微图案牵引显微镜的图案生成
Summary
我们描述了测量细胞牵引力的标准方法的改进，该方法基于微接触印刷，具有软水凝胶上细胞外基质蛋白点阵列的单个减法图案化步骤。这种方法允许更简单，更一致地制造岛状图案，这对于控制细胞簇形状至关重要。
Abstract
微图案牵引显微镜允许控制单个细胞和细胞簇的形状。此外，在微米长度尺度上图案的能力允许使用这些图案化的接触区来测量牵引力，因为每个微图案点允许形成单个焦点粘附，然后使柔软的底层水凝胶变形。这种方法已被用于广泛的细胞类型，包括内皮细胞，平滑肌细胞，成纤维细胞，血小板和上皮细胞。
本综述描述了允许细胞外基质蛋白以预定大小和间距的常规点阵列印刷到聚丙烯酰胺水凝胶上的技术的演变。由于微米级图案很难直接打印到软基板上，因此首先在刚性玻璃盖玻片上生成图案，然后在凝胶化过程中使用图案转移到水凝胶上。首先，描述了在盖玻片上生成小点阵列的原始微接触打印方法。第二步是去除大部分图案以留下小点岛，以控制这些图案点阵列上细胞和细胞簇的形状。
接下来，描述了这种方法的演变，该方法允许使用单个减法图案化步骤生成点岛。这种方法对用户来说大大简化了，但缺点是制造图案所需的母模寿命缩短。最后，描述了为分析位移点图像和随后的细胞产生的牵引场而开发的计算方法，并提供了这些分析包的更新版本。
Introduction
大多数细胞表型对其环境施加牵引力。这些牵引力是由细胞的收缩细胞骨架产生的，这是肌动蛋白和肌球蛋白以及其他丝状生物聚合物和交联蛋白1，2，3，4的网络。细胞内产生的力可以传递到细胞外环境或相邻细胞，主要通过跨膜蛋白如整合素和钙粘蛋白，分别为5，6。细胞如何扩散或收缩 – 以及与这些运动相关的牵引力的大小 – 是与其环境进行密切对话的结果，这在很大程度上取决于细胞外基质中存在的蛋白质的类型和数量（ECM）7，8以及ECM的硬度。事实上，牵引力显微镜已成为了解细胞对局部刺激（如基底刚度，施加的机械应力和应变或与其他细胞接触）的反应的宝贵工具。该信息与癌症和哮喘等疾病的理解直接相关9，10，11，12。
需要一个可用于测量已知材料特性的基板的力诱导变形的系统来计算牵引力。这些变化必须随着时间的推移而跟踪，这需要成像和图像处理技术。用于确定细胞牵引力的首批方法之一是观察和分析接种有细胞的胶原蛋白水凝胶的收缩，尽管这种方法只有半定量13。另一种更精细的方法是通过确定由硅胶薄片14变形引起的力来测量单个电池施加的牵引力。后来，开发了更多的定量测量技术，这些方法还允许使用软水凝胶，如聚丙烯酰胺（PAA）12，15，16。当使用这些软材料时，牵引力可以通过嵌入水凝胶中的随机置换珠的力诱导位移和凝胶16，17的机械性能来确定。另一个进步来自由软聚二甲基硅氧烷（PDMS）制成的微柱阵列的发展，以便可以使用梁理论18测量其挠度并将其转换为力。
最后，开发了用于微图案化软水凝胶的方法，因为这些方法可以控制细胞粘附的接触区域。通过测量微图案在细胞接触区域内的变形，可以很容易地计算出牵引力，因为不需要无力参考图像19。该方法已被广泛采用，因为它允许将微米大小的离散荧光蛋白粘附点的常规阵列间接图案化到PAA凝胶上，以测量细胞牵引力20。为了计算这些力，已经开发了一种图像处理算法，该算法可以在不需要用户输入的情况下跟踪每个微图案点的运动。
虽然此方法对于创建点图案的整个网格很简单，但是当需要点的孤立补丁（或岛）图案时，它更复杂。当需要控制细胞簇的形状和一定程度的大小时，微图案化岛屿是有用的。为了创建这些岛屿，上述微接触印刷方法需要两个不同的步骤：i）使用一个PDMS图章在盖玻片上创建点的高保真图案，然后ii）使用第二个不同的PDMS图章来去除大部分这些点，留下孤立的点21岛。使用这种原始方法创建岛屿的难度由于在过程的第一步中制作一致的网格模式本身就具有挑战性而变得更加复杂。缩微印刷邮票由一系列圆形微柱组成，其直径对应于所需的点大小。然后将这些印章涂上均匀的蛋白质层，然后在经过处理的盖玻片上施加精确压力，以创建所需的图案。一方面，对印章施加太大的压力会导致蛋白质转移不均匀，并且由于柱子之间的屈曲或下垂导致与玻璃接触，从而导致图案保真度差。另一方面，施加太小的压力会导致蛋白质转移很少或没有，并且图案保真度差。由于这些原因，需要一种传输过程，该过程可用于在短短一步内始终如一地创建孤立的点岛的高质量微图案。
本文描述了一种将微米级荧光蛋白粘附点的岛间接微图案化到PAA凝胶上的方法，该凝胶比以前开发的方法更加一致和通用。虽然较旧的间接微图案化方法依赖于蛋白质图案从PDMS图章转移到中间底物，但这里介绍的方法使用PDMS图章代替蛋白质去除的容器，而不是添加。这是通过首先从根本上改变所使用的PDMS图章的结构来完成的。在这种方法中，邮票不是制作由均匀间隔的圆柱图案组成的邮票，而是由均匀间隔的圆孔图案组成。
有了这个新结构，这些PDMS印章的表面可以用戊二醛处理，如前所述20，29，30，使印章能够与蛋白质共价键合。当用于均匀涂有荧光蛋白的玻璃盖玻片上时，这些经过戊二醛处理的PDMS印章用于去除盖玻片表面上的大部分蛋白质，仅留下由印章上微米大小孔的位置预先确定的所需点图案。此更改提高了生成由近乎连续的点网格组成的模式的成功率，并且只需一步即可创建孤立的点岛。
Protocol
1. 创建硅胶母带 注意：用于PDMS印章重复成型的硅母版的设计，创建和故障排除的大部分过程已在前面21中介绍过，因此这里将仅介绍这种新方法的关键差异。 使用 AutoCAD 或类似设计软件创建光掩模的设计。在光掩模的一侧涂上一层薄薄的玻璃，涂上一层薄薄的铬，以控制紫外线的散射。设计光掩模，使紫外线穿过它照射到所选的光刻胶?…
Representative Results
采用减法微图案化方法制备了杨氏模量为E = 3.6 kPa，泊松比为0.445的PAA水凝胶。水凝胶的厚度约为100μm，这允许它们使用此处使用的成像设置进行成像，同时还可以防止细胞感应凝胶下方的刚性盖玻片，这将在专注于细胞刚性感测的研究中引起问题23，33。许多其他刚度水平（高达30 kPa）的凝胶已经成功地使用间接微图案化方法…
Discussion
