O. A. Banda was supported by funding from a NSF IGERT SBE2 fellowship (1144726), startup funds provided by the University of Delaware, and the National Institutes of Health/National Cancer Institute IMAT Program (R21CA214299). JHS is supported by funding from the National Institutes of Health/National Cancer Institute IMAT Program (R21CA214299) and the National Science Foundation CAREER Award Program (1751797). Microscopy access was supported by grants from the NIH-NIGMS (P20 GM103446), the NSF (IIA-1301765) and the State of Delaware. The structured illumination microscope was acquired with funds from the State of Delaware Federal Research and Development Grant Program (16A00471). The LSM880 confocal microscope used for two-photon laser scanning lithography was acquired with a shared instrumentation grant (S10 OD016361).

<ol>
	<li>Rauskolb, C., Sun, S., Sun, G., Pan, Y., Irvine, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal+tension+inhibits+Hippo+signaling+through+an+Ajuba-Warts+complex.">Cytoskeletal tension inhibits Hippo signaling through an Ajuba-Warts complex.</a> Cell. 158 (1), 143-156 (2014).</li><li>Huang, S., Chen, C. S., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Cyclin+D1,+p27Kip1,+and+Cell+Cycle+Progression+in+Human+Capillary+Endothelial+Cells+by+Cell+Shape+and+Cytoskeletal+Tension.">Control of Cyclin D1, p27Kip1, and Cell Cycle Progression in Human Capillary Endothelial Cells by Cell Shape and Cytoskeletal Tension.</a> Molecular Biology of the Cell. 9 (11), 3179-3193 (1998).</li><li>Plotnikov, S. V., Pasapera, A. M., Sabass, B., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+Fluctuations+within+Focal+Adhesions+Mediate+ECM-Rigidity+Sensing+to+Guide+Directed+Cell+Migration.">Force Fluctuations within Focal Adhesions Mediate ECM-Rigidity Sensing to Guide Directed Cell Migration.</a> Cell. 151 (7), 1513-1527 (2012).</li><li>&#193;lvarez-Gonz&#225;lez, B., Meili, R., Bastounis, E., Firtel, R. A., Lasheras, J. C., Del &#193;lamo, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+balance+of+cortical+tension+and+axial+contractility+enables+fast+amoeboid+migration.">Three-dimensional balance of cortical tension and axial contractility enables fast amoeboid migration.</a> Biophysical Journal. 108 (4), 821-832 (2015).</li><li>Provenzano, P. P., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+signaling+through+the+cytoskeleton+regulates+cell+proliferation+by+coordinated+focal+adhesion+and+Rho+GTPase+signaling.">Mechanical signaling through the cytoskeleton regulates cell proliferation by coordinated focal adhesion and Rho GTPase signaling.</a> Journal of Cell Science. 124 (8), 1195-1205 (2011).</li><li>Reilly, G. C., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+extracellular+matrix+properties+regulate+stem+cell+differentiation.">Intrinsic extracellular matrix properties regulate stem cell differentiation.</a> Journal of Biomechanics. 43 (1), 55-62 (2010).</li><li>Wen, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+of+matrix+stiffness+and+protein+tethering+in+stem+cell+differentiation.">Interplay of matrix stiffness and protein tethering in stem cell differentiation.</a> Nature Materials. 13 (10), 979-987 (2014).</li><li>Lee, J., Abdeen, A. A., Tang, X., Saif, T. A., Kilian, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+guidance+of+integrin+mediated+traction+stress+during+stem+cell+differentiation.">Geometric guidance of integrin mediated traction stress during stem cell differentiation.</a> Biomaterials. 69, 174-183 (2015).</li><li>Steward, A. J., Kelly, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+regulation+of+mesenchymal+stem+cell+differentiation.">Mechanical regulation of mesenchymal stem cell differentiation.</a> Journal of Anatomy. 227 (6), 717-731 (2015).