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

Summary

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

Abstract

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.

Introduction

Thin deformable polydimethylsiloxane (PDMS) membranes are widely used in a variety of custom-made stretching devices^1,2,3, as PDMS is a well-established biocompatible inert material compatible with microscopy imaging. Here, as many others^4,5,6,7,8,9, 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 "stretching post" (Figure 1A-C), a uniform and isotropic deformation is achieved. The biological sample is located at the ring'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 cells^11,12, 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 proteins^14,15. 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 stretching^13,16, 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 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 shapes^14,16.

Protocol

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 be three-dimensionally (3D) printed but may deform after repeated use; aluminum-based posts will not suffer from this drawback. Make sure to anodize aluminum-based posts to prevent oxidation. Upright configuration allows for a larger amplitude of movement. In addition, an upright configuration provides direct imaging of the sample without interface, which is optically favorable in the case of the deformable PDMS membrane substrate. Instead, in an inverted configuration, the illumination beam goes through the PDMS membrane (Figure 1F); since upon stretch application, the PDMS membrane will move, the immersion media for the objective should be carefully chosen (recommended water immersion types, air or glycerol; oil immersion is not recommended due to incompatibility with PDMS substrates). Commercial holders can be modified using extra screw holes; beware of using plastic or Teflon-based screw to avoid dust while screwing. It can be convenient to design a holder that enables the stretching post to be fitted on top of the stage, as displayed in Figure 1E. The FreeCAD file provided in this protocol is compatible with the stage of an Eclipse Ni-E upright microscope.
2. Design stretch rings (Figure 1C) compatible in size with the stretching post (Figure 1A,B). The FreeCAD files provided in this protocol are compatible with the corresponding posts (to cover the rings while handling samples, their diameter can be chosen to be compatible with the size of a Petri dish).
   NOTE: The ring can also be aluminum-based (anodized) or 3D-printed. Include gaskets of adequate diameters to vacuum-seal the stretching post and rings.
3. Optional: Fabricate a vacuum controller. An electrical map of the vacuum control box used in this protocol is provided in supplementary information (see Supplementary Folder 2). It permits manual control of the vacuum level applied to the stretching device.
   NOTE: Commercial vacuum controllers can also provide similar functions and can usually be accompanied by control software enabling the application of a variety of signal types (sinusoidal, triangular, etc.) (Fluigent, Cobalt).

2. Fabrication of the stretchable membrane

1. Design circular plates (recommended material: polymethacrylate, PMMA). Ensure that these plates are wider than the rings and compatible in size with the spin coater (Figure 2A). See Table 1 for suggested dimensions and Supplementary Folder 3.
   NOTE: For post 1, a 12 cm diameter was used; for post 2, an 18-21 cm diameter. Prior to and between each use, the plates should be carefully cleaned using nonwoven wipes and 96% ethanol. It is important to avoid PDMS accumulation in the plates, as well as on borders and underneath.
2. Prepare PDMS 1:10 and mix well to obtain a very homogeneous emulsion (use recommended stirring spoons).
   NOTE: For 12 cm, prepare 4.5 g per plate; for 18 cm plates, 8 g per plate. For multiple plates preparation, prepare extra PDMS, for instance 10 mL more than necessary for 10 plates. For 10 plates of 12 cm, prepare 50 g of component A and 5 g of component B. It is handy to use large receptacles to easily pipette. The receptacle must not contain dust or any other dirt that might impede proper PDMS spinning and curing. Use gloves of compatible material (avoid latex; vinyl gloves are appropriate) to ensure PDMS curing.
3. Degas for a minimum of 1 h using a vacuum chamber.
   NOTE: Alternatively, the PDMS can be centrifuged at 0.2 × g for 5 min using a 50 mL tube, but beware that it is complicated to aspirate the PDMS mixture from a tube with the dispensing syringe (necessary for step 2.4). Do not degas for too long (no more than 2 h) with direct light to avoid pre-curing PDMS.
4. PDMS spin coating on top of the PMMA plates
   1. Dispense 4.2 mL or 7.5 mL of degassed PDMS on top of the PMMA plates.
   2. Spin-coat the plates using the following recommended settings:
      1. Spin coat for 5 s at 500 rpm with an acceleration of 100 rpm/s.
      2. Spin coat for 1 min at 500 rpm with an acceleration of 300 rpm/s.
         NOTE: It can be convenient to mark the center of the PMMA plate on its bottom side to ensure the centering of the plates during spinning and homogeneous spinning. Step 2.4.2.1 is not compulsory especially if the acceleration parameter cannot be adjusted; it helps spread the PDMS before spinning. This protocol yields PDMS membranes with a thickness of around 140 µm (measured with interferometry; see Supplementary Figure 1 and Table 2). Calibrations must be performed prior to experiments in case the spin coater model or parameters lead to different membrane characteristics.
5. Store each plate in a Petri dish to avoid dust accumulation and polymerize overnight at 65 °C inside the oven.
   NOTE: Beware of using temperature-resistant dishes. The curing condition will affect the PDMS deformability for a given vacuum level applied.