本文介绍了一种间接图案化PAA水凝胶的改进方法。此方法基于以前使用过的方法20，35，36，37，38，39，40，41，42。主要的变化是PDMS印章现在用于去除蛋白质并将所需的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢波士顿大学机械工程系的Paul Barbone博士在数据分析方面的有益讨论和帮助。这项研究得到了NSF拨款CMMI-1910401的支持。
Materials
| (3-aminopropyl)trimethoxysilane | Sigma Aldrich | #281778 | |
| 1.5 mL Microcentrifuge tube | Fisher Scientific | #05-408-129 | |
| 15 mL conical tube | Fisher Scientific | #05-539-12 | |
| 4 x 4 in 0.060 Quartz LR Chrome Photomask | Advance Reproductions Corporation | N/A | Custom-designed mask |
| 6 Well Plates | Fisher Scientific | #07-200-83 | |
| Acetone | Fisher Scientific | #A18P-4 | |
| Acrylamide Solution, 40% | Sigma Aldrich | #A4058 | |
| AlexaFluor 488 | Thermo Fisher | #A20000 | |
| Aminonium Persulfate | Fisher Scientific | #BP179-25 | |
| Bisacrylamide | Fisher Scientific | #PR-V3141 | |
| Ethanol | Greenfield Global | #111000200C1GL | |
| Glass Coverslips, 25 mm round | Fisher Scientifc | #12-545-102 | |
| Glass Coverslips, 30 mm round | Warner | #64-1499 | |
| Hamamatsu ORCA-R2 Camera | Hamamatsu | #C10600-10B | |
| Human Plasma | Valley Biomedical | #HP1051P | Used to isolate fibronectin |
| Hydrochloric Acid, 1.0 N | Millipore Sigma | #1.09057 | |
| ImageJ | Wayne Rasband | #1.53n | |
| Interchangeable Coverslip Dish Set | Bioptechs | #190310-35 | |
| Kim Wipes | Fisher Scientific | #06-666-11C | |
| Mask Alinger | Karl Suss | #MA6 | |
| Matlab 2021 | Mathworks | #R2021a | |
| MetaMorph Basic | Molecular Devices | #v7.7.1.0 | |
| N-hydroxysuccinimide ester | Sigma Aldrich | #130672-5G | |
| NucBlue Live Cell Stain | Thermo Fisher | #R37605 | |
| Olympus IX2-ZDC Inverted Microscope | Olympus | #IX81 | |
| PD-10 Desalting Columns | GE Healthcare | #52-1308-00 | |
| Photoresist Spinner Hood | Headway Research | #PWM32 | |
| Plasma Cleaner | Harrick | #PDC-001 | |
| Plasma Etcher | TePla | #M4L | |
| Prior Lumen 200Pro Light Source | Prior Scientific | #L200 | |
| Silicon Wafers, 100 mm | University Wafer | #809 | |
| SU-8 2005 | Kayaku Advanced Materials Inc. | #NC9463827 | |
| SU-8 Developer | Kayaku Advanced Materials Inc. | #NC9901158 | |
| Sylgard 184 Silicone Elastomer | Essex Brownell | #DC-184-1.1 | |
| Tetramethylethylenediamine | Fisher Scientific | #BP150-20 | |
| Trichloro(1H,1H,2H,2H-perfluorooctyl)silane | Sigma Aldrich | #448931 | |
| UAPON-40XW340 Objective | Olympus | #N2709300 | |
| UV Flood Exposure | Newport | #69910 | |
| Wafer Carrier Tray, 110 x 11 mm | Ted Pella, Inc. | #19395-40 |
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Cite This Article
Bunde, K. A., Stamenović, D., Smith, M. L. Pattern Generation for Micropattern Traction Microscopy. J. Vis. Exp. (180), e63628, doi:10.3791/63628 (2022).