</li><li>Lv, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+regulation+of+stem+cell+differentiation+by+matrix+stiffness.">Mechanism of regulation of stem cell differentiation by matrix stiffness.</a> Stem Cell Research &amp; Therapy. 6 (1), 103 (2015).</li><li>Tijore, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Cytoskeletal+Tension+in+the+Induction+of+Cardiomyogenic+Differentiation+in+Micropatterned+Human+Mesenchymal+Stem+Cell.">Role of Cytoskeletal Tension in the Induction of Cardiomyogenic Differentiation in Micropatterned Human Mesenchymal Stem Cell.</a> Advanced Healthcare Materials. 4 (9), 1399-1407 (2015).</li><li>Lombardi, M. L., Knecht, D. A., Dembo, M., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+force+microscopy+in+Dictyostelium+reveals+distinct+roles+for+myosin+II+motor+and+actin-crosslinking+activity+in+polarized+cell+movement.">Traction force microscopy in Dictyostelium reveals distinct roles for myosin II motor and actin-crosslinking activity in polarized cell movement.</a> Journal of Cell Science. 120 (9), 1624-1634 (2007).</li><li>Legant, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidimensional+traction+force+microscopy+reveals+out-of-plane+rotational+moments+about+focal+adhesions.">Multidimensional traction force microscopy reveals out-of-plane rotational moments about focal adhesions.</a> Proceedings of the National Academy of Sciences. 110 (3), 881-886 (2013).</li><li>Sabass, B., Gardel, M. L., Waterman, C. M., Schwarz, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+traction+force+microscopy+based+on+experimental+and+computational+advances.">High resolution traction force microscopy based on experimental and computational advances.</a> Biophysical Journal. 94 (1), 207-220 (2008).</li><li>Munevar, S., Wang, Y., Dembo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+Force+Microscopy+of+Migrating+Normal+and+H-ras+Transformed+3T3+Fibroblasts.">Traction Force Microscopy of Migrating Normal and H-ras Transformed 3T3 Fibroblasts.</a> Biophysical Journal. 80 (4), 1744-1757 (2001).</li><li>Pushkarsky, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastomeric+sensor+surfaces+for+high-Throughput+single-cell+force+cytometry.">Elastomeric sensor surfaces for high-Throughput single-cell force cytometry.</a> Nature Biomedical Engineering. 2 (2), 124-137 (2018).</li><li>Bergert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+reference+free+traction+force+microscopy.">Confocal reference free traction force microscopy.</a> Nature Communications. 7, (2016).</li><li>Schwarz, U. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cellular+forces+at+focal+adhesions+using+elastic+micro-patterned+substrates.">Measurement of cellular forces at focal adhesions using elastic micro-patterned substrates.</a> Materials Science and Engineering: C. 23 (3), 387-394 (2003).</li><li>Tan, J. L., Tien, J., Pirone, D. M., Gray, D. S., Bhadriraju, K., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+lying+on+a+bed+of+microneedles:+An+approach+to+isolate+mechanical+force.">Cells lying on a bed of microneedles: An approach to isolate mechanical force.</a> Proceedings of the National Academy of Sciences. 100 (4), 1484-1489 (2003).</li><li>Desai, R. a., Yang, M. T., Sniadecki, N. J., Legant, W. R., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+Post-Array-Detectors+(mPADs):+an+Approach+to+Isolate+Mechanical+Forces.">Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces.</a> Journal of Visualized Experiments. , 1-5 (2007).</li><li>Banda, O. A., Sabanayagam, C. R., Slater, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference-Free+Traction+Force+Microscopy+Platform+Fabricated+via+Two-Photon+Laser+Scanning+Lithography+Enables+Facile+Measurement+of+Cell-Generated+Forces.">Reference-Free Traction Force Microscopy Platform Fabricated via Two-Photon Laser Scanning Lithography Enables Facile Measurement of Cell-Generated Forces.