Three-Dimensional Traction Force Measurement using Planar Epifluorescence Microscopy for Cell Mechanics Studies

Abstract

Traction force microscopy (TFM) has become a standard biophysical tool for quantitatively measuring mechanical forces in cell-cell and cell-extracellular matrix interactions. Currently, 3D TFM techniques require a three-dimensional imaging modality, e.g., a confocal microscope, to capture the three-dimensional cell-imparted motion fields of a substrate embedded with fiducial marker particles. While full, volumetric three-dimensional imaging techniques provide the greatest spatial resolution in all three directions, their high upfront cost has kept their proliferation into the mainstream communities limited as compared to epifluorescence microscopy. To provide the community with full access to three-dimensional measurements without the burden of requiring a truly volumetric imaging modality, we present a new 3D TFM technique to measure the three-dimensional displacement of maker particles using a planar epifluorescence microscope. The known mechanical properties and three-dimensional deformation measurement of the substrate are used to compute three-dimensional tractions in a standard manner using a previously established forward approach. With the use of different color fluorescent beads, deconvolution and position-based single particle tracking (P-SPT), we can accurately and reliably reconstruct 3D deformation fields from two-dimensional epifluorescence images. As a test case, we measure the three-dimensional tractions produced by breast adenocarcinoma cells and mammary epithelial cells. Our techniques' ability to use epifluorescence microscopy to measure three-dimensional deformation makes traction force measurement more accessible to a much broader biological community.