Design of a Novel Equi-Biaxial Stretcher for Live Cellular and Subcellular Imaging.

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

Design of a Novel Equi-Biaxial Stretcher for Live Cellular and Subcellular Imaging - Fig 2

<p>(<i>A</i>) Nonlinear relationship between the indenter depth and the corresponding change in surface area of a demarcated region on the membrane; this calibration curve was subsequently used to prescribe strain waveforms as a function of indenter depth. Data points represent the average of n = 3 calibrations with different membranes; standard deviation bars (not shown) are smaller than symbols. (<i>B</i>) Representative sinusoidal waveform applied to membrane (amplitude, 20% strain; frequency, 0.0464 Hz), the input waveform (solid line) and the observed change in surface area (open circles) are shown demonstrating close agreement between input and measurement. (<i>C</i>) Frequency response of the device is flat for stretch frequencies between 0.01–1.0 Hz; sinusoidal waveforms with prescribed strain amplitudes of 20% (filled triangles) and 40% (open squares) are shown with reference lines, error bars represent the standard deviations (N = 3). (<i>D</i>) <i>Left</i>: characteristic image of marker beads used to track micro strain; <i>Right</i>: detection algorithm showing beads at baseline (green) and after applied strain (magenta), specific beads are tracked and the areas enclosed by their polygons are used to determine the corresponding micro strain. (<i>E</i>) Change in micro strain closely follows the prescribed macrostrain, line of identity is shown for reference; error bars represent standard deviations (n = 9).</p

Intracellular calcium level responses of primary bovine fibroblasts to single sinusoidal equi-biaxial strains.

<p>Cells were loaded with Fluo4 calcium indicator dye and subjected to strains of 3 or 10% change in membrane surface area (ΔSA). (A) Following single 6 s long stretch (dotted line), cells stretched to 10% ΔSA showed large transient increases in fluorescence relative to baseline (mean of 77 cells), while cells stretched to 3% ΔSA showed little response (mean of 44 cells). Images of cells stretched to 10% ΔSA revealed differences between neighboring cells in the timing of their response to stretch, with some cells responding immediately and other requiring up to 10 s to respond. (B) The changes in fluorescence were significantly greater with 10% ΔSA than with 3% ΔSA (p<0.001).</p

Response of intracellular calcium levels in bovine fibroblasts up to one hour of continuous sinusoidal stretching with maximum amplitude of 10% change in membrane surface area.

<p>Average ratios of Fura2 fluorescence at 340 and 380 nm excitation wavelengths were computed in each of 11 cells. Individual cell responses varied (A), but there was an overall trend of increasing Fura2 ratios over time, with significant increases present at 20 and 60 min relative to baseline (B). **, p<0.01; ***, p<0.001</p