</a> ACS Applied Materials &amp; Interfaces. 11 (20), 18233-18241 (2019).</li><li>Guo, J., Keller, K. A., Govyadinov, P., Ruchhoeft, P., Slater, J. H., Mayerich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+flow+in+augmented+networks+(AFAN):+an+approach+to+generating+three-dimensional+biomimetic+microfluidic+networks+with+controlled+flow.">Accurate flow in augmented networks (AFAN): an approach to generating three-dimensional biomimetic microfluidic networks with controlled flow.</a> Analytical Methods. 11 (1), 8-16 (2019).</li><li>Heintz, K. A., Mayerich, D., Slater, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided,+Laser-based+Fabrication+of+Vascular-derived+Microfluidic+Networks.">Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks.</a> Journal of Visualized Experiments. (119), 1-10 (2017).</li><li>Heintz, K. A., Bregenzer, M. E., Mantle, J. L., Lee, K. H., West, J. L., Slater, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+3D+Biomimetic+Microfluidic+Networks+in+Hydrogels.">Fabrication of 3D Biomimetic Microfluidic Networks in Hydrogels.</a> Advanced Healthcare Materials. 5 (17), 2153-2160 (2016).</li><li>Slater, J. H., Miller, J. S., Yu, S. S., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+Multifaceted+Micropatterned+Surfaces+with+Laser+Scanning+Lithography.">Fabrication of Multifaceted Micropatterned Surfaces with Laser Scanning Lithography.</a> Advanced Functional Materials. 21 (15), 2876-2888 (2011).</li><li>Slater, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulation+and+Modulation+of+the+Cellular+Architecture+of+a+User-Chosen+Cell+of+Interest+Using+Cell-Derived,+Biomimetic+Patterning.">Recapitulation and Modulation of the Cellular Architecture of a User-Chosen Cell of Interest Using Cell-Derived, Biomimetic Patterning.</a> ACS Nano. 9 (6), 6128-6138 (2015).</li><li>Shukla, A., Slater, J. H., Culver, J. C., Dickinson, M. E., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+Surface+Patterning+Promotes+Mesenchymal+Stem+Cell+Differentiation.">Biomimetic Surface Patterning Promotes Mesenchymal Stem Cell Differentiation.</a> ACS Applied Materials &amp; Interfaces. 8 (34), 21883-21892 (2016).</li><li>Slater, H. J., Banda, A. O., Heintz, A. K., Nie, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+Surfaces+for+Cell+Engineering.">Biomimetic Surfaces for Cell Engineering.</a> Carbon Nanomaterials for Biomedical Applications. , 543-569 (2016).</li><li>. Slater Lab Code Repositories Available from: <a target="_blank" href="https://github.com/SlaterLab">https://github.com/SlaterLab</a> (2019)</li><li>Culver, J. C., Hoffmann, J. C., Poch&#233;, R. A., Slater, J. H., West, J. L., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+biomimetic+patterning+in+hydrogels+to+guide+cellular+organization.">Three-dimensional biomimetic patterning in hydrogels to guide cellular organization.</a> Advanced Materials. 24 (17), 2344-2348 (2012).</li><li>Toyjanova, J., Bar-Kochba, E., L&#243;pez-Fagundo, C., Reichner, J., Hoffman-Kim, D., Franck, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution,+large+deformation+3D+traction+force+microscopy.">High resolution, large deformation 3D traction force microscopy.</a> PLoS ONE. 9 (4), 1-12 (2014).</li><li>Pradhan, S., Keller, K. A., Sperduto, J. L., Slater, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+Laser-Based+Hydrogel+Degradation+and+Applications+in+Cell+and+Tissue+Engineering.">Fundamentals of Laser-Based Hydrogel Degradation and Applications in Cell and Tissue Engineering.</a> Advanced Healthcare Materials. 6 (24), 1-28 (2017).</li><li>Tibbitt, M. W., Shadish, J. A., DeForest, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photopolymers+for+Multiphoton+Lithography+in+Biomaterials+and+Hydrogels.">Photopolymers for Multiphoton Lithography in Biomaterials and Hydrogels.</a> Multiphoton Lithography. , 183-220 (2016).</li></ol>

