The authors would like to thank Dr. Paul Barbone from the Boston University Department of Mechanical Engineering for helpful discussions and assistance with data analysis. This study was supported by NSF grant CMMI-1910401.

1. Creation of silicone masters
NOTE: Most of the process of the design, creation, and troubleshooting of silicon masters for the repeated molding of PDMS stamps has been covered previously21, so only key differences in this new approach will be described here.
<ol>
	<li>Create the design for the photomask using AutoCAD or similar design software. Coat one side of the photomask, a thin piece of glass, with a thin layer of chrome to control UV light sc...

The authors have no conflicts of interest to disclose.

An improved method of indirectly patterning PAA hydrogels is described in this paper. This approach builds on methods that have been used previousely20,35,36,37,38,39,40,41,42. The primary change is that PDMS stamps are no...

Most cell phenotypes exert traction forces on their environment. These traction forces are generated by a cell&#39;s contractile cytoskeleton, which is a network of actin and myosin, and other filamentous biopolymers and crosslinking proteins1,2,3,4. Forces generated within the cell can be transmitted to the extracellular environment or adjacent cells, primarily via transmembrane proteins such as integrins and cadherins, respectively5,6. How a cell spreads or contracts-and the magnitudes of the traction forces associated with those movements-is the result of an intimate conversation with its environment, which largely depends on the type and quantity of protein present in the extracellular matrix (ECM)7,8 and the stiffness of the ECM. Indeed, traction force microscopy has become an invaluable tool for understanding cell responsiveness to local stimuli such as substrate stiffness, imposed mechanical stresses and strains, or contact with other cells. This information is directly relevant to the understanding of diseases such as cancer and asthma9,10,11,12.
A system that can be used to measure force-induced deformation of a substrate of known material properties is required to calculate traction forces. These changes must be tracked over time, requiring both imaging and image processing techniques. One of the first methods used to determine cellular traction forces was the observation and analysis of the contraction of collagen hydrogels seeded with cells, though this method was only semiquantitative13. Another, more refined method was to measure the traction forces exerted by single cells by determining the forces resulting from the deformation of a thin sheet of silicone14. Later on, more quantitative measurement techniques were developed, and these methods also allowed for the use of soft hydrogels such as polyacrylamide (PAA)12,15,16. When using these soft materials, traction forces could be determined from the force-induced displacement of randomly displaced beads embedded in the hydrogel and the mechanical properties of the gel16,17. Another advancement came with the development of micropost arrays made of soft polydimethylsiloxane (PDMS) so that their deflection could be measured and converted to force using the beam theory18.
Finally, methods for micropatterning soft hydrogels were developed as these approaches allow control of the contact areas for cell adhesion. By measuring the deformation of the micropattern within a cell&#39;s contact area, traction forces could easily be calculated because a force-free reference image is not required19. This method has been widely adopted as it allows for the indirect patterning of a regular array of micron-sized, discrete fluorescent protein adhesion points onto PAA gels for the measurement of cellular traction forces20. To calculate these forces, an image-processing algorithm, which can track the movements of each micropatterned dot without requiring user input, has been developed21.
While this method is simple for creating entire grids of dot patterns, it is more complicated when patterns of isolated patches (or islands) of dots are desired. Micropatterned islands are useful when control of shape, and to some extent of size, of clusters of cells is needed. To create these islands, the aforementioned method of microcontact printing necessitates two distinct steps: i) using one PDMS stamp to create a high-fidelity pattern of dots on a coverslip, and then ii) using a second different PDMS stamp to remove most of those dots, leaving behind isolated islands of dots21. The difficulty in creating islands with this original method is compounded by the fact that making consistent grid patterns in the first step of the process is challenging on its own. Microprinting stamps are composed of an array of circular microposts, the diameter of which corresponds to the desired dot size. These stamps are then coated with an even layer of protein and then stamped with a precise amount of pressure onto treated coverslips to create the desired pattern. On the one hand, applying too much pressure to the stamp can result in uneven protein transfer and poor pattern fidelity due to pillar buckling or sagging between pillars, leading to contact with the glass. On the other hand, applying too little pressure results in little to no protein transfer and poor pattern fidelity. For these reasons, a transfer process that can be used to consistently create high-quality micropatterns of isolated islands of dots in just one step is desired.
Herein, a method is described for the indirect micropatterning of islands of micron-sized fluorescent protein adhesion points onto a PAA gel that is more consistent and versatile than previously developed methods. Whereas older indirect micropatterning methods rely on the transfer of protein patterns from a PDMS stamp to an intermediate substrate, the method introduced here uses PDMS stamps instead as a vessel for protein removal, not addition. This is done by first fundamentally changing the structure of the PDMS stamps used. Rather than making stamps that are composed of a pattern of evenly spaced circular pillars, stamps are made up of a pattern of evenly spaced circular holes in this method.
With this new structure, the surface of these PDMS stamps can then be treated with glutaraldehyde as described previously20,29,30, making the stamp able to bond covalently with protein. When used on a glass coverslip evenly coated with fluorescent protein, these glutaraldehyde-treated PDMS stamps are used to remove most of the protein on the surface of the coverslip, leaving behind only the desired pattern of dots predetermined by the location of micron-sized holes on the stamp. This change increases the success rate for generating patterns made up of a near-continuous grid of dots and for creating isolated islands of dots through only one step.

