方法文章

Equibiaxial Stretching Device for High Magnification Live-Cell Confocal Fluorescence Microscopy

DOI：

10.3791/67520

⸱

2025年6月13日

 ,  ,  ,  ,  ,  ,  , 
1Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and Technology (BIST), 2University of Barcelona (UB), 3Department of Physics, King's College London, 4ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 5Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 6Institució Catalana de Recerca i Estudis Avançats (ICREA)

本文内容

摘要

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Here, we present a versatile equibiaxial stretching device that is pneumatically actuated and compatible with high-resolution microscopy.

摘要

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Cells are continuously exposed to mechanical forces in physiological and pathological situations, including a variety of tensile and compressive stresses. There is very active research exploring how cells and tissues respond and adapt to such stresses and how these responses integrate with mechanochemical signaling. This has generated a need for sophisticated tools compatible with standard cell culture protocols and microscopy methods, reproducing physiological stresses in in-vitro studies. This study presents the design, function, and characterization of a stretching device compatible with high-resolution optical and fluorescence microscopy. Numerous stretching devices, either pneumatic- or motor-based, have been developed and used in the field. We present one of these systems in detail, including design guidelines, a variety of applications, and all the tools to fabricate a similar setup. The system is based on a deformable polydimethylsiloxane (PDMS) membrane, stretched equibiaxially upon vacuum application, rendering a homogeneous, reproducible, and controlled sample strain. It provides a variety of tensile stresses, from punctual and immediate stretch to repeated stretch-release cycles of controlled amplitude and frequency. Substrate coating with adhesion proteins allows seeding cells bearing fluorescent reporters in the stretching device and performing live-imaging of these cells upon stretch using high magnification fluorescence microscopes. Compressive stresses can also be applied by letting the sample adapt to stretch and subsequently releasing it or by seeding the sample on a pre-stretched substrate before stretch release. Additional topographical patterning of the PDMS substrate enables imaging of the same sample in different microscopy modes (such as fluorescence and electron microscopy). Polyacrylamide gels can also be attached to the PDMS membrane, resulting in the stretching of cells seeded on substrates of different stiffnesses. Overall, by applying controlled tensile stresses on live samples, this stretching device, coupled with high-quality fluorescence microscopy, can address a large variety of questions in mechanobiology.

引言

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Thin deformable polydimethylsiloxane (PDMS) membranes are widely used in a variety of custom-made stretching devices1,2,3, as PDMS is a well-established biocompatible inert material compatible with microscopy imaging. Here, as many others4,5,6,7,8,9, the simple concept of applying a vacuum is used to deform the membrane to assemble a robust and controllable equibiaxial stretching ....

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方案

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1. Designing the stage and stretching post (Figure 1)

  1. Design a stretching post compatible in dimension with the stage size/holders and objective configuration. The two specific stretching posts are displayed in Figure 1A,D.
    NOTE: The FreeCAD files provided in this protocol (Supplementary Folder 1) are compatible with the following Nikon microscopes and stages: Eclipse Ni-E upright equipped with a NI-SH-E stage, and Eclipse Ti-E/B inverted equipped with a TI-S-ER stage, Figure 1D. The post can b....

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结果

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Calibration and radiality
Proper characterization of the strain obtained is a guarantee for reproducibility and control of the experiments. While fluorescent beads can be added to each sample to precisely record the strain, a previous calibration step ensures proper planning for the experiment, knowing the magnitude of strain the experimentalist wants to apply to the sample before the actual stretching. Strain will not only depend on the vacuum applied but will vary depending on stretch-ring dimensio.......

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讨论

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A large number of stretching devices have been used to apply mechanical stretch to biological samples, mimicking physiological conditions in the body24,25. Numerous setups are available in the literature, and observation of the sample response is usually performed by coupling stretching devices to atomic force26 or fluorescence microscopy27,11.

The desi.......

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披露

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We do not declare any conflicts of interest.

致谢

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This work was supported by funding from "la Caixa" Foundation (grant 406 LCF/PR/HR20/52400004 to X.T., J.V.R., and P.R.-C. and fellowships LCF/BQ/DR19/11740009 to 407 I.G.-M. and LCF/BQ/DR19/11740009 to M.M.J.), the Spanish Ministry of Science and Innovation 408 (PID2022-142672NB-I00 to P.R.-C., PID2021-128635NB-I00 to X.T., and PID2022-141040NA-I00 409 to I.A.), the Generalitat de Catalunya (2021 SGR 01425 to X.T. and P.R.-C.), The prize "ICREA 410 Academia" for excellence in research to P.R.-C., Fundació la Marató de TV3 (201936-30-31 to 411 P.R.-C.), the European Union (ERC, MECHANOSYNTH, grant 101097753 to P.R-C. and ERC, Adv-412 883739 E....