The authors have nothing to disclose.

The goal of this protocol is to provide a workflow that alleviates much of the difficulty associated with the generation and analysis of TFM data. Once prepared, the photopatterned hydrogels are simple to use, requiring only knowledge of standard tissue culture practices and fluorescence microscopy. The reference-free aspect enables carefree navigation on cell-laden hydrogels and eliminates cumbersome image processing steps such as image registration between reference and deformed images. The resulting analysis is nearly completely automated and data from individual COIs can be analyzed from start to finish in less than 10 min.
The primary challenges in this protocol stem from the fabrication of the photopatterned hydrogel. Because most of the reagents used in the protocol are synthesized in-house, we expect that users with little experience using synthetic hydrogel materials may be somewhat deterred from attempting this protocol. That said, all of the PEGylated components used in this protocol are synthesized from nearly identical protocols using one-pot reactions and many of the components can be purchased from commercial vendors.
This protocol also requires some mastery over the use of laser-scanning microscopes. The laser-scanning parameters need to be optimized for every different microscope, as laser intensities, objective lenses, and other microscope components will vary, even between microscopes of the same model. A simple approach to determining the best microscope settings for photo-patterning is to perform a panel of scans with varied settings. Any setting that affects the total laser exposure received by the sample will affect the size and quality of the patterned markers. This includes: scan speed, pixel size, averaging number, laser power/intensity/fluence, magnification, and numerical aperture32,33. It is often easiest to adjust laser power and scan speed and so it is recommended to start with those two parameters. It should also be noted that the software used to generate regions files was designed specifically for the brand of microscopes we use and may need to be augmented to accommodate other microscope makes and models. With the settings provided in the protocol, the user should expect to generate ellipsoidal markers with 3D gaussian-like intensity profiles of full-width half-max dimensions of 0.84 &plusmn; 0.11 &mu;m in XY and 3.73 &plusmn; 0.30 &mu;m in Z. The object detection algorithm uses a center-of-mass of intensity to identify marker centroids and will perform best when markers present a definite gradient of intensity.
Properly rinsing the hydrogel and protecting it from light and from contaminants is critically important for cell culture. As mentioned in the protocol, some of the patterning components may crash out of solution and deposit on the surface of the hydrogel. If these components are not completely rinsed from the surface, cell adhesion may be unpredictably affected. If exposed to UV spectrum light, even in small doses, while the hydrogel is soaking in the patterning solution, the patterning solution will begin to polymerize and will yield poor results. Finally, airborne particulates or unfiltered particulates will complicate analysis, especially if they are fluorescent. Special care should be taken to prevent these contaminants from entering the sample at all steps in the protocol.
The analysis code predicts reference locations of deformed fiducial markers by predicting the original array from non-deformed markers in the same row or column as the deformed marker. While this greatly simplifies analysis, it also imposes some restrictions on what can or cannot be analyzed. At a minimum, each deformed marker being analyzed should have 2 or more members of both its column and row of markers which are not deformed so that an accurate prediction can be made. A row is defined by the group of dots printed in a single line scan on the microscope, and a column is defined by markers patterned on a single vertical axis using a z-stack function. This limits analysis to single cells or small cell clusters based on the length and depth of each patterned area. It is important to note that this is not remedied by placing two patterned regions perfectly next to each other. This is because although the XY components may line up, it is not feasible to perfectly line up the Z positional components of the arrays, unless the sample surface is perfectly level with both the focal plane and the stage. The best way to avoid this limitation is to employ additional rounds of patterning that specifically dictate adhesion ligand placement. If aligned properly, cell adhesion can be restricted to usable areas on the patterns.
The analysis code has been optimized for square arrays where markers are spaced approximately 3.5 &mu;m apart in Z and 2.1 &mu;m in X and Y. The final spacing will vary based on patterning fidelity and swelling, but this should not greatly impact performance if in-row spacing is consistent (inter-row spacing variability will not affect tracking). While the code may run to completion on differently prescribed arrays, its current state may yield poor results if the arrays do not match the parameters listed here. Our current goals for the analysis code are to include support for variable spacing as well as triangular arrays, which may improve traction reconstruction accuracy and reduce regional bias as compared to square arrays.
To conclude, we provide an approach to TFM that allows for facile collection and analysis of TFM data without perturbing cellular function. Our goal with this approach is to provide a more accessible and non-intrusive TFM approach, without compromising the resolution and quality of TFM data. We expect that this methodology will promote the convergence of biochemical knowledge of cellular phenotypes with the observed physical properties of cells.