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		<tr>
			<td>(3-aminopropyl)trimethoxysilane</td>
			<td>Sigma Aldrich</td>
			<td>#281778</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>#05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>Fisher Scientific</td>
			<td>#05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>4 x 4 in 0.060 Quartz LR Chrome Photomask</td>
			<td>Advance Reproductions Corporation</td>
			<td>N/A</td>
			<td>Custom-designed mask</td>
		</tr>
		<tr>
			<td>6 Well Plates</td>
			<td>Fisher Scientific</td>
			<td>#07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>#A18P-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide Solution, 40%</td>
			<td>Sigma Aldrich</td>
			<td>#A4058</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 488</td>
			<td>Thermo Fisher</td>
			<td>#A20000</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminonium Persulfate</td>
			<td>Fisher Scientific</td>
			<td>#BP179-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Bisacrylamide</td>
			<td>Fisher Scientific</td>
			<td>#PR-V3141</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Greenfield Global</td>
			<td>#111000200C1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Coverslips, 25 mm round</td>
			<td>Fisher Scientifc</td>
			<td>#12-545-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Coverslips, 30 mm round</td>
			<td>Warner</td>
			<td>#64-1499</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu ORCA-R2 Camera</td>
			<td>Hamamatsu</td>
			<td>#C10600-10B</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Plasma</td>
			<td>Valley Biomedical</td>
			<td>#HP1051P</td>
			<td>Used to isolate fibronectin</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid, 1.0 N</td>
			<td>Millipore Sigma</td>
			<td>#1.09057</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Wayne Rasband</td>
			<td>#1.53n</td>
			<td></td>
		</tr>
		<tr>
			<td>Interchangeable Coverslip Dish Set</td>
			<td>Bioptechs</td>
			<td>#190310-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Kim Wipes</td>
			<td>Fisher Scientific</td>
			<td>#06-666-11C</td>
			<td></td>
		</tr>
		<tr>
			<td>Mask Alinger</td>
			<td>Karl Suss</td>
			<td>#MA6</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab 2021</td>
			<td>Mathworks</td>
			<td>#R2021a</td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph Basic</td>
			<td>Molecular Devices</td>
			<td>#v7.7.1.0</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide ester</td>
			<td>Sigma Aldrich</td>
			<td>#130672-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue Live Cell Stain</td>
			<td>Thermo Fisher</td>
			<td>#R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX2-ZDC Inverted Microscope</td>
			<td>Olympus</td>
			<td>#IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>PD-10 Desalting Columns</td>
			<td>GE Healthcare</td>
			<td>#52-1308-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoresist Spinner Hood</td>
			<td>Headway Research</td>
			<td>#PWM32</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick</td>
			<td>#PDC-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Etcher</td>
			<td>TePla</td>
			<td>#M4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Prior Lumen 200Pro Light Source</td>
			<td>Prior Scientific</td>
			<td>#L200</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon Wafers, 100 mm</td>
			<td>University Wafer</td>
			<td>#809</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2005</td>
			<td>Kayaku Advanced Materials Inc.</td>
			<td>#NC9463827</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>Kayaku Advanced Materials Inc.</td>
			<td>#NC9901158</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer</td>
			<td>Essex Brownell</td>
			<td>#DC-184-1.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine</td>
			<td>Fisher Scientific</td>
			<td>#BP150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloro(1H,1H,2H,2H-perfluorooctyl)silane</td>
			<td>Sigma Aldrich</td>
			<td>#448931</td>
			<td></td>
		</tr>
		<tr>
			<td>UAPON-40XW340 Objective</td>
			<td>Olympus</td>
			<td>#N2709300</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Flood Exposure</td>
			<td>Newport</td>
			<td>#69910</td>
			<td></td>
		</tr>
		<tr>
			<td>Wafer Carrier Tray, 110 x 11 mm</td>
			<td>Ted Pella, Inc.</td>
			<td>#19395-40</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

