Name: stiffness measurement of soft silicone substrates for mechanobiology studies using a widefield fluorescence microscope
Uploaded: 2018-07-03T14:00:00
Description: Watch this Scientific Journal Video about Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope at JoVE.com

We thank Margaret Gardel for generously allowing the use of the rheometer. We acknowledge support from the NIH (1R15GM116082) that enabled this work.

1. Fabrication of Soft Silicone Substrate
<ol>
	<li>Weigh out 1.75 g of the A component and 1.75 g of the B component (A:B = 1:1) from the soft silicone elastomer kit using (polystyrene) weighing trays.</li>
	<li>Add the A component to the B component in the weighing tray and mix them together for 5 min using an appropriate applicator stick.</li>
	<li>Add the above mixture to a 35 mm Petri dish. Allow the mixture to spread evenly across the Petri dish for a couple of minutes. 
	Note: The choice of the Petri dish d...

<ol>
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While the sphere indentation method is easy to implement, careful attention must be paid to the choice of indentor and the thickness of the soft silicone sample. The equation used to calculate the Young&#39;s modulus is valid under a set of conditions11and these are typically satisfied when the thickness of the silicone sample is &#62; 10% of the indentor radius and &#60; ~13x the indentor radius. We found that a silicone thickness of 5 - 10x the indentor radius was a good choice, wherein the samp...

Cells in soft tissues reside in a micro-environment whose stiffness is in the kilopascal range1, in contrast to tissue culture dishes whose stiffness is several orders of magnitude higher. Early experiments with cells on extracellular matrix protein-coated soft substrates showed that the substrate stiffness influences how cells move on as well as adhere to the extracellular matrix beneath2,3. In fact, the substrate stiffness fundamentally influences the cell function4&#160;in a manner similar to pervasive biochemical signals. Polyacrylamide gels (coated with extracellular matrix proteins) are (water-permeating) hydrogels that have been extensively used as cell culture substrates for mechanobiology studies5. Polydimethylsiloxane (PDMS), the most common silicone (polysiloxane), has been widely used as a stiff silicone with megapascal-range stiffness for micron-scale fabrication6. More recently, soft silicone substrates with stiffness in the more physiologically relevant kilopascal range have been employed as cell culture substrates for mechanobiology studies7,8.
Several methods have been used to measure the stiffness of flexible substrates, including atomic force microscopy, macroscopic deformation of whole samples upon stretching, rheology, and indentation using spheres and spherically tipped microindentors9. While each technique has its own advantages and disadvantages, indentation with a sphere is an especially simple yet fairly accurate method that only requires the access to a widefield fluorescence microscope. Indentation with a metallic sphere has been used to measure the stiffness of hydrogels in prior work3,9,10. Early work that demonstrated the importance of substrate stiffness to cell movement utilized this method to determine hydrogel substrate stiffness3. More recently, confocal microscopy has also been used for an elegant characterization10.
Here, we present a step-by-step protocol for preparing a soft silicone substrate, coupling fluorescent beads (and an extracellular matrix protein such as collagen I) just to the top surface, imaging an indenting sphere and the top surface using phase and fluorescence imaging, respectively, and finally analyzing the images to compute the Young&#39;s modulus of the silicone substrate. The soft silicone substrate prepared in this manner can be readily used for traction force microscopy experiments. The use of stiff silicone (instead of a Petri dish) as the base for the soft silicone also enables mechanobiology studies using an external stretch. Where warranted, practical considerations necessary for avoiding possible complications are also indicated.

<table border="0" cellpadding="0" cellspacing="0" width="949">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:353px;">CY 52-276 A/B silicone elastomer kit</td>
			<td style="width:191px;">&#160;Dow Corning</td>
			<td style="width:144px;">CY 52-276</td>
			<td style="width:263px;">Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific Pierce EDC</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td>PI22980</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific&#160;Pierce Sulfo-NHS crosslinker</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td>PI-24510</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carboxyl fluorescent pink particles, 0.4-0.6 &#181;m, 2 mL</td>
			<td>Spherotech, Inc.</td>
			<td>CFP-0558-2</td>
			<td>Store at 4&#176;C, do not freeze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.0 mm Acid washed Zirconium beads</td>
			<td style="width:191px;">OPS Diagnostics LLC</td>
			<td>BAWZ 1000-250-33</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:353px;">Deep UV chamber with ozone evacuator</td>
			<td style="width:191px;">Novascan Technologies, Inc.</td>
			<td style="width:144px;">PSD-UV4, OES-1000D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Wide field fluorescence microscope</td>
			<td style="width:191px;">Leica Microsystems</td>
			<td style="width:144px;">DMi8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Collagen I, from rat tail</td>
			<td style="width:191px;">Corning</td>
			<td style="width:144px;">354236</td>
			<td>Stock concentration = 4 mg/ml; store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">ImageJ-NIH</td>
			<td style="width:191px;">N/A</td>
			<td style="width:144px;">N/A</td>
			<td>public-domain software</td>
		</tr>
	</tbody>
</table>

stiffness measurement of soft silicone substrates for mechanobiology studies using a widefield fluorescence microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Using the protocol detailed above, we prepared soft silicone in a 35 mm Petri dish, cured it at 70 &#176;C for 30 min and coupled fluorescent microbeads (and collagen I) to the top surface as schematically depicted in Figure 1. Deep UV has been used previously for the eventual protein coupling to substrates13. Note that (I) the curing conditions used here are specific to this soft silicone and (II) the indentation measurement is pe...

