N-cadherin-functionalized polymer-tethered multi-bilayer: a cell surface-mimicking substrate to probe cellular mechanosensitivity.

Fate and function of anchorage-dependent cells depend on a variety of environmental cues, including those of mechanical nature. Previous progress in the understanding of cellular mechanosensitivity has been closely linked to the availability of artificial cell substrates of adjustable viscoelasticity, allowing for a direct correlation between substrate stiffness and cell response. Exemplary, polymeric gel substrates with polymer-conjugated cell-substrate linkers provided valuable insight into the role of mechanical signals during cell migration in an extracellular matrix environment. In contrast, less is known about the role of external mechanical signals across cell-cell interfaces, in part, due to the limitations of traditional polymeric substrates to mimic the remarkable dynamics of cell-cell linkages. To overcome this shortcoming, we introduce a cell surface-mimicking cell substrate of adjustable stiffness, which is comprised of a polymer-tethered lipid multi-bilayer stack with N-cadherin linkers. Unlike traditional polymeric cell substrates with polymer-conjugated linkers, this novel artificial cell substrate is able to replicate the dynamic assembly/disassembly of cadherin linkers into linker clusters and the long-range movements of cadherin-based cell-substrate linkages observed at cell-cell interfaces. Moreover, substrate stiffness can be changed by adjusting the number of bilayers in the multi-bilayer stack, thus enabling the analysis of cellular mechanosensitivity in the presence of artificial cell-cell linkages. The presented biomembrane-mimicking cell substrate provides a valuable tool to explore the functional role of mechanical cues from neighboring cells.

The IsoStretcher: An isotropic cell stretch device to study mechanical biosensor pathways in living cells.

Mechanosensation in many organs (e.g. lungs, heart, gut) is mediated by biosensors (like mechanosensitive ion channels), which convert mechanical stimuli into electrical and/or biochemical signals. To study those pathways, technical devices are needed that apply strain profiles to cells, and ideally allow simultaneous live-cell microscopy analysis. Strain profiles in organs can be complex and multiaxial, e.g. in hollow organs. Most devices in mechanobiology apply longitudinal uniaxial stretch to adhered cells using elastomeric membranes to study mechanical biosensors. Recent approaches in biomedical engineering have employed intelligent systems to apply biaxial or multiaxial stretch to cells. Here, we present an isotropic cell stretch system (IsoStretcher) that overcomes some previous limitations. Our system uses a rotational swivel mechanism that translates into a radial displacement of hooks attached to small circular silicone membranes. Isotropicity and focus stability are demonstrated with fluorescent beads, and transmission efficiency of elastomer membrane stretch to cellular area change in HeLa/HEK cells. Applying our system to lamin-A overexpressing fibrosarcoma cells, we found a markedly reduced stretch of cell area, indicative of a stiffer cytoskeleton. We also investigated stretch-activated Ca(2+) entry into atrial HL-1 myocytes. 10% isotropic stretch induced robust oscillating increases in intracellular Fluo-4 Ca(2+) fluorescence. Store-operated Ca(2+) entry was not detected in these cells. The Isostretcher provides a useful versatile tool for mechanobiology.

Differential interference contrast microscopy using light-emitting diode illumination in conjunction with dual optical traps.

Differential interference contrast (DIC) microscopy is a common mode of biological light microscopy used to achieve maximal resolution and contrast with label-free, weakly absorbing specimens such as cells. Maintaining the polarization state of the illuminating light is essential for the technique, and this requirement can conflict with optical trapping. We describe how to optimize DIC imaging using a light-emitting diode illumination source in a microscope while integrating a dual optical trap into the set up. Every time a polarized light beam reflects off or transmits through a dichroic mirror in the beam path, its polarization state will change if it is not polarized exactly parallel (p) or perpendicular (s) to the plane of incidence. We observe wavelength-dependent optical rotation and depolarization effects in our illumination light upon reflection from/transmission through dichroic mirrors in the beam path, resulting in significant degradation of image quality. We describe a method to compensate for these effects by introducing quarter-waveplates and a laser clean-up filter into the imaging pathway. We show that this approach achieves a full recovery of image quality.