PAA hydrogels with the Young&#39;s modulus of E = 3.6 kPa and the Poisson&#39;s ratio of &#957; = 0.445 were made for use by this subtractive micropatterning method. The hydrogels were made to be ~100 &#181;m thick, which allows them to be imaged with the imaging setup used here while also preventing the cells from sensing the rigid coverslip below the gel, which would cause problems in studies focused on cellular rigidity sensing23,33. Gels of ...

Watch this Scientific Journal Video about Pattern Generation for Micropattern Traction Microscopy at JoVE.com

Pattern Generation for Micropattern Traction Microscopy

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 ...

This protocol allows for the consistent creation of high-fidelity protein micro patterns of whatever shape is desired, notably isolated islands of pattern for study of cell clusters. This technique can be used to make isolated island micro patterns of any shape and size in only one step, whereas previously making such patterns required two separate steps. Begin by mixing PDMS in the correct curing agent to base ratio as described in the manufacturer's instructions.Incubate at room temperature and pressure for 15 minutes, then degas the mixture under vacuum for 15 minutes. Pour the PDMS into the master mold and transfer it to an incubator set to 37 degrees Celsius to cure overnight. Remove the master mold from the incubator and allow it to cool down to room temperature.While the master mold is cooling down, sonicate 25 millimeter coverslips in ethanol for 10 minutes. Use as many coverslips as the number of stamps being prepared. Thoroughly rinse the coverslips with DI water and dry them using a filtered air gun.Treat the coverslips with plasma for one minute using the plasma cleaner under a high vacuum. Slowly release the vacuum after treatment to prevent the coverslips from moving inside the chamber. Coat each coverslip with 100 microliters of the fluorescent labeled protein solution in a room devoid of direct sunlight or overhead lighting.Cover the samples for extra protection from light and incubate them for 20 minutes at room temperature. Thoroughly rinse each coverslip with DI water. Remove excess water from the surface by gently tapping the sides of each coverslip onto a paper towel or a similar absorbent material.Allow the coverslips to dry completely by leaving them uncovered in the dark for at least 30 minutes. While the protein coated coverslips dry, remove the PDMS stamp from the master mold by cutting it with a scalpel or any sharp blade. Treat the PDMS stamps with plasma for two minutes under a high vacuum.Place the stamps in a container with a lid under the fume hood, then coat each stamp with a thin layer of 10%3-APTMS diluted in 100%ethanol. Cover the container with the stamps and incubate at room temperature for five minutes. Thoroughly rinse each stamp on both sides with DI water.Place the stamps in a clean container and coat them with 2.5%glutaraldehyde prepared in DI water. Cover the stamps and incubate them at room temperature for 30 minutes. Rinse the stamps thoroughly with DI water.Remove excess water from the surface as previously described and allow the stamps to dry uncovered for 30 minutes. If after 30 minutes the protein-coated coverslips or the stamps are not completely dry, use a filtered air gun to dry them completely. Push the pattern side of the stamps onto the coverslips with enough pressure for them to make complete contact.Leave it undisturbed for 15 minutes. Then carefully peel the PDMS stamps off the coverslips. Using a fluorescent microscope with the appropriate filter, check the fidelity of the patterned coverslips.Use the patterned coverslips immediately or store them away from direct light until further use. Before preparing the PAA hydrogel precursor, remove the acrylic acid NHS ester from the refrigerator to reach room temperature before being opened. Prepare an interchangeable coverslip