Name: Equibiaxial Stretching Device for High Magnification Live-Cell Confocal Fluorescence Microscopy (Video) | JoVE Journal
Uploaded: 2025-06-13T13:40:00.000+00:00
Duration: 8 min 41 s
Description: Learn how to use a pneumatic equibiaxial stretching device for live-cell mechanobiology research. This versatile method enables high-resolution microscopy of cellular responses to mechanical stimuli.

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This work was supported by funding from &#34;la Caixa&#34; 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 &#34;ICREA 410 Academia&#34; for excellence in research to P.R.-C., Fundaci&#243; la Marat&#243; 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 Epifold to X.T.). Views and opinions expressed are however those of the authors(s) only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. A.E.M.B. was supported by a Sir Henry Wellcome Fellowship (210887/Z/18/Z). The authors acknowledge funding from the Juan de La Cierva fellowship supported by MCIU/AEI/10.13039/501100011033 and the European Social Fund Plus (FSE+) (JDC2023-051559-I to V.V). IBEC is a recipient of a Severo 415 Ochoa Award of Excellence from MINCIN. We thank V Gonz&#225;lez-Tarrag&#243; for assistance with the stretch system and Evelyn Coderch for technical support. We would also like to acknowledge the support given by the Unitat de Criomicrosc&#242;pia Electr&#242;nica TEM/SEM (Centres Cient&#237;fics i Tecnol&#242;gics de la Universitat de Barcelona, CCiTUB) and the MicroFabSpace and Microscopy Characterization Facility, Unit 7 of ICTS &#39;NANBIOSIS&#39; from CIBER-BBN at IBEC. We also thank Maria Garcia Parajo to facilitate communication to the ICFO mechanical workshop and Tecnifill for the smooth assistance in parts engineering.&#160;

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</ol>

We do not declare any conflicts of interest.

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...

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 device. By clamping the membrane inside a circular ring and sealing the ring on top of a &#34;stretching post&#34; (Figure 1A-C), a uniform and isotropic deformation is achieved. The biological sample is located at the ring&#39;s center on the free-standing PDMS membrane, which enables imaging using fluorescence microscopy. Here, two custom-made setups are presented, compatible with two microscope stage models (Figure 1D-F) and highly versatile in terms of biological applications. While the overall design is simple and transposable to other stages, the dimensions of the setup must be carefully chosen to fit the imaging requirements. Once achieved, the strain profile of the device must be characterized and validated.
Apart from the setup itself, a simple method accompanied by a Matlab code to characterize strain magnitude, homogeneity, and isotropy is presented. The stretching device can subsequently be used to apply equibiaxial tensile stresses to synthetic systems10, single cells11,12, or monolayers13, while recording with high spatiotemporal resolution the biological response of the sample using fluorescence microscopy. The membrane can be coated with different extracellular matrix adhesion proteins14,15. Extra features can be added according to the needs of the biological study. For instance, polyacrylamide gels can be bonded to the PDMS membrane to tune the substrate rigidity while stretching13,16, and the PDMS membranes can be fabricated with a reference numbered grid for posterior imaging of the same region of interest within a sample12. Fixed stretched samples can also be processed by immunostaining&#160;and subsequently restretched for fluorescence imaging17. The applied stretch pattern can be controlled, ranging from a single immediate stretch pulse to cyclic strain of varying magnitudes, frequencies or shapes14,16.