Traction force microscopy (TFM) is the process of approximating cellular tractions using interpolated displacement fields of fiducial markers generated by an adherent and contractile cell. Using TFM, the influence of mechanical cues in the extracellular environment on important cellular processes such as proliferation, differentiation, and migration can be investigated1,2,3,4,5,6,7,8,9,10,11,12. Unfortunately, many existing approaches can be difficult to implement or require familiarity with highly specialized analytical and computational tools making TFM difficult for inexperienced researchers to use. We describe a methodology to generate a TFM platform that eliminates some of the difficulty in analysis while also providing high-throughput data acquisition.
Of the existing TFM approaches, the most commonly used for quantifying material strain involves incorporation of small fluorescent markers (typically nano- or micrometer-sized fluorescent beads) into a deformable hydrogel, such as polyacrylamide (PAA) or poly(ethylene glycol) diacrylate (PEGDA)13,14,15. These bead-based approaches provide the ability to densely cluster fiducial markers around a cell of interest to maximize displacement sampling. Unfortunately, the distribution of the beads throughout the hydrogel cannot be directly controlled so the spatial organization is random. This random placement leads to problems such as beads which are too close to each other to accurately resolve, or so spread that patches of the substrate yield low quality data. The inability to predict where fiducial markers lie in the absence of cells also creates a constraint that, for every collected set of cell traction data, an additional reference image of the underlying markers in a relaxed state must also be captured. The reference image is required so that displacement in the stressed image can be approximated as the difference between the stressed and unstressed images. To achieve a relaxed state, the cells being measured are either chemically relaxed or completely removed. This process often prevents acquisition of further experimental measurements, inhibits long-term cell studies, and limits throughput. A reference image also requires image registration techniques to accommodate for drift which may have occurred during experimentation, often leading to cumbersome manual matching of stress state images to reference images.
Other TFM methods deemed reference-free, implement some form of control over the distribution of fiducial markers, either by high resolution lithography, microcontact printing, or micromolding16,17,18,19,20. Reference-free TFM is achieved through the assumption that the relaxed state for each fiducial marker can be predicted based on how marker positions were prescribed during the fabrication process. These methods allow for complete capture of a cell&rsquo;s tension state within a single image capture in which fiducial marker displacements are measured in comparison to an implied reference than can be inferred from the fiducial marker geometry. While consistency in marker placement is typically achieved using these platforms, they generally suffer from their own shortcomings relative to the widely used bead-based approaches including: 1) decreased traction resolution; 2) decreased accuracy of out-of-plane displacements (in some cases a complete inability to measure); and 3) decreased customizability of platform substrates and materials (e.g., ligand presentation, mechanical properties).
To address these shortcomings, we designed a new reference-free TFM platform. The platform utilizes multiphoton activated chemistry to crosslink a small volume of a fluorophore into specific 3D locations within the hydrogel that serve as fiducial markers to measure material strain. In this way, we have designed a platform that operates similarly to bead-based approaches but with the significant benefit that fiducial markers are organized into gridded arrays allowing for reference-free material strain tracking. This reference-free property affords many advantages. First and foremost, it allows for non-intrusive monitoring of cellular traction states (i.e., circumvents the need to relax or remove cells to acquire reference positions of displaced fiducial markers). This was our primary goal in designing this system, as we intended to incorporate other downstream analytical methods in tandem with TFM, which can be difficult with destructive end-point TFM approaches. Second, using an implied reference based on gridded arrays allows for near-complete automation of displacement analysis. The regularity of the arrays creates a predictable workflow where the occurrence of exceptional cases (i.e., sample cell data containing unanticipated artifacts such as suboptimal marker spacing or registration mismatches) can be maintained at a minimum. Third, forgoing the need to acquire a reference image provides the freedom to monitor many cells on a single sample over extended periods of time. This contrasts with traditional bead-based approaches, where, depending on the fidelity of the microscope&rsquo;s automated stage movements, errors in positioning can accumulate and increase the difficulty of properly registering reference images to cell tension images. Overall, this platform facilitates higher throughput in collecting cellular tension data.
With this protocol, we hope to familiarize the readers with the two-photon, laser scanning lithography technique that we implemented to generate this reference-free TFM platform to measure in-plane and out-of-plane traction components generated by cells seeded on the surface. Not covered in this protocol is the synthesis of some of the monomeric components. In general, these reactions include nearly identical &ldquo;one-pot&rdquo; synthesis reaction schemes described previously21, and alternatives to these products can also be purchased. We also aim to familiarize readers with the software-based tools we generated to promote the use of commercially-available laser-scanning microscopes as 3D printing tools and to facilitate analysis of fiducial marker displacements.

