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

我们感谢玛格丽特卡洛斯·加德尔慷慨地允许使用流变仪。我们承认 NIH (1R15GM116082) 支持这项工作。

1. 软硅基板的制备
<ol><li>使用 (聚苯乙烯) 称重托盘, 从软硅胶弹性体套件中称量1.75 克的 a 组分和1.75 克 (a: b = 1:1)。</li>
	<li>将一个组件添加到称量托盘中的 B 组件中, 并用适当的涂抹棒将其混合在一起5分钟。</li>
	<li>将上述混合物添加到35毫米培养皿中。允许混合物均匀地分布在培养皿上几分钟。 注: 培养皿直径的选择和软硅胶的用量将决定软硅的厚度。在这里, 厚度将在3.5 毫米左右;有关在<s...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+movement+is+guided+by+the+rigidity+of+the+substrate.">Cell movement is guided by the rigidity of the substrate.</a> Biophysical Journal. 79, 144-152 (2000).</li><li>Discher, D. E., Janmey, P., Wang, Y. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+Substrate.">Tissue cells feel and respond to the stiffness of their Substrate.</a> Science. 310, 1139-1143 (2005).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, 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=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+toward+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses.</a> Methods in Cell Biology. 83, 29-46 (2007).</li><li>Johnston, I. D., McCluskey, D. K., Tan, C. K. L., Tracey, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+characterization+of+bulk+Sylgard+184+for+microfluidics+and+microengineering.">Mechanical characterization of bulk Sylgard 184 for microfluidics and microengineering.</a> Journal of Micromechanics and Microengineering. 24 (3), 035017 (2014).</li><li>Style, R. W., 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=Traction+force+microscopy+in+physics+and+biology.">Traction force microscopy in physics and biology.</a> Soft Matter. 10 (23), 4047-4055 (2014).</li><li>Lee, E., 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=Deletion+of+the+cytoplasmic+domain+of+N-cadherin+reduces,+but+does+not+eliminate,+traction+force-transmission.">Deletion of the cytoplasmic domain of N-cadherin reduces, but does not eliminate, traction force-transmission.</a> Biochemical and Biophysical Research Communications. 478 (4), 1640-1646 (2016).</li><li>Frey, M. T., Engler, A., Discher, D. E., Lee, J., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+methods+for+measuring+the+elasticity+of+gel+substrates+for+cell+culture:+microspheres,+microindenters,+and+atomic+force+microscopy.">Microscopic methods for measuring the elasticity of gel substrates for cell culture: microspheres, microindenters, and atomic force microscopy.</a> Methods Cell Biol. 83, 47-65 (2007).</li><li>Lee, D., Rahman, M. M., Zhou, Y., Ryu, S. <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+confocal+microscopy+indentation+method+for+hydrogel+elasticity+measurement.">Three-dimensional confocal microscopy indentation method for hydrogel elasticity measurement.</a> Langmuir. 31 (35), 9684-9693 (2015).</li><li>Dimitriadis, E. K., Horkay, F., Maresca, J., Kachar, B., Chadwick, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+elastic+moduli+of+thin+layers+of+soft+material+using+the+atomic+force+microscope.">Determination of elastic moduli of thin layers of soft material using the atomic force microscope.</a> Biophysical Journal. 82 (5), 2798-2810 (2002).</li><li>Hertz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#220;ber+die+Beru&#776;hrung+fester+elastischer+Ko&#776;rper.">&#220;ber die Beru&#776;hrung fester elastischer Ko&#776;rper.</a> Journal f&#252;r die reine und angewandte Mathematik. 92, 156-171 (1882).</li><li>Azioune, A., Carpi, N., Tseng, Q., Th&#233;ry, M., Piel, M., Cassimeris, L., Tran, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+micropatterns:+a+direct+printing+protocol+using+deep+UVs.">Protein micropatterns: a direct printing protocol using deep UVs.</a> Microtubules: In Vivo. , 133-146 (2010).</li><li>Bashirzadeh, Y., Qian, S., Maruthamuthu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-intrusive+measurement+of+wall+shear+stress+in+flow+channels.">Non-intrusive measurement of wall shear stress in flow channels.</a> Sensors and Actuators A: Physical. 271, 118-123 (2018).</li><li>Muhamed, I., Chowdhury, F., Maruthamuthu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+tools+to+study+cellular+mechanotransduction.">Biophysical tools to study cellular mechanotransduction.</a> Bioengineering (Basel). 4 (1), 12 (2017).</li><li>Dumbali, S. 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球形压痕法易于实现, 但要注意 indentor 的选择和软硅试样的厚度。用于计算杨氏模量的方程式在一组条件下有效11, 当硅胶样品的厚度 > 10% 的 indentor 半径和 < ~ 13 x indentor 半径时, 通常会满足这些要求。我们发现, 硅树脂厚度为 5-10x 的 indentor 半径是一个很好的选择, 其中试样厚度不太高 (即目标工作距离不成为限制), 计算刚度也不太敏感的硅胶厚度的精确值。球形 indentor 的?...