<table><tbody><tr><td>Acetate mask</td><td>JD Photodata</td><td></td><td></td></tr><tr><td>Acrylamide</td><td>Biorad</td><td>161-0140</td><td>40% Acrylamide solution</td></tr><tr><td>APS</td><td>Sigma Aldrich</td><td>A3678</td><td>Ammonium persulfate for electrophoresis, &amp;ge;98%</td></tr><tr><td>Aptes</td><td>Sigma Aldrich</td><td>281778</td><td>(3-Aminopropyl)triethoxysilane</td></tr><tr><td>Autonomous microfluidic pump</td><td>Elveflow Cobalt</td><td>Cobalt Dual</td><td>Cobalt Dual</td></tr><tr><td>Bisacrylamide</td><td>Biorad</td><td>161-0142</td><td>2% Bis Solution</td></tr><tr><td>Blade</td><td>Fisher Scientific</td><td>11733919</td><td>Didactic Group Hoja did&amp;aacute;ctica X100 LAME BISTOURI STERILE N&amp;deg;23&amp;nbsp; LAMD23&amp;nbsp;</td></tr><tr><td>Clips</td><td>Sprintis</td><td>AKXX000001</td><td>Bull Dog Clips, 32 mm, made of metal, capacity 10 mm, nickel-plated</td></tr><tr><td>Corona treater</td><td>Electrotechnicproducts</td><td>BD-20AC, SKU:12051A</td><td>BD-20AC LABORATORY CORONA TREATER</td></tr><tr><td>Cups</td><td>VWR</td><td>611-1368</td><td>VWR, Beaker Cups, Disposable, 250 mL</td></tr><tr><td>DAPI</td><td>Roche</td><td>10236276001</td><td>DAPI staining</td></tr><tr><td>Ethanol 96%</td><td>Panreac Qu&amp;iacute;mica, SLU</td><td>131085-1212</td><td>Etanol 96% v/v para an&amp;aacute;lisis, ACS- 2,5 L</td></tr><tr><td>Fibronectin</td><td>Sigma</td><td>F0895</td><td>Fibronectin, From Human plasma, liquid&amp;nbsp; 0.1%</td></tr><tr><td>FluoSpheres Carboxylate-Modified Microspheres, 0.1 &amp;micro;m</td><td>Life Technology</td><td>F8801, F8803</td><td>Yellow green and Red fluorescent beads for resolution assessment</td></tr><tr><td>FluoSpheres Carboxylate-Modified Microspheres, 0.2 &amp;micro;m</td><td>Fisher Scientific</td><td>F8809, F8810</td><td>Yellow green and Red fluorescent beads for strain calibration</td></tr><tr><td>Glutaraldehyde</td><td>Sigma Aldrich</td><td>G6403</td><td>Glutaraldehyde solution 50% in H&lt;font class=&quot;font6&quot;&gt;&lt;sub&gt;2&lt;/sub&gt;&lt;/font&gt;&lt;font class=&quot;font5&quot;&gt;O&lt;/font&gt;</td></tr><tr><td>Interferometer</td><td>Veeco&amp;nbsp;</td><td>Wyko NT1100</td><td>Interferometer to measure PDMS membranes thicknesses</td></tr><tr><td>Isopropanol</td><td>Panreac Qu&amp;iacute;mica, SLU</td><td>131090-1212&amp;nbsp;</td><td>2-Propanol (Reag. Ph. Eur.) para an&amp;aacute;lisis, ACS, ISO</td></tr><tr><td>Laiton weighs</td><td></td><td></td><td>Custom made</td></tr><tr><td>Lint free tissues</td><td>Berkshire</td><td>DURX670, DR670090920</td><td>Nonwoven-wipes</td></tr><tr><td>Lubricant (vaseline)</td><td>Sigma</td><td>16415</td><td>Vaseline</td></tr><tr><td>Mask Aligner</td><td>S&amp;uuml;ss MicroTec&amp;nbsp;</td><td>MJB4&amp;nbsp;</td><td>Mask aligner for photolithography on PMMA plates</td></tr><tr><td>Mowiol</td><td>Calbiochem - Merck</td><td>475904</td><td>MOWIOL 4-88 Reagent</td></tr><tr><td>NIKON microscope spinning disk confocal</td><td>Nikon</td><td>Eclipse Ti-E/B</td><td>equipped with Nikon Ti-S-ER&amp;nbsp; stage</td></tr><tr><td>NIKON Microscope Upright</td><td>Nikon</td><td>Eclipse Ni-E</td><td>equipped with Nikon NI-SH-E stage</td></tr><tr><td>Objective 60x</td><td>Nikon</td><td>CFI Apo NIR 60X W</td><td>NIR Apo 60X/1.0w DIC N2 WD 2.8</td></tr><tr><td>PBS solution</td><td>Gibco</td><td>14200067</td><td>DPBS, 10x</td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>2401673921</td><td>Sylgard - 184 Silicone Elastomer KIT DN 109</td></tr><tr><td>Petri dish ring cover (big)</td><td>Proquinorte</td><td>93060</td><td>Plato de cultivo celular TPP SKU: 07177925 diametro 100</td></tr><tr><td>Petri dish ring cover (small)</td><td>Proquinorte</td><td>93060</td><td>Plato de cultivo celular TPP SKU: 07177924</td></tr><tr><td>Plasma cleaner</td><td>Harrick&amp;nbsp;</td><td>PDC-002</td><td>Oxygen Plasma Generator</td></tr><tr><td>PMMA plates and rings</td><td></td><td></td><td>see excel specification table in supplementary documents</td></tr><tr><td>Prolong</td><td>Invitrogen</td><td>P36930 /P36934</td><td>ProLong Gold antifade reagent</td></tr><tr><td>Repel-Silane</td><td>GE Healthcare</td><td>17-1332-01</td><td>PlusOne Repel-Silane ES</td></tr><tr><td>Rings /rubber ring</td><td>Tecnifil</td><td></td><td>Custom made</td></tr><tr><td>Spin coater</td><td>Laurel</td><td>WS-650Sz</td><td>Spin coater</td></tr><tr><td>SPY650-DNA</td><td>Spirochrome</td><td>SC 501</td><td>SPY650-DNA</td></tr><tr><td>Steering spoon</td><td>VWR</td><td>231-0416</td><td>Eco short disposable spatula, double-ended spoon and scoop, PP and EcoPure additive</td></tr><tr><td>SU8 resin 2100</td><td>Microchem</td><td>Y111075</td><td>SU8-2100</td></tr><tr><td>SU8-Developer</td><td>Sigma-Aldrich</td><td>484431</td><td>Propylene Glycol Monomethyl Ether acetate</td></tr><tr><td>TEMED</td><td>Sigma-Aldrich</td><td>T9281-50ML</td><td>N,N,N&#039;,N&#039;-Tetramethylethylenediamine, Bioreagent suitab. Electrophoresis reagent</td></tr><tr><td>Trichloro(1H,1H,2H,2H-perfluorooctyl)silane&amp;nbsp;</td><td>Sigma Aldrich</td><td>448931</td><td>&amp;nbsp;Trichloro(1H,1H,2H,2H-perfluorooctyl)silane</td></tr><tr><td>Tweezers</td><td>IESMAT</td><td>T5039&amp;nbsp;</td><td>Dumont HP Tweezers 7 Stainless Steel. 0.17 x 0.10mm tip.(Agar Scientific Ltd)</td></tr><tr><td>Vacuum controller</td><td>GARCAP/Parker</td><td>&amp;nbsp;990-005203-005&amp;nbsp;</td><td>Controlador de vacio&amp;nbsp;</td></tr><tr><td>Vacuum controller fluigent</td><td>Fluigent</td><td>LU-FEZ-N800</td><td>LineUp Flow EZ (n800mbar)</td></tr><tr><td>White bench paper</td><td>Raima</td><td>931.1001</td><td>&amp;nbsp;Paper Raima 42 x 52</td></tr></tbody></table>

equibiaxial stretching device for high magnification live-cell confocal fluorescence microscopy

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

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.

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...

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

Cells are continuously exposed to mechanical forces in physiological and pathological situations, including a variety of tensile and compressive stresses. ...

equibiaxial-stretching-device-for-high-magnification-live-cell

Research

JoVE Journal

Bioengineering

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

We illustrate the use of a constant force axial optical tweezers to explore the mechanical properties of short DNA molecules.  By stretching DNA axially, we minimize steric hindrances and artifacts arising in conventional lateral manipulation, allowing us to study DNA molecules as short as ~100 nm.

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

Live Cell Imaging during Mechanical Stretch

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

Super-Resolution Live Cell Imaging of Subcellular Structures

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Super-resolution microscopy can provide a detailed insight into the organization of components within the synaptonemal complex in meiosis. Here, we demonstrate a protocol to resolve individual proteins of the Caenorhabditis elegans synaptonemal complex.

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Here, we present a novel device that applies isotropic and uniaxial strain waveforms to cells while supporting culture on substrates with tunable stiffness and customizable protein matrices. This simple, economical platform enables researchers to investigate how mechanical cues regulate cellular function in both fundamental studies and disease-relevant contexts.

A Novel Platform for In Vitro Cellular Stretching and Imaging

Learn how to use a pneumatic equibiaxial stretching device for live-cell mechanobiology research. This versatile method enables high-resolution microscopy of cellular responses to mechanical stimuli.

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

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