<table><tbody><tr><td>Acrodisc Syringe Filter, 0.2 &amp;mu;m Supor Membrane, Low Protein Binding</td><td>Pall</td><td>PN 4602</td><td>Allows for filtering of macromer solutions prior to base gel synthesis and subsequent lithography steps.</td></tr><tr><td>Acrylate-Silane Functionalized #1.5 Coverslips</td><td>&lt;em&gt;in-house&lt;/em&gt;</td><td>&lt;em&gt;in-house&lt;/em&gt;</td><td>Acrylates allow binding of base hydrogel to the glass surface to immobilize the hydrogels. See reference: 21-24</td></tr><tr><td>Axio-Observer Z1 w/Apotome</td><td>Zeiss</td><td></td><td>Widefield microscope with structured illumination module used to capture images for TFM.</td></tr><tr><td>Chameleon Vision ii</td><td>Coherent Inc.</td><td></td><td>Equipped on laser-scanning microscope used for multiphoton Lithography.</td></tr><tr><td>Double Coated Tape, 9500PC, 6.0 mil</td><td>3M</td><td></td><td>Binds acrylate-silane functionalized coverslips to Petri dishes.</td></tr><tr><td>Flexmark90 PFW Liner</td><td>FLEXcon</td><td>FLX000620</td><td>Allows lining of double coated tape enabling feeding of tape into plotter.</td></tr><tr><td>LSM-880</td><td>Zeiss</td><td></td><td>Laser-Scanning microscope used for Multiphoton Lithography.</td></tr><tr><td>MATLAB</td><td>Mathworks</td><td>R2018a</td><td>Runs custom scripts to generate lithography instructions for microscope and for analysis of TFM data.</td></tr><tr><td>Model SC Plotter</td><td>USCutter</td><td>SC631E</td><td>Cuts double coated tape into rings to bind coverslips to petri dishes.</td></tr><tr><td>Objective C-Apochromat 40x/1.20 W Corr M27</td><td>Zeiss</td><td></td><td>Equipped on both widefield microscope and laser-scanning microscope to be used for both lithography and TFM.</td></tr><tr><td>PEG-AF633</td><td>&lt;em&gt;in-house&lt;/em&gt;</td><td>&lt;em&gt;in-house&lt;/em&gt;</td><td>Fluorophore-labeled acrylate PEG variant for creating fiducial markers. See reference: 21</td></tr><tr><td>PEG-DA</td><td>&lt;em&gt;in-house&lt;/em&gt;</td><td>&lt;em&gt;in-house&lt;/em&gt;</td><td>Base material for hydrogels. See reference: 21</td></tr><tr><td>PEG-RGDS</td><td>&lt;em&gt;in-house&lt;/em&gt;</td><td>&lt;em&gt;in-house&lt;/em&gt;</td><td>RGDS peptide-labeled mono-acrylate PEG variant for promoting cell-adhesion. See reference: 21</td></tr><tr><td>Petri Dishes</td><td>CELLTREAT</td><td>229638</td><td>8mm holes are cut into the center of each dish using a coring bit to fit base hydrogels.</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>3097358-1004</td><td>For creating spacers to control base hydrogel thickness (aka PDMS).</td></tr><tr><td>Syringe, Leur-Lok, 1 mL</td><td>BD</td><td>309628</td><td>Allows for filtering of macromer solutions prior to base gel synthesis and subsequent lithography steps.</td></tr><tr><td>UV Lamp</td><td>UVP</td><td>Blak-Ray&amp;reg; B-100AP</td><td>Polymerizes base hydrogel.</td></tr><tr><td>1-vinyl-2-pyrrolidinone (NVP)</td><td>Sigma-Aldrich</td><td>V3409-5G</td><td>Radical accelerant and co-monomer. Improves pegylated fluorophore incorporation during lithography.</td></tr></tbody></table>

fabrication and implementation of a reference-free traction force microscopy platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

