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Cells engage in mechanical force exchange with their extracellular environment through tension generated by the cytoskeleton. A method combining laser scanning confocal microscopy (LSCM) and digital volume correlation (DVC) enables tracking and quantification of cell-mediated deformation of the extracellular matrix in all three spatial dimensions. Time-lapse confocal imaging of migrating 3T3 fibroblasts on fibronectin (FN)-modified polyacrylamide gels of varying thickness reveals significant in-plane (x, y) and normal (z) displacements, and illustrates the extent to which cells, even in nominally two-dimensional (2-D) environments, explore their surroundings in all three dimensions. The magnitudes of the measured displacements are independent of the elastic moduli of the gels. Analysis of the normal displacement profiles suggests that normal forces play important roles even in 2-D cell migration.digital volume correlation ͉ laser scanning confocal microscopy ͉ three-dimensional T he measurement of cellular traction forces has been of increasing interest since the discovery that the mechanical properties of the cellular microenvironment can direct many important cellular processes including spreading, migration, and differentiation (1-4). It is now widely accepted that mechanical properties must be considered along with chemical signals if we are to understand how cells integrate environmental cues to modulate their behavior (5-8). The correlation between cell-induced deformations in materials and biochemical signaling and regulation, particularly focal adhesion formation and clustering, has been investigated through the use of a variety of techniques including surface wrinkling, displacement-tracking using traction force microscopy (TFM), and bending of pillar arrays (9-15). These methods have yielded substantial insight into cellular behavior, but are inherently restricted to two-dimensional (2-D) analysis and interpretation of cell-matrix interactions. Furthermore, these approaches calculate stresses by comparing images before and after cell detachment (10), thus providing only snapshots of cell behavior rather than dynamic analyses of the processes by which cells explore their microenvironments.In this report, we demonstrate the capability to dynamically track and quantify cellular traction forces in three dimensions (3-D). Mechanical interactions between 3T3 fibroblasts and FN-modified polyacrylamide gels are quantified dynamically by computing the displacement and traction fields generated by motile cells. Use of a recently developed digital volume correlation (DVC) method (16) allows 3-D displacements and traction fields to be determined directly from volumetric confocal image stacks, and obviates the need for complex inverse formulations (10). The method has a temporal resolution that permits confocal imaging over time scales relevant for the migration of anchorage dependent cells, such as endothelial cells and fibroblasts (17).The 3-D character of this approach relies on the use of laser scanning confoca...

Biological cells sense and respond to mechanical forces, but how such a mechanosensing process takes place in a nonlinear inhomogeneous fibrous matrix remains unknown. We show that cells in a fibrous matrix induce deformation fields that propagate over a longer range than predicted by linear elasticity. Synthetic, linear elastic hydrogels used in many mechanotransduction studies fail to capture this effect. We develop a nonlinear microstructural finite-element model for a fibre network to simulate localized deformations induced by cells. The model captures measured cell-induced matrix displacements from experiments and identifies an important mechanism for long-range cell mechanosensing: loss of compression stiffness owing to microbuckling of individual fibres. We show evidence that cells sense each other through the formation of localized intercellular bands of tensile deformations caused by this mechanism.

The interactions between biochemical processes and mechanical signaling play important roles during various cellular processes such as wound healing, embryogenesis, metastasis, and cell migration. While traditional traction force measurements have provided quantitative information about cell matrix interactions in two dimensions, recent studies have shown significant differences in the behavior and morphology of cells when placed in three-dimensional environments. Hence new quantitative experimental techniques are needed to accurately determine cell traction forces in three dimensions. Recently, two approaches both based on laser scanning confocal microscopy have emerged to address this need. This study highlights the details, implementation and advantages of such a three-dimensional imaging methodology with the capability to compute cellular traction forces dynamically during cell migration and locomotion. An application of this newly developed three-dimensional traction force microscopy (3D TFM) technique to single cell migration studies of 3T3 fibroblasts is presented to show that this methodology offers a new quantitative vantage point to investigate the three-dimensional nature of cell-ECM interactions.

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