Watch this Scientific Journal Video about Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope at JoVE.com

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

This method can be used to categorize substrates used to study key phenomena in mechanobiology, the cell migration and first generation. The main advantage of this technique is that it requires the use of a wide-field microscope, a piece of equipment that is commonly available in many labs. This method can be applied to measure the Young's modulus of silicon or polyacrylamide gel, as well as any of the soft isotropic linear elastic material.The substrate's prepared and characterized by this method can also be used for self-stretching experiments with the stiff silicone base. To fabricate the soft silicone substrate after weighting out about 1.75 grams each of components A and B from the soft silicone elastomer kit, add most of the weighed portion of component A to the B component polystyrene weighing tray and use an appropriate applicator stick to mix the two components together for five minutes. Transfer the mixture to a 35 millimeter Petri dish and allow the substrate to spread evenly across the Petri dish for a few minutes.Next, place the Petri dish in a vacuum chamber for 15 minutes to remove any air bubbles and pre-heat a hot plate. When the hot plate reaches 70 degrees Celsius, place a glass slide onto the hot plate, and place the Petri dish onto the glass side. Then, let the silicone cure at 70 degrees Celsius for 30 minutes.To couple the soft silicone with fluorescent beads, place the cured silicone in a deep UV chamber, five to ten centimeters away from the UV lamp and expose the sample to deep UV light for five minutes. While the silicone is being treated, dissolve 19 milligrams of EDC in 500 microliters of deionized water in a 1.5 milliliter microcentrifuge tube. And vortex the tube to dissolve the chemical.Next, add 500 microliters of deionized water to 11 milligrams of sulfo-NHS in a second 1.5 milliliter tube. And vortex to dissolve the chemical. Then, combine the EDC and sulfo-NHS solutions into a single microcentrifuge tube.Now, add 30 microliters of an appropriate suspension of fluoro-4 conjugated carboxylate-modified micro-beads and 0.02 milligrams of rat tail Collagen 1 to the mixture. After briefly vortexing, place a piece of Parafilm onto the lid of a second smaller diameter lid and transfer one milliliter of the EDC-NHS bead Collagen 1 mixture to the Parafilm. Invert the Petri dish containing the soft silicone over the mixture, supporting the dish on both sides with two glass slides, so that the soft silicone surface contacts the bead solution without directly touching the surface of the smaller diameter lid below.Then, cover the sample with aluminum foil for 30 minutes at room temperature, before washing the silicone two times, with two milliliters of PBS per wash and let the silicon cure upright in two milliliters of fresh PBS at 37 degrees Celsius overnight. The next morning, use pointed tweezers to drap five millimeter zirconium sphere indentors onto the soft silicone under at least one milliliter of PBS away from the edges of the silicone layer and at least five indentor diameters away from the location of the other indentors. Next, place the dish of soft silicon onto a wide-field microscope stage and use the 10x objective to obtain a phase image of a whole indentor without any visible defects.After saving the image, select the appropriate fluorescent channel for visualizing the fluorescent micro-beads. With the indentor center near the right edge of the frame, decrease the z-coordinates until the fluorescent micro-beads under the sphere indentor's center go just out of focus. Then, acquire a z-stack with an image for every 0.5 micrometer z-increment until the micro-beads in the top layer of the silicone near the left edge of the imaging frame come in and then out of focus.To calculate the silicone stiffness, first, open the indentor phase image in ImageJ and click analyze and set scale and check unit of length to confirm that the length is set to pixels. Using the line tool, click and hold onto a point on the indentor edge, moving the cursor to a point on the diametrically opposite edge, to measure the diameter of the indentor and note the length in pixels on the status bar of the ImageJ main window before releasing the cursor. Next, open the file menu and select import an image sequence to allow an image in the z-stack of fluorescent micro-bead images to be selected and click okay to open the stack.Using the line tool, draw a line across a well-defined micro-bead in one image and click analyze plot profile in live to obtain the updated line scan intensity across the bead, while selecting different frames. The frame that gives the highest value of the maximum intensity can then be selected as the frame in focus. Fluorescence images of the micro-beads in the top surface of the silicone taken at an x-y frame position, places the region under the indentor to the far-right side of the frame.The region on the far-left side of the frame is then selected as the region away from the indentor. In this representative set of intensity line scans measured across a single micro-bead, the focus varies in z-increments of 0.5 micrometers. The z-value corresponding to the in-focus image can then be objectively selected based on the z-value corresponding to the line scan with the highest maximum intensity.Here, the Young's modulus of the silicone for various A:B component mixture ratios, was obtained based on the method as just demonstrated. After watching this video, you should have a good understanding of how to prepare soft silicone substrates, cup the fluorescent beads to them, and measure the stiffness of the substrate using a wide-field fluorescent microscope.

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