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

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.