dish set by sterilizing it with 70%ethanol, then incubate it under UV light in a biosafety cabinet for at least 30 minutes before use.Under the fume hood, add 1.25 milliliters of 40%acrylamide prepared in DI water to a 15 milliliter conical tube. To the same tube, add 175 microliters of bis-acrylamide solution prepared in DI water. Then add 500 microliters of 10X PBS, followed by 2.915 milliliters of DI water.Pipette 969 microliters of this precursor into a 1.5 milliliter microcentrifuge tube and store the rest at four degrees Celsius for up to two weeks. Measure 50 to 100 milligrams of APS in a fresh microcentrifuge tube and dilute it to 100 milligrams per milliliter in DI water. Open the NHS ester in the hood and carefully measure up to three milligrams of NHS in a microcentrifuge tube.Dilute the NHS ester to one milligram per milliliter in 1X PBS. Under the fume hood, add two microliters of TEMED to the microcentrifuge tube containing the 969 microliter aliquot of PAA precursor. Add 15 microliters of one molar HCL to decrease the pH of the hydrogel solution and avoid hydrolysis of the NHS ester.Add 10 microliters of the NHS ester solution to the tube. Perform the following steps in a biosafety cabinet. Carefully place the 30 millimeter coverslip within the middle part of the coverslip dish set with the 3-APTMS and glutaraldehyde treated side up and screw the plastic ring on top.Set up the patterned coverslip with minimum light exposure to prepare for the next step. Pipette five microliters of APS solution into the PAA precursor aliquot tube, then invert and mix. Immediately pipette 35 microliters of this solution onto the 30 millimeter coverslip.Place the patterned coverslip on the solution with the protein side down. Be careful not to create air bubbles in the hydrogel. Protect the hydrogel from light and allow it to polymerize for 90 minutes.Once the hydrogel has polymerized, use a razor blade or scalpel to remove the upper coverslip, ensure that the coverslip does not fall back or slide off the gel once removed to avoid ruining the pattern on the surface of the gel. To passivate any remaining NHS ester in the hydrogel, add two milliliters of sterile PBS to the gel and incubate at 37 degrees Celsius for 45 minutes. Immediately prepare the gels for experiments or store them overnight in sterile PBS at four degrees Celsius until further use.The hydrogels were indirectly patterned with fluorescent fibronectin to visualize and measure cellular traction forces within clusters and create island patterns of predetermined size and shape. The pattern quality was directly related to the fidelity of the master mold from which the PDMS stamp was cast. This method could fabricate isolated, well-defined island patterns of predetermined shape.The fibronectin adhesion dots were present only within the desired area of the island. These isolated islands of adhesion dots further allowed better control of cluster shape. Coating PDMS stamps with two little 3-APTMS is critical because it will limit the effectiveness of the removal method, while too much will create an orange residue on the stamp surface.The patterns created here can be used to determine cellular traction forces in both individual cells and clusters, which gives insight into their ability to maintain mechanical homeostasis.

pattern-generation-for-micropattern-traction-microscopy

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2.1K Views.  Boston University. This protocol allows for the consistent creation of high-fidelity protein micro patterns of whatever shape is desired, notably isolated islands of pattern for study of cell clusters. This technique can be used to make isolated island micro patterns of any shape and size in only one step, whereas previously making such patterns required two separate steps. Begin by mixing PDMS in the correct curing agent to base ratio as described in the manufacturer's instructions.Incubate at room temp...