3. Membrane sandwiching in rings

1. Design of the mounting material.
   1. Design PMMA rings of internal/external diameter to lay the spin-coated PDMS circular membrane (Figure 2A). See the proposed dimensions in Table 1 and Supplementary Folder 3.
   2. Design a mounting support (Figure 2B) on top of which the ring will fit, and the height will also determine the pre-stretch when mounting the membrane in the ring.
      NOTE: The mounting support can be a simple solution based on existing labware (e.g., a Petri dish). A more elegant solution is to 3D-print the support (see FreeCAD file of this part provided in this protocol, see Supplementary Folder 3), as the height can be user-defined and easily varied (a 10 mm height was used in this protocol for the small rings). The center part height of the support should be chosen smaller than the half-ring height so that the PDMS substrate does not touch the support when mounting (see further use in step 3.4.2). A 3D printer using polylactic acid (PLA) filaments (see Table of Materials) is suitable for this. A cutting ring can also be designed, 3D printed as well, to serve as a guide for the scalpel (Figure 2A).
   3. Design and fabricate brass weights that are compatible in size to exert weight on the ring while mounting the membrane (Figure 2C) and lead to reproducible pre-tension.
      NOTE: The FreeCAD files provided here (see Supplementary Folder 3) match the ring dimensions presented in this protocol.
2. Remove the PDMS membrane from the PMMA plate.
   1. Prepare PMMA rings on top of a sheet of white bench paper.
      NOTE: It is important that the bench and paper remain flat to avoid the paper touching the PDMS membrane during mounting.
   2. Carefully go around the edge of the plate with a blade (once or twice) and then trace the same edge with sharp tweezers to ensure that the PDMS has a clean cut along its entire circumference (Figure 2A).
   3. Use tweezers to carefully lift the membrane at one edge. Then, gently peel the edge of the PDMS sheet with the thumbs to lift the membrane evenly at the edge. Detach the edge of the membrane from the plate along half of the plate circumference (Figure 2A).
      NOTE: Pouring some drops of 96% ethanol below the PDMS membrane will help to lift the membrane without breaking it.
3. Dry the PDMS membrane.
   1. Wipe 96% ethanol around the whole top surface of the PMMA ring previously prepared (step 3.2.1), using the fingers to spread it evenly.
   2. Carefully remove the PDMS membrane, lifting from the previously lifted edge (explained in step 3.2.3), and place it symmetrically on the ethanol-covered PMMA ring. Remove any wrinkles from the PDMS, but make sure not to stretch the membrane; only straighten it.
      NOTE: Applying pre-stretch at this stage will affect the PDMS deformability for a given vacuum level applied.
   3. Once the ethanol is completely dried, ensure that the PDMS membrane is tightly stuck to the PMMA ring.
4. Assemble the stretch ring.
   1. Take another empty PMMA ring. Place it on top of the mounting ring and use metal clips to seal them together, sandwiching the PDMS membrane between the two rings (PDMS membrane sandwich, Figure 2B).
   2. Place the bottom half of the stretch ring onto the mounting support designed in step 3.1.2 (Figure 2B).
   3. Place the PDMS ring sandwich on the stretch ring and push it down firmly until it reaches all the way down. The PDMS substrate does not touch the mounting support. Secure the gasket in the correct position (Figure 2B).
   4. Place the circular brass weights on top of the sample (Figure 2C). This applies a fixed pre-tension similar to that of all mounted rings, ensuring further reproducibility when calibrating the system.
   5. Place the top half of the metal stretch ring on top, with screw holes aligned, place all the screws, and tighten (Figure 2C).
      NOTE: At this stage, the PDMS membrane has holes, so do not try to unmount or go backward in the protocol.
   6. Cut the PDMS around the ring gently with a blade (Figure 2C).
   7. Cover (e.g., with a Petri dish) to protect the membrane from dust (Figure 2C).
      NOTE: If the PDMS needs to be functionalized with a polyacrylamide gel, proceed to section 5. For initial calibration measurements, proceed to section 6. If the PDMS ring is to be used for cell seeding, proceed to section 7.

