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...

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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A three-dimensional (3-D) full-field measurement technique was developed for measuring large deformations in optically transparent soft materials. The technique utilizes a digital volume correlation (DVC) algorithm to track motions of subvolumes within 3-D images obtained using fluorescence confocal microscopy. In order to extend the strain measurement capability to the large deformation regime (>5%), a stretch-correlation algorithm was developed and implemented into the Fast Fourier Transform (FFT)-based DVC algorithm. The stretch-correlation algorithm uses a logarithmic coordinate transformation to convert the stretchcorrelation problem into a translational correlation problem under the assumption of small rotation and shear. Estimates of the measurement precision are provided by stationary and translation tests. The proposed measurement technique was used to measure large deformations in a transparent agarose gel sample embedded with fluorescent particles under uniaxial compression. The technique was also employed to measure non-uniform deformation fields near a hard spherical inclusion under far-field uniaxial compression. Introduction of the stretch-correlation algorithm greatly improved the strain measurement accuracy by providing better precision especially under large deformation. Also, the deconvolution of confocal images improved the accuracy of the measurement in the direction of the optical axis. These results shows that the proposed technique is well-suited for investigating cell-matrix mechanical interactions as well as for obtaining local constitutive properties of soft biological materials including tissues in 3-D.

In the United States over 1.7 million cases of traumatic brain injury are reported yearly, but predictive correlation of cellular injury to impact tissue strain is still lacking, particularly for neuronal injury resulting from compression. Given the prevalence of compressive deformations in most blunt head trauma, this information is critically important for the development of future mitigation and diagnosis strategies. Using a 3D in vitro neuronal compression model, we investigated the role of impact strain and strain rate on neuronal lifetime, viability, and pathomorphology. We find that strain magnitude and rate have profound, yet distinctively different effects on the injury pathology. While strain magnitude affects the time of neuronal death, strain rate influences the pathomorphology and extent of population injury. Cellular injury is not initiated through localized deformation of the cytoskeleton but rather driven by excess strain on the entire cell. Furthermore we find that, mechanoporation, one of the key pathological trigger mechanisms in stretch and shear neuronal injuries, was not observed under compression.

Control of the spatial arrangement of proteins on surfaces is essential in a number of emerging technologies, including protein microarrays, biosensors, 1 tissue engineering, and regenerative medicine. 2 Patterning is also a powerful tool in cell biology, wherein cell arrays are used to elucidate the factors that mediate migration, proliferation, and cell-cell interactions. 3 Although photolithography holds a preeminent place as a patterning method in the microelectronics industry, optical lithography of proteins has been hampered by the need either to use traditional chemical photoresists or to modify proteins chemically by attachment of photoreactive functional groups; both methods can compromise protein function. 4Production of a protein "photoresist" without the need for post-translational chemical modification would require an intrinsically photoreactive protein. Recently, the incorporation of photo-reactive, non-canonical amino acids into proteins via site-specific 5 and residuespecific techniques has been reported. 6 Here we describe the microbial expression of artificial proteins bearing the photosensitive non-canonical amino acid para-azidophenylalanine (pN 3 Phe). The recombinant proteins, designated artificial extracellular matrix proteins with aryl azides (aECM-N 3 ), belong to a family of engineered proteins designed to exhibit mechanical properties similar to those of native elastins 7 and to support adhesion of mammalian cells through cell-binding domains (CS5 or RGD) derived from fibronectin ( Fig 1A). 8 These proteins can be crosslinked efficiently upon irradiation at 365 nm. The physical properties of the crosslinked films can be controlled by changing the pN 3 Phe content, and thin films can be patterned on surfaces via photolithographic techniques. We demonstrate the utility of the method by creating cell arrays through selective cell attachment to photolithographically prepared protein patterns. aECM-N 3 variants were expressed in Escherichia coli cultures supplemented with pN 3 Phe (Supporting Information). Incorporation of pN 3 Phe into the recombinant proteins relies on activation of the photosensitive amino acid by the phenylalanyl-tRNA synthetase (PheRS) of the bacterial expression host. The PheRS used for this study was a previously characterized mutant with relaxed substrate specificity. 9 This method results in statistical decoding of phenylalanine (Phe) codons placed at regular intervals in the coding sequence. 9 Proteins were expressed in a Phe-auxotrophic E. coli strain and purified by exploiting the temperaturedependent phase behavior of proteins that contain elastin-like repeats. 10 Incorporation efficiency was determined by integration of the aromatic proton signals in the 1 H NMR spectra of the purified proteins; the extent of Phe replacement varied from 13% to 53%, depending on the concentration of pN 3 Phe in the expression medium (Supporting Information). Understanding the response of the photoreactive protein to irradiation is crucial for highresolution pattern formation....

Over the past decade, investigators have attempted to establish the pathophysiological mechanisms by which non-penetrating injuries damage the brain. Several studies have implicated either membrane poration or ion channel dysfunction pursuant to neuronal cell death as the primary mechanism of injury. We hypothesized that traumatic stimulation of integrins may be an important etiological contributor to mild Traumatic Brain Injury. In order to study the effects of forces at the cellular level, we utilized two hierarchical, in vitro systems to mimic traumatic injury to rat cortical neurons: a high velocity stretcher and a magnetic tweezer system. In one system, we controlled focal adhesion formation in neurons cultured on a stretchable substrate loaded with an abrupt, one dimensional strain. With the second system, we used magnetic tweezers to directly simulate the abrupt injury forces endured by a focal adhesion on the neurite. Both systems revealed variations in the rate and nature of neuronal injury as a function of focal adhesion density and direct integrin stimulation without membrane poration. Pharmacological inhibition of calpains did not mitigate the injury yet the inhibition of Rho-kinase immediately after injury reduced axonal injury. These data suggest that integrin-mediated activation of Rho may be a contributor to the diffuse axonal injury reported in mild Traumatic Brain Injury.