independent control of strain in two directions of a membrane, enabling unequal biaxial stretching and realtime microscopy during actuation.MethodsThe stretching was achieved by two independent pneumatic channels controlled by electrical signals. We used finite element simulations to compute the membrane’s strain field and particle tracking algorithms based on image processing techniques to validate the strain fields and measure the cell orientation and morphology.ResultsThe device can supply uniaxial, equibiaxial, and unequal biaxial stretching up to 15% strain in each direction at a frequency of 1Hz, w...

Experiments have shown that mechanical stress can regulate many cellular processes. However, in most cases, the exact regulatory mechanisms are still not well understood. One approach in improving our understanding of such mechanically induced regulation is the quantitative study of cell deformation under an externally applied stress. In this paper, an axisymmetric finite-element model is developed and used to study the deformation of single, suspended fibroblasts in an optical stretcher in which a stretching force is applied onto the surface of the cell. A feature of our physical model is a viscoelastic material equation whose parameters vary spatially to mimic the experimentally observed spatial heterogeneity of cellular material properties. Our model suggests that cell size is a more important factor in determining the maximal strain of the optically stretched fibroblasts compared to the thickness of the actin cortical region. This result could explain the higher deformability observed experimentally for malignant fibroblasts in the optical stretcher. Our model also shows that maximal stress propagates into the nuclear region for malignant fibroblasts whereas for normal fibroblasts, it does not. We discuss how this may impact the transduction of cancer signaling pathways.