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材料

本文使用的材料清单
姓名公司目录编号评论
醋酸酯面膜京东光数据
丙烯酰胺比奥拉德161-014040% 丙烯酰胺溶液
APS的西格玛·奥尔德里奇A3678用于电泳的过硫酸铵，≥98%
阿普特斯西格玛·奥尔德里奇281778（3-氨基丙基）三乙氧基硅烷
自主微流控泵Elveflow 钴钴双钴双
双丙烯酰胺 Bisacrylamide比奥拉德161-01422% 双溶液
叶片费雪科学11733919教学小组 Hoja 做了áctica X100 跛脚 BISTOURI 无菌 N&°C;23 LAMD23 
剪辑斯普林蒂斯AKXX000001Bull Dog 夹子，32 毫米，金属制成，容量 10 毫米，镀镍
电晕处理器电工产品BD-20AC， 货号：12051ABD-20AC实验室电晕处理机
杯大众611-1368VWR， 烧杯，一次性，250 mL
DAPI的罗 氏10236276001DAPI染色
乙醇 96%Panreac Quí云母，SLU131085-1212乙醇 96% v/v para análisis，ACS- 2.5 升
纤连蛋白西格马F0895纤连蛋白，来自人血浆，液体 0.1%
FluoSpheres 羧酸盐改性微球，0.1 &m生命科技F8801、F8803用于分辨率评估的黄绿色和红色荧光珠
FluoSpheres 羧酸盐改性微球，0.2 和微粒;m费雪科学F8809、F8810用于应变校准的黄绿色和红色荧光珠
戊 二 醛西格玛·奥尔德里奇G6403型H2O 中的戊二醛溶液 50%
干涉仪维科 威科NT1100干涉仪测量 PDMS 膜厚度
异丙醇Panreac Quí云母，SLU131090-1212 2-丙醇 （Reag. Ph. Eur.） para análisis、ACS、ISO
莱顿称重定制
无绒纸巾伯 克 希尔DURX670、DR670090920无纺布湿巾
润滑剂（凡士林）西格马16415凡士林
蒙版对齐器Süss MicroTec MJB4 用于 PMMA 板上光刻的掩模对准器
莫维奥尔Calbiochem - 默克475904MOWIOL 4-88 试剂
尼康显微镜转盘共聚焦尼康日食 Ti-E/B配备尼康Ti-S-ER 阶段
尼康显微镜立式尼康日蚀镍电配备尼康 NI-SH-E 载物台
物镜 60 倍尼康CFI Apo 近红外 60X W近红外 Apo 60X/1.0w DIC N2 WD 2.8
PBS解决方案吉布科14200067DPBS，10 倍
PDMS的道康宁2401673921Sylgard - 184 硅胶弹性体套件 DN 109
培养皿环盖（大）普罗基诺特93060Plato de cultivo celular TPP SKU： 07177925直径 100
培养皿环盖（小）普罗基诺特93060Plato de cultivo celular TPP SKU： 07177924
等离子清洗机哈里克 PDC-002型氧等离子发生器
PMMA 板和环请参阅补充文档中的 Excel 规格表
延长Invitrogen第 36930 号/第 36934 号ProLong Gold 抗淬灭试剂
排斥-硅烷GE医疗17-1332-01PlusOne 抗斥硅烷 ES
环/橡胶圈泰克尼菲尔定制
旋涂机月桂WS-650Sz旋涂机
SPY650-DNA螺色素南卡罗来纳州 501SPY650-DNA
转向勺大众231-0416Eco 短一次性抹刀、双头勺子和勺子、PP 和 EcoPure 添加剂
SU8树脂2100微化学Y111075苏8-2100
SU8-显影剂西格玛-奥尔德里奇484431丙二醇单甲醚醋酸酯
特梅德西格玛-奥尔德里奇T9281-50毫升N，N，N'，N'-四甲基乙二胺，生物试剂 suitab。电泳试剂
三氯（1H，1H，2H，2H-全氟辛基）硅烷 西格玛·奥尔德里奇448931 三氯（1H，1H，2H，2H-全氟辛基）硅烷
镊子IESMATT5039 Dumont HP 镊子 7 不锈钢。0.17 x 0.10 毫米尖端。（琼脂科学有限公司）
真空控制器GARCAP/派克 990-005203-005 Controlador de vacio 
真空控制器 fluigent流畅的陆-FEZ-N800阵容流量 EZ （n800mbar）
白色台式纸雷马931.1001 纸 Raima 42 x 52

参考文献

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  1. Huang, L., Mathieu, P. S., Helmke, B. P. A stretching device for high-resolution live-cell imaging. Ann Biomed Eng. 38 (5), 1728-1740 (2010).
  2. Meza, D., Abejar, L., Rubenstein, D. A., Yin, W. A shearing-stretching device that ca....

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