Force Sensing with Elastic Micropillars
Elastic micropillars are a technique used to quantify cellular traction forces when cells are in contact with a substrate. The micropillar technique may be used to measure the forces present in the early phases of cell spreading, dynamics after extended periods of time or the effects of substrate stiffness on traction forces.
Micropillar arrays are formed from Polydimethylsiloxane (PDMS) mixed with a curing agent (Sylgard 184) which is deposited on to silicon moulds containing a repeating pattern of micron-wide holes with varying depth. To achieve a desired Young’s modulus (2 MPa) PDMS is mixed with its curing agent at a 10:1 ratio and cured at 70ᵒC for 12 hours. To vary the rigidity of the substrate, micropillar spring constants are varied though the dimensions of the pillars (in this case the height) allowing for arrays with spring constants 0.5-10 nN/µm. (Eq.1)
Eq.1. The relation between deflection, Δx and force where the spring constant k is determined by pillar properties E, Young’s modulus, D Pillar diameter and L, pillar length.
Pillars used for Time vs. Deflection plots and the analysis of peak forces applied by the stellate cells during spreading.
Cellular forces are determined with fibronectin coated micropillar arrays by analysing the deflection of the vertical pillars in image sequences obtained via brightfield microscopy. Through the application of Hooke’s Law the force is directly proportional to the deflection of a pillar and thus can be calculated using pillars of a known dimension/spring constant.
We use custom algorithms to quantify the forces that cells exert on pillars. (A) Microscopic image of a pillar array with a cell placed on the pillars. (B) Vector-map that represents the magnitude and direction of the force applied by the cell on each pillar. (C) Heat map for the force generated by this cell on the pillars
Time vs. Pillar deflection traces of cells on 6 micron pillars. Each trace shows the calculated peak force value.
Tracking of micropillars in acquired image sequences is conducted with an ImageJ plugin which allows pixel displacements to be recorded. Applying known pixel sizes to displacement values allows the pillar displacement to be calculated, this is combined with the known spring constant to calculate the force acting on a pillar (Eq.1).
Immunofluorescence or in-vivo cell imaging can be combined with elastic pillars to investigate the location and function of specific proteins involved in mechanosensing.
Cell spreading on 6 micron pillars with image acquisition at 2 second intervals for 15 minutes (video 100X faster).
Immunofluorescence images of cells on 1um pillars. The images show the location of paxillin (green) and myosin (red).
Deflection of pillars during cell spreading
Deflection of pillars during cell contraction