Nanonet Force Microscopy
Cells migrate through extracellular matrices, often through formation of integrin-mediated focal adhesions. This allows the cells to sense the external environment by exerting forces and thus allowing cells to achieve tensional homeostasis. Mechanical forces are important for biological processes ranging from migration to remodeling of ECM in tumor environment to maintaining tissue homeostasis and organ development. Measurement and calibration of these forces can provide insight in cell mechanobiology and pathology of diseases.
At STEP lab, we have devised a suspended fiber-based force measurement platform (Nanonet Force Microscopy, NFM) using ECM-mimicking anisotropic fibers. NFM estimates cell forces from deflection of fibers (inwards or outwards) as cells tug or push on them. We deposit large diameter fibers (μm) that act as supporting structures (‘base fibers’). Smaller diameter fibers are deposited orthogonal to base fibers at desired spacing and fused at the intersections to achieve fixed-fixed boundary conditions. Our method establishes force vectors that originate from the focal adhesion sites and are directed along the major actin stress fibers (force bearing elements). A combination of beam mechanics and optimization techniques allows us to estimate forces by minimizing the error between finite element model predictions and measured experimental fiber deflections. NFM can be used to study the two-broad classification of forces: innate contractility inside-out (IO) forces and externally applied forces outside-In (OI). The unique fiber based platform has allowed us to demonstrate the following specific applications:
Single Cell Forces after Electroporation
Exogenous high-voltage pulses increase cell membrane permeability through a phenomenon known as electroporation. This process may also disrupt the cell cytoskeleton causing changes in cell contractility; however, the contractile signature of cell force after electroporation remains unknown. Here, single-cell forces post-electroporation are measured using suspended extracellular matrix-mimicking nanofibers that act as force sensors. Ten, 100 μs pulses are delivered at three voltage magnitudes (500, 1000, and 1500 V) and two directions (parallel and perpendicular to cell orientation), exposing glioblastoma cells to electric fields between 441 V cm–1 and 1366 V cm–1. Cytoskeletal-driven force loss and recovery post-electroporation involves three distinct stages. Low electric field magnitudes do not cause disruption, but higher fields nearly eliminate contractility 2–10 min post-electroporation as cells round following calcium-mediated retraction (stage 1). Following rounding, a majority of analyzed cells enter an unusual and unexpected biphasic stage (stage 2) characterized by increased contractility tens of minutes post-electroporation, followed by force relaxation. The biphasic stage is concurrent with actin disruption-driven blebbing. Finally, cells elongate and regain their pre-electroporation morphology and contractility in 1–3 h (stage 3). With increasing voltages applied perpendicular to cell orientation, we observe a significant drop in cell viability. Experiments with multiple healthy and cancerous cell lines demonstrate that contractile force is a more dynamic and sensitive metric than cell shape to electroporation. A mechanobiological understanding of cell contractility post-electroporation will deepen our understanding of the mechanisms that drive recovery and may have implications for molecular medicine, genetic engineering, and cellular biophysics.