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).

1. Photopolymerizing a PEGDA base hydrogel
<ol>
	<li>Gathering Reagents
	<ol>
		<li>Collect lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 3.4 kDa poly(ethylene) glycol diacrylate (PEGDA), n-vinyl pyrrolidone (NVP), AlexaFluor 488 labeled PEGDA (PEG-488), AlexaFluor 633 labeled PEGDA (PEG-633), and PEGylated RGDS peptide (PEG-RGDS) from their respective freezers and bring each to room temperature.</li>
		<li>In separate amber microcentrifuge tubes, measure 3 mg of LAP, 10 mg of PEGDA, 5 mg of ...

<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. 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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...

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 &#956;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>in-house</td>
			<td>in-house</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>in-house</td>
			<td>in-house</td>
			<td>Fluorophore-labeled acrylate PEG variant for creating fiducial markers. See reference: 21</td>
		</tr>
		<tr>
			<td>PEG-DA</td>
			<td>in-house</td>
			<td>in-house</td>
			<td>Base material for hydrogels. See reference: 21</td>
		</tr>
		<tr>
			<td>PEG-RGDS</td>
			<td>in-house</td>
			<td>in-house</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&#174; 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>
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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 c...

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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 ...

Traction force microscopy is used to measure cell generated forces. This protocol simplifies the collection analysis of traction force data, in order to make it more accessible to inexperienced users. The main advantage of this technique over existing reference-free traction force microscopy platforms, is that multiphoton lithography allows a quick and easy patterning setting redesign according to individual experimental needs.There are many key steps in the fabrication and implementation of this new reference-free traction platform that are easier to understand through visual representation rather than text alone. For photopolymerization of the base hydrogel, first add three microliters of the pre-polymer solution onto a thin, flat sheet of perfluoroalkoxy alkanes, and place flat 150 micrometer thick PDMS strips surrounding, but not in contact with, the drop of pre-polymer solution. Place an acrylate silanized coverslip on the PDMS, with the pre-polymer droplet centered under the coverslip to flatten the pre-polymer droplet to the thickness of the PDMS spacers.Expose the sandwiched pre-polymer solution to UV light for about one minute. When a hydrogel is fully formed, carefully separate the coverslip from the PDMS spacers. Use high performance double-sided acrylic adhesive to attach an open bottom Petri dish to the coverslip.Take special care when adhering the hydrogel-laden coverslip to the Petri dish. A wet dish or too little pressure during the application will allow leaks to form, ruining the sample. Apply pressure to the adhesive contact surface to create a complete seal between the coverslip, adhesive, and Petri dish, taking care not to crack the glass.Use sterile filtered PBS to rinse the hydrogel. Then add eight microliters of NVP to 800 microliters of the lab solution prepared for the hydrogel synthesis. To convert a binary image to a digital mask, open MATLAB and open and run the runscript.m MATLAB script. Select the binary TIF file for conversion and select a folder to save the region's files. Input the desired final size in microns of the selected image.The input image will be scaled and dimensioned to match these parameters. To create a single pixel array, in the Region of Interest Generator Options, uncheck the Remove tic box under Small Regions/Single Pixels, check the Squares tic box and uncheck Use under Horizontal Break Lines. Then, click OK.The OVL file will be found in the previously specified folder.Open the file in the microscope software and load the desired regions that controlled the laser shutter during two photon laser scanning lithography. The following steps should be performed in conditions with minimal light. For fabrication of fiducial marker arrays under low light conditions, thoroughly mix 200 microliters of the previously prepared NVP lab solution with 20 milligrams of Alexa Fluor 633 and remove all of the PBS from the dish containing the hydrogel.Add the PEG-633 mixture to the hydrogel as a droplet that completely encompasses the base hydrogel and load the Petri dish onto the sample holder of the microscope stage. Then cover the dish and allow the patterning solution to soak into the hydrogel for at least 30 minutes, protected from light. To configure the microscope for imaging, select the appropriate lasers and filters for visualizing Alexa Fluor 488.Locate the hydrogel using the PEG-488 signal and block the collection of longer emission wavelengths from the detector. Use vertical and horizontal tile scans to locate the center of the hydrogel in the XY plane and zero the stage in this position. Use line scans in the Z-stack function to locate the surface of the hydrogel and zero the focus in this position.Level the hydrogel by repeating the line scans away from the XY center to identify the surface position, adjusting the set screws for the microscope stage, as necessary. Within the microscope software workspace, create a separate experiment file for photopatterning and set the multi-photon power to 1.8%and the scanning speed to six. Adjust the size of the image frame and pixels to achieve a pixel size of 0.1 micrometer per pixel and an aspect ratio of 100 to one and load the regions file into the region's tab.Use a macro to set all of the regions to acquire and turn on the Z-stack function. Set the spacing to 3.5 micrometers for a total depth of 28 micrometers and total number of Z slices of nine. Then use the Positions function to set specific locations on the hydrogel where the fiducial marker arrays will be photopatterned.As the soak time approaches the 30 minute mark, acquire successive Z-stack line scans of the surface of the hydrogel every five minutes to check for swelling based on changes in the location of the surface relative to the zeroed focus position. If no change in the surface position has occurred over the five minute interval, run the patterning settings and use the 488 nanometer laser to verify that the surface of the hydrogel did not move during the patterning. Then remove the hydrogel from the microscope, aspirate the PEG-633 solution from the Petri dish, and rinse the dish with sterile filtered PBS.To acquire a cell and fiducial marker images, return the Petri dish to the sample holder to locate the patterned areas. Locate a cell of interest and acquire a transmitted or fluorescent image of the cell. Then acquire a Z-stack of the patterned array beneath the cell as demonstrated, and acquire a second set of transmitted or fluorescent images of the cell.When collecting an image stack, the resulting images should display a regular array of patterned features that oscillate in intensity as a function of the Z position within the image stack. The tracking. m script provides several diagnostic images, including a Z-projection of the fluorescent fiducial markers to assess the pre-processing quality, a plot of the detected centroids, color coded as a function of the Z position to assess the object detection quality, and a plot of the tracks representing the detected marker centroids that have been linked into columns in the Z direction to assess the object tracking quality.The DISP 3D. m script provides a plot of intensity as a function of the Z position for each column of detected features in a given image stack to assess the quality of the fiducial marker intensity profiles. Both DISP 3D.m and DISP shear. m together provide histograms of the measured displacement noise in each of the Cartesian coordinate dimensions as well as interpolated heat maps of displacement. In addition, the interp final3D_2.m provides heat maps of the surface tractions calculated using the outsourced code. The most important thing to remember during this procedure is that solutions containing LAP are sensitive to light and should be protected as much as possible from ambient light sources. Remember that NVP is a volatile organic compound and should always be handled in a chemical flow hood.

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

Research

JoVE Journal

Bioengineering

6.4K visualizações.  University of Delaware. A microscopia de força de tração é usada para medir forças geradas por células. Este protocolo simplifica a análise de coleta de dados de força de tração, a fim de torná-lo mais acessível a usuários inexperientes. A principal vantagem dessa técnica em relação às plataformas de microscopia de força de tração sem referência existentes, é que a litografia multifotal permite um redesign rápido e fácil de configuração de padronização de acordo com as necessidades experi...