软组织细胞位于微环境中, 其刚度在 kilopascal 范围为1, 与组织培养皿的硬度为几级以上。早期的细胞外基质蛋白涂层软基板的实验表明, 基底刚度影响细胞的移动以及坚持在2,3下的细胞外基质。事实上, 基底刚度从根本上影响细胞功能4 , 其方式类似于弥漫的生化信号。聚丙烯酰胺凝胶 (涂有细胞外基质蛋白) 是 (水渗透) 凝胶, 已广泛用作细胞培养基质的美国匹茨堡研究5。烷是最常见的有机硅 (聚硅氧烷), 广泛应用于兆帕斯卡硬度为微米级制造6的硬硅树脂。最近, 在生理上相关的 kilopascal 范围内有刚性的软硅基板被使用作为细胞培养基质为美国匹茨堡研究7,8。
采用了几种方法测量柔性基板的刚度, 包括原子力显微镜、拉伸、流变和压痕时全试样的宏观变形, 球面和球尖 microindentors 9..虽然每种技术都有自己的优缺点, 但用球体压痕是一种特别简单而又相当精确的方法, 只需要进入 widefield 荧光显微镜。在前工作3、9、10中, 用金属球体压痕测量水凝胶的刚度。早期的工作表明基底刚度对细胞运动的重要性, 利用这种方法来确定水凝胶基板刚度3。最近, 共焦显微术也被用于一个优雅的特征10。
在这里, 我们提出了一个逐步的协议, 以准备一个软的硅基板, 耦合荧光珠 (和细胞外基质蛋白, 如胶原 i) 只是到顶端表面, 成像一个缩进球和顶面使用阶段和荧光成像, 分别对图像进行分析, 计算出硅基板杨氏模量。这种方式制备的软硅基板可以方便地用于牵引力显微实验。使用硬硅树脂 (而不是培养皿) 作为软硅胶的基础也使美国匹茨堡研究使用外部拉伸。如有必要, 还指出了避免可能出现的并发症所需的实际考虑。

<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

人体软组织通常在 kilopascal (kPa) 范围内有刚度。因此, 硅胶和水凝胶柔性基质已被证明是有益的基质, 培养细胞在物理微环境中, 部分模拟体内条件。在这里, 我们提出了一个简单的协议, 以表征杨氏模量的各向同性线性弹性基板通常用于美国匹茨堡研究。该协议包括在培养皿或硬硅树脂上制备软硅基板, 用荧光珠涂覆硅基板的顶面, 使用毫米级球体缩进顶面 (按重力), 成像荧光用荧光显微镜对缩进硅胶表面上的珠子进行分析, 并对所合成的图像进行解析, 计算出硅基板的杨氏模量。将基体的顶面与模数胞外基质蛋白耦合 (除了荧光珠), 使硅基板易于用于细胞电镀和随后的研究使用牵引力显微镜实验。使用硬质硅胶, 而不是培养皿, 作为软硅胶的基础, 使使用美国匹茨堡研究涉及外部拉伸。该协议的一个具体优点是, widefield 荧光显微镜在许多实验室中是常见的, 是本程序所需的主要设备。我们通过测量不同弹性模量的软硅基板的杨氏模量来证明该协议。

使用上面详细的协议, 我们准备了软硅胶在一个35毫米培养皿, 治愈它在70°c 30 分钟和耦合的荧光微球 (和胶原 i) 到顶端表面, 如图 1所示。深紫外线已用于最终的蛋白质耦合到基板13。请注意, (I) 这里使用的固化条件是特定于这种软硅胶和 (II) 压痕测量是在第二天进行, 因为软硅胶有望在一天的过程中进一步治疗。
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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

kilopascal 范围内的刚性基底对于研究细胞对生理相关的微环境刚度的反应是有益的。使用一个 widefield 荧光显微镜, 年轻的软硅胶凝胶的弹性模量可以确定使用一个合适的球压痕。

用 Widefield 荧光显微镜研究软硅基美国匹茨堡的刚度测量

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.

Research

JoVE Journal

Bioengineering

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科研

生物工程

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Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

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Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

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We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

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The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

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It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

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Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

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Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

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Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...