4. Variation of PDMS membrane fabrication: pattern a reference grid

1. Prepare a PMMA plate (prior to PDMS spin-coating) "patterned" with a SU8-based grid created using photolithography (Figure 2D). For this, design a pattern of interest to create a photomask film (acetate mask).
   NOTE: The pdf file provided in this protocol (see Supplementary Folder 4) creates a grid with alternate numbers; see Figure 2D. The grid can be created using simple software such as Inkscape given the size of the pattern (or alternatives such as Clewin or FreeCAD). The design can be printed by external companies on photography films (see Table of Materials).
2. Photolithography protocol on the PMMA plate (see Supplementary Folder 3 for PMMA optical properties).
   1. Perform an oxygen plasma treatment of the PMMA plates for 20 min at 7 W with a constant oxygen flow of 0.8 bar.
      NOTE: Make sure possible plastic protection of the plates is removed and PMMA plates are completely transparent.
   2. Bake for 5 min at 95 °C.
   3. Cool the PMMA plate using a nitrogen gun, and then place 4 mL of SU-8 2100 resin onto the 12 cm PMMA plate (adjust the volume if using different plate sizes).
   4. Spin with the following parameters (or adjust the spin parameters to create a layer approximately 10 µm high): 5 s at 500 rpm with an acceleration of 100 rpm/s followed by 30 s at 4000 rpm with an acceleration of 300 rpm/s.
   5. Bake for 2.5 min at 95 °C.
   6. Place the PMMA plate on a mask aligner in the presence of the acetate mask and use the following parameters: without i-line. Broadband exposure (between 260 and 460 nm), 25 mW/cm², exposure of 7.5 s.
   7. Bake for 3.5 min at 95 °C.
   8. Develop the pattern for 1 min with propylene glycol monomethyl ether acetate.
   9. Wash extensively with isopropanol.
   10. Perform visual verification of the pattern quality using a low magnification objective (2-10x) in a brightfield microscope.
       NOTE: Silanization of the PMMA patterned plate increases the longevity of the pattern for repeated use. For this, activate the patterned PMMA plate for 30 s with oxygen plasma treatment and then perform a 1 h treatment with Trichloro(1H,1H,2H,2H-perfluorooctyl)silane under vacuum in a chemical hood.
3. Use the patterned PMMA plate as in steps 2.2 to 2.5 to spin coat the PDMS stretchable membrane.
   NOTE: The patterned PDMS can be visualized in a brightfield microscope, as mentioned previously, as well as in a scanning electron microscope (Figure 2D). Beware which side of the pattern is facing up while mounting the PDMS membrane in the stretch ring to obtain the pattern in the desired orientation.

5. Attaching a polyacrylamide (PAA) gel on top of the PDMS membrane

NOTE: This step can be performed either on the PDMS membrane attached to the PMMA plate (see section 2) or on the previously mounted stretch rings (see section 3). Work under a chemical hood; for the steps involving heating, it is convenient to use a small oven fitting under the chemical hood, see Table of Materials; otherwise, keep the samples in a hermetic box in the oven).

