Total Internal Reflection Fluorescence Microscopy, Cell Adhesion, and Cell Traction Force Measurements on Soft Silicone Gels

Abstract

Substrate rigidity impacts cellular behaviors such as migration, gene expression, and cell fate. Total Internal Reflection Fluorescence (TIRF) microscopy enables selective visualization of the dynamics of substrate adhesions, vesicle trafficking, and biochemical signaling at the cell-substrate interface. Here we apply high-refractive-index silicone gels to perform TIRF microscopy on substrates with a wide range of physiological elastic moduli, measure traction forces exerted by cells on the substrate, and perform analysis of the cell spreading and the biochemical signaling that drives this process. We successfully implemented TIRF on mouse endothelial cells plated on ∼30 micron thick layers of silicone gels with a refractive index of 1.49 and obtained high quality images. We also functionalized gel surfaces and covalently bound to them 40nm far-red fluorescent beads that were distributed nearly evenly with a high surface density. Measurements of displacements of the beads under cell traction forces made it possible to obtain high-resolution substrate deformation maps that were converted to traction force maps. We show mouse embryonic fibroblasts (MEFs) spreading over a significantly larger area on silicone gels of moderate physiological stiffness, 30 kPa, versus those grown on softer substrates of 0.5 and 3.5 kPa, in agreement with cells grown on polyacrylamide substrates of similar stiffness. We developed a novel microfluidic technique to measure the elastic moduli of thin gel layers and applied it to characterizing gels made using Sylgard 184 silicone with elastic moduli from 0.4 to 300 kPa. We prepared gel substrates with elastic moduli covering nearly the entire physiological range in different wells of a 6-well plate. Western-blot analysis of harvested cells revealed varying levels of phosphorylated Focal Adhesion Kinase (FAK) in MEFs grown on silicone gels with different elastic moduli.