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. 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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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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>
</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 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 ...

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

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Bioengineering

6.4K 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

Visualization of Recombinant DNA and Protein Complexes Using Atomic Force Microscopy

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1) 1,2. Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. 
The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation 3,such as Ni2+ or Mg2+, which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase 4 and a repair enzyme 5, bound to individual DNA strands. These experiments provide significant insight into the protein-protein and DNA-protein biophysical interactions taking place at the molecular level. Imaging individual macromolecular particles with AFM can be useful for determining particle homogeneity and for identifying the physical arrangement of constituent components of the imaged particles. While the present method was developed for visualization of GR-chaperone protein complexes 1,2 and DNA strands to which the GR can bind, it can be applied broadly to imaging DNA and protein samples from a variety of sources.

A tapping mode atomic force microscope (AFM) method for the visualization of plasmid DNA, cytoplasmic proteins, and DNA-protein complexes is described. The method includes alternate approaches for preparing samples for AFM imaging following biochemical manipulation. DNA containing specific protein interacting regions are observed in near-physiologic buffer conditions.

Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

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 ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and&#160;wide-field&#160;illumination&#160;microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.&#160;

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...

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 ...

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...