ABSTRACTIn this work we demonstrate the integration of magnetic tweezers (MT) with substrate deformation tracking microscopy (DTM) and traction force microscopy (TFM) for the investigation of extracellular matrix rheology and human epidermal keratinocyte mechanobiology in the context of human blistering skin diseases. Two model bead-on-gel experiments are described in which an MT device is used to apply a prescribed force or displacement waveform to a fibronectin-coated superparamagnetic bead attached to a type I collagen gel containing a layer of covalently attached red-fluorescent microspheres. Serial fast time-lapse DIC and epifluorescence image acquisitions are used to capture displacements of the bead and microspheres, respectively, in response to the applied force or displacement. Due to the large number of acquired images and the dynamic behavior of substrate microspheres observed during the experiment, new quantitative methods are developed for the tracking and filtering of microsphere displacement data, the selection of L2 regularization parameters used for TFM analysis, and the identification of time intervals within the overall image set that can be approximated as being subject to elastostatic conditions. Two major proof-of-concept applications are described in which integrated MT-DTM/TFM experiments are used to (i) estimate the elastic properties of a fibrillar type I collagen gel substrate and (ii) demonstrate how a force applied to a focal adhesion contact on the apical surface of a living keratinocyte is directly transmitted to basal cell-matrix anchoring junctions as observed by substrate deformations and incremental traction stresses that develop within the collagen subjacent to the cell.
Integration of Magnetic Tweezers and Traction Force Microscopy for the Exploration of Matrix Rheology and Keratinocyte Mechanobiology: Model Force- and Displacement-Controlled Experiments