Throughout the protocol, there are a number of checkpoints providing feedback to assess the quality of the patterning procedure. To provide some insight concerning how to assess progress at each of these checkpoints, we provide representative results of an actual experiment. The results highlight the application of this protocol performed on a photopatterned hydrogel prepared for TFM analysis of human umbilical vein endothelial cells (HUVECs). The results include raw image data as well as processed data outputs at each critical step.
The first checkpoint occurs at step 4, once the fiducial marker array has been photopatterned in the hydrogel. When collecting an image stack, the resulting images should display a regular array of patterned features (Figure 1A-C) that oscillate in intensity as a function of z-position within the image stack. If the patterned surface was not perfectly level, different regions of each image slice may seem out of phase with each other with respect to these oscillations; this is expected and should not affect analysis except in extreme cases.
The remaining checkpoints utilize outputs from each of the scripts run during analysis of a given COI. The tracking.m script provides several diagnostic images including a z-projection of fluorescent fiducial markers (Figure 2A) to assess pre-processing quality, a plot of detected centroids color coded as a function of z-position (Figure 2B) to assess object detection quality, and a plot of tracks representing detected marker centroids which have been linked into columns in the z-direction (Figure 2C) to assess object tracking quality.
For accurate 3D centroid detection necessary to calculate reference coordinates and displacements, fluorescent fiducial markers should resemble ellipsoidal shapes with intensity decreasing radially from the center. The disp3D.m script provides a plot of intensity as a function of z-position for each column of detected features in a given image stack (Figure 3A,B) to assess the quality of the fiducial marker intensity profiles. Both disp3D.m and dispShear.m together provide histograms of measured displacement noise in each of the Cartesian coordinate dimensions (Figure 4A-C) as well as interpolated heat maps of displacement (Figure 4E,F). Finally, the interpFinal3D_2.m provides heat maps of surface tractions calculated using outsourced code (Figure 5)31.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60383/60383fig01.jpg" /> 
Figure 1: Typical patterning results. 
(A) Fluorescent images of fiducial markers displaying intensity fluctuations through the Z-dimension from the hydrogel surface to 12.4 &#956;m within the hydrogel. (B) A processed volumetric rendering reveals the ellipsoidal shape of the fiducial markers. (C) Sectioned profile views of the volumetric rendering display raw marker data. A,C: Scale bar =5 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60383/60383fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60383/60383fig02.jpg" /> 
Figure 2: Diagnostic outputs from the particle tracking algorithm. 
The object detection and tracking software outputs several diagnostic images including (A) a Z-weighted projection of the fluorescent fiducial markers and (B) a scatter plot of marker positions with color coded Z-position. (C) An additional output displays a z-weighted projection, as in (A), with 2D detections from each frame linked into columns. (D) A closer look at (B) more clearly shows individual 2D detections color coded by z-position. A: Scale bar =20 &#956;m. C: Scale bar =5 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60383/60383fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60383/60383fig03.jpg" /> 
Figure 3: Diagnostic outputs from the 3D displacement algorithm. 
(A, B) Line plots of marker intensity as a function of frame position display oscillations between markers grouped in the vertical direction. These line plots allow for qualitative assessment of grid alignment with the imaging plane, as well as overall quality of the patterned fiducial markers. (A) Overlapping oscillations in marker intensity confirm acceptable alignment of the imaging plane with the patterned fiducial marker arrays. Including empty frames at the top of a Z-stack allows for automated calculation of a noise threshold in the processing software. (B) Poor overlap between oscillations is often indicative of poor alignment of the patterned grids with the imaging plane but may also suggest that the grid themselves are misaligned and may yield poor results. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60383/60383fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60383/60383fig04.jpg" /> 
Figure 4: Diagnostic outputs from the 2D and 3D displacement algorithms. 
(A-C) Histograms of measured displacements in regions considered &#8216;non-deformed&#8217; (i.e., displacement noise) provide a quantitative assessment of the accuracy of calculated reference lines for approximating marker reference positions. Displacement fields provide a visual representation of both (D) in-plane (shear) and (E) out-of-plane (normal) measured displacements. D: Scale bar =20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60383/60383fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60383/60383fig05.jpg" /> 
Figure 5: Diagnostic Outputs from the Traction Conversion Algorithm. 
(A-C) Traction stresses can be calculated based on interpolated displacement fields to determine the (B) total traction, (C) shear traction, and (D) normal traction. A: Scale bar =20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60383/60383fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform at JoVE.com

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

fabrication-implementation-reference-free-traction-force-microscopy

Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.5K Views.  University of Delaware. This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Video: Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

Biology

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...

Neuroscience

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...