1. PDMS membrane pre-coating (Figure 2E)
   1. Prepare the coating solution by mixing 96% ethanol with 10% (3-Aminopropyl)triethoxysilane (APTES) in a microcentrifuge tube (e.g., 20 µL of APTES in 180 µL of ethanol).
   2. Activate the PDMS membranes with a corona plasma discharger for 1 min (or activate for 30 s using an oxygen plasma cleaner).
   3. Place 200 µL of the APTES/ethanol solution at the center of the membrane.
   4. Leave the membrane at 65 °C inside the oven for 1 h.
   5. Wash twice with PBS. For the second wash, leave the PBS for 10 min while shaking.
   6. Prepare the 3% glutaraldehyde (GA) solution in PBS. Place 200 µL of GA per membrane on the same region of the membrane treated with APTES.
   7. Wait for 25 min (no longer than 30 min).
   8. Wash twice with PBS, the second time while shaking.
   9. Aspirate the liquid and place the ring in a declined position, until PBS is totally dried.
2. Coverslip silanization
   1. Prepare 6 mL of Repel Silane and pour it into a Petri dish.
   2. Place square 18 mm glass coverslips with care, one by one, into the liquid.
   3. Incubate the coverslips for 5 min.
   4. Prepare two beakers; one containing 96% ethanol and the other ultrapure water.
   5. Remove the coverslip from the Repel Silane with tweezers, and wash first in 96% ethanol, followed by a wash in ultrapure water (the coverslip should come out "dry" from the water wash).
   6. Let dry for an additional 30 min at the border of a Petri dish.
3. Polyacrylamide (PAA) gel attachment to PDMS membrane
   1. Arrange half of the Repel Silane treated coverslips at the bottom of a Petri dish.
   2. Prepare a PAA gel mix of the desired rigidity^16,18.
   3. Place a drop of 10 µL of gel mix on top of each coverslip.
   4. Place another Repel Silane treated coverslip on top to spread the gel. Do not align the two coverslips. Ensure that they are arranged in a star shape to facilitate easy separation. Press gently so the gel is flattened but does not exceed the coverslip area.
   5. Wait for 45 min to 1 h.
   6. Separate the coverslips gently using tweezers. Ensure that the gel remains attached to one of the coverslips.
   7. Take the coverslip to which the gel is attached and place the gel in contact with the PDMS area previously treated with GA (Figure 2E).
   8. Press with two fingers from the bottom and the top for 5 s.
   9. Incubate at 37 °C overnight.
   10. Next day, fill the ring with PBS for 30 min.
   11. Gently detach the top coverslip with tweezers.
   12. Draw the perimeter of the PAA gel with a permanent marker from the other side of the PDMS membrane. This helps to locate the sample in the following steps.
   13. Aspirate the PBS.
   14. Perform a functionalization step to facilitate attachment of the desired extracellular matrix protein to the gel¹⁵.

6. Stretching device operation, strain calibration, and resolution assessment

1. Prepare a PDMS stretch ring with fluorescent beads for strain calibration.
   1. Prepare a 1:50000 dilution of 0.1 µm or 0.2 µm fluorescent bead (see Table of Materials) in phosphate buffer saline (PBS) solution by serial dilution (1:500 intermediate solution of 1 mL in PBS with 2 μL of the beads stock solution followed by final dilution of 10 μL from the intermediate solution in 1 mL of PBS).
      NOTE: A high bead density onto the membrane is optimal for the calibration step; do not sonicate the solution, as bead clumps help the operation during calibration.
   2. Place a 10-30 µL drop in the center of the PDMS.
      NOTE: Mounting support can be conveniently designed with a cross-to-center sample deposition
   3. Leave to dry at no higher than 40 °C to avoid post-curing confounding issues.
2. Prepare a PDMS stretch ring with fluorescent beads for resolution assessment.
   1. Follow the protocol by Cole and al.¹⁹ to prepare the bead solution using beads with diameters smaller than the diffraction limit, typically 0.1 µm diameter beads.
      ​(see Table of Materials). Carefully sonicate the bead solutions at each intermediate step.
   2. Use corona treatment at the center of the ring to activate the PDMS substrate.
   3. Immediately spread 20 µL of the bead solution on top of the treated PDMS central area and leave it to dry.
      NOTE: In this case, the bead solution is highly diluted and incubated on the hydrophilic PDMS to ensure extensive spreading and obtain single separated 0,1 µm beads adsorbed on the substrate.
3. Mounting the stretching device onto the microscope stage holder (Figure 1F).
   1. Mount the stretching post in the microscope.
   2. Generously apply lubricant (Vaseline) on top of the circular area of the post (middle part, Figure 1D, F).
      NOTE: Use of the lubricant is mandatory for the PDMS substrate to overcome frictions and slide on the post, and it is necessary to refresh the lubricant between each sample. Vaseline is used as a very convenient lubricant in the setup presented in this protocol.
   3. Mount the stretch ring prepared in step 6.1 in the stretching post and screw the top cap to seal the assembly.
   4. Connect the vacuum controller to the vacuum source and the stretching post (Figure 3A).
      1. Connect the rear of the vacuum control box to the vacuum source (Figure 3A).
      2. Connect the front of the box to the stretch-post (Figure 1B, D, F).
         NOTE: The vacuum source can be the vacuum line available in the infrastructure of the building. A moderate vacuum is required for a sufficient stretch but depends on the stretch ring diameter (a smaller strain will be obtained at the same vacuum level for a smaller diameter). Alternatively, vacuum pumps are commercially available (Fluigent or Elveflow provide autonomous vacuum pumps); in this case, follow manufacturer instructions for connection to the vacuum source. Do not use a vacuum supply without a vacuum-controlled source (such as the custom-made vacuum control box presented in this protocol or the commercially available systems)
4. Strain calibration
   1. Choose a field of interest that includes a high bead density all over the field of view and recognizable bead clumps for the user (see Figure 3B).
   2. Acquire an image "at rest" of the chosen field of view (save the position).
   3. Apply stretch at a low vacuum level (in this setup, -20 mbar). The sample will move in all directions (X-Y-Z).
   4. Refocus first in the z-plane.
   5. Move the stage in x-y directions to find the same region of interest imaged at rest, fine-focus the image of the beads in the stretched state, and acquire an image.
      NOTE: If the sample is very off-centered, the region of interest will move significantly, depending on the strain magnitude. The displacement will be larger away from the center, but the strain magnitude is independent of the location of the sample on the substrate. Record images without binning, with a 2048x2048 resolution.
   6. Release the strain. Repeat steps 6.4.3-6.4.5 with a higher vacuum level (-30 mbar, -40 mbar, etc.).
      NOTE: For calibration purposes, always return to the rest state before restretching (see Supplementary Figure 3A), as PDMS has a hysteresis behavior upon deformation.
   7. Mount a second stretch ring and repeat steps 6.4.1-6.4.6. Perform this calibration for at least 3 different stretch-rings.
   8. Store the acquired images in a folder organized by sample.
   9. In each folder, name the reference bead image acquired at rest reference_str1, and the images stretched at increasing vacuum stretched_str1, stretched_str2, stretched_str3, etc.
      NOTE: This step constitutes the core calibration of the system. Depending on the user's needs, additional calibration using similar bead samples but using other vacuum application protocols should be performed. Apply a strain protocol resembling the strain applied to the biological sample (see representative results section).
5. Use the Matlab code to quantify the strain²⁰.
   1. Download the whole Matlab folder at the following link https://github.com/xt-prc-lab/Le_Roux_et_al_2024_JOVE
      NOTE: The main code to be run is named gel_strain and calls other Matlab code files that must be kept as provided, stored in the same folder as the main code.
   2. Open Matlab and open the code gel_strain.
   3. Set the parameters (see Table 3, Code_Parameter for recommended settings).
   4. Define the parent and experiment folder paths as indicated in the code.
      NOTE: If the folder does not exist, the code will show a warning pop-up; select add to path and browse till the experimental folder where images are stored.
   5. Run the gel_strain code.
   6. Open stretched_str1 as requested by the code.
   7. Open reference_str1 as requested by the code.
      NOTE: It is convenient to process all the stretched images at different vacuum levels at once as indicated in this protocol; all the files must be stored in the same experimental folder but the code only requests to open the first stretched image. Image names should finish with strX names, X being incremental numbers 1, 2, 3, etc, as indicated in Point 6.4.9 of this chapter.
   8. Verify that the results folder appears with the 4 subfolders: Croppeddata containing the cropped and aligned data; Displacements containing the bead displacement file; Images containing the plot of the radial displacements and strain; Strains containing the spreadsheet of the strain matrix obtained for each vacuum level.
      1. Calculate the median of the strain matrix for each vacuum level.
      2. Repeat for each sample (3 minimum).
      3. Plot the median strain matrix versus the vacuum level for the 3 samples to obtain the calibration curve (Figure 3C).
         NOTE: A few parameters can be modified in case the bead image is not properly processed from the code. To do this, open the file set_settings_stretch in Matlab and modify Settings.Resolution and Settings.Overlap according to recommendations in code comments (also see Table 3). Depending on the experimental needs, the strain applied to the biological sample can be varied (cyclic strain, larger strain, etc): a calibration of these conditions is recommended (See examples in Supplementary Figure 2 and Supplementary Figure 3).
6. Resolution assessment (follow the protocol by Cole et al.¹⁹)
   1. Mount the stretch ring prepared in step 6.2.
   2. Image a field of view containing single bead dots (image a stack with Z-step distance relevant to the objective used).
   3. Use MetroloJ (Fiji plugin), as indicated in Cole et al., to characterize the X-Y-Z resolution of the stretch setup (Figure 3D and Supplementary Figure 4).

7. Cell seeding and stretching

1. Coating of the PDMS with