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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.

Traction Force Microscopy (TFM) is a powerful approach for quantifying cell-material interactions that over the last two decades has contributed significantly to our understanding of cellular mechanosensing and mechanotransduction. In addition, recent advances in three-dimensional (3D) imaging and traction force analysis (3D TFM) have highlighted the significance of the third dimension in influencing various cellular processes. Yet irrespective of dimensionality, almost all TFM approaches have relied on a linear elastic theory framework to calculate cell surface tractions. Here we present a new high resolution 3D TFM algorithm which utilizes a large deformation formulation to quantify cellular displacement fields with unprecedented resolution. The results feature some of the first experimental evidence that cells are indeed capable of exerting large material deformations, which require the formulation of a new theoretical TFM framework to accurately calculate the traction forces. Based on our previous 3D TFM technique, we reformulate our approach to accurately account for large material deformation and quantitatively contrast and compare both linear and large deformation frameworks as a function of the applied cell deformation. Particular attention is paid in estimating the accuracy penalty associated with utilizing a traditional linear elastic approach in the presence of large deformation gradients.

Mechanobiology relates cellular processes to mechanical signals, such as determining the effect of variations in matrix stiffness with cell tractions. Cell traction recorded via traction force microscopy (TFM) commonly takes place on materials such as polyacrylamideand polyethylene glycol-based gels. Such experiments remain limited in physiological relevance because cells natively migrate within complex tissue microenvironments that are spatially heterogeneous and hierarchical. Yet, TFM requires determination of the matrix constitutive law (stress-strain relationship), which is not always readily available. In addition, the currently achievable displacement resolution limits the accuracy of TFM for relatively small cells. To overcome these limitations, and increase the physiological relevance of in vitro experimental design, we present a new approach and a set of associated biomechanical signatures that are based purely on measurements of the matrix's displacements without requiring any knowledge of its constitutive laws. We show that our mean deformation metrics (MDM) approach can provide significant biophysical information without the need to explicitly determine cell tractions. In the process of demonstrating the use of our MDM approach, we succeeded in expanding the capability of our displacement measurement technique such that it can now measure the 3D deformations around relatively small cells (∼10 micrometers), such as neutrophils. Furthermore, we also report previously unseen deformation patterns generated by motile neutrophils in 3D collagen gels.traction force microscopy | confocal microscopy | large deformations | neutrophil M echanical cues within the cellular microenvironment regulate numerous fundamental functions including cell adhesion, deformation, and generation of traction (1-6). Analysis of cellular force generation, and its role in regulating homeostasis across a variety of cellular phenotypes and experimental platforms, has received much attention over the last three decades (7-13). Experimental quantification of cellular forces has produced several cell traction measurement techniques, ranging from surface wrinkle detection and flexure of micropillars to traction force microscopy (TFM) (12,(14)(15)(16)(17)(18)(19)(20). In TFM, measured cell-induced displacements are converted into tractions using various mathematical frameworks (14,15,17,18,21,22). Both twoand 3D TFM techniques have steadily increased in sophistication and now feature high-spatial displacement resolution and advanced computational formalisms to connect this displacement information to complex material constitutive laws (17,23,24).To successfully perform TFM, it is critical to know the stressstrain constitutive behavior of the matrix surrounding the cell. Although many TFM substrates feature relatively simple artificial gel constructs, such as polyacrylamide and polyethylene glycol, these constructs are impenetrable by cells and obviate measures obtained while cells are in a 3D setting (as would be the case within a bodily ...

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Native cell-material interactions occur on materials differing in their structural composition, chemistry, and physical compliance. While the last two decades have shown the importance of traction forces during cell-material interactions, they have been almost exclusively presented on purely elastic in-vitro materials. Yet, most bodily tissue materials exhibit some level of viscoelasticity, which could play an important role in how cells sense and transduce tractions. To expand the realm of cell traction measurements and to encompass all materials from elastic to viscoelastic, this paper presents a general, and comprehensive approach for quantifying 3D cell tractions in viscoelastic materials.This methodology includes the experimental characterization of the time-dependent material properties for any viscoelastic material with the subsequent mathematical implementation of the determined material model into a 3D traction force microscopy (3D TFM) framework. Utilizing this new 3D viscoelastic TFM (3D VTFM) approach, we quantify the influence of viscosity on the overall material traction calculations and quantify the error associated with omitting timedependent material effects, as is the case for all other TFM formulations. We anticipate that the 3D VTFM technique will open up new avenues of cell-material investigations on even more physiologically relevant time-dependent materials including collagen and fibrin gels.

The mechanical interaction between Schwann cells (SCs) and their microenvironment is crucial for the development, maintenance and repair of the peripheral nervous system. In this paper, we present a detailed investigation on the mechanosensitivity of SCs across a physiologically relevant substrate stiffness range. Contrary to many other cell types, we find that the SC spreading area and cytoskeletal actin architecture were relatively insensitive to substrate stiffness with pronounced stress fibre formation across all moduli tested (0.24-4.80 kPa). Consistent with the presence of stress fibres, we found that SCs generated large surface tractions on stiff substrates and large, finite material deformations on soft substrates. When quantifying the three-dimensional characteristics of the SC traction profiles, we observed a significant contribution from the out-of-plane traction component, locally giving rise to rotational moments similar to those observed in mesenchymal embryonic fibroblasts. Taken together, these measurements provide the first set of quantitative biophysical metrics of how SCs interact with their physical microenvironment, which are anticipated to aid in the development of tissue engineering scaffolds designed to promote functional integration of SCs into post-injury in vivo environments.

The pyridine nucleotides (NADþ, NADPþ) are the major coenzymes participate in multiple redox processes in living cells. Both NADþ and NADPþ are not fluorescent and almost structurally identical, so it is difficult to directly distinguish NADþ or NADPþ via optical methods (such as fluorescence and Raman spectroscopy). We report here a sensitive probe of NADþ/NADPþ based on fluorescent silver nanoclusters with dual emission band. The silver nanoclusters with an initial fluorescent emission peak at 410 nm were synthesized by etching large size silver nanoparticles. With the addition of NADþ/ NADPþ solution, due to the strong coupling (charge-transfer) between silver nanoclusters and ligands (NADþ/NADPþ), a new fluorescence emission peak of the silver nanoclusters was found and raised at 550 nm and the fluorescence intensity was dependent on the ratio between NADþ and NADPþ. The time-resolved fluorescence decay (at 550 nm) of silver nanoclusters showed a single-exponential decay lifetime of 3.9 ns caused by the strong coupling between silver nanoclusters and ligands (NADþ/NADPþ). Meanwhile, the 410 nm emission was also selectively enhanced by the different ratio of NADþ /NADPþ molecules. The intensity ratio of fluorescence emission at 410nm and 550nm may be useful to monitor the levels of NADþ /NADPþ in aqueous solutions, cellular extracts and living cells. Candidate mechanisms and the analysis of time resolved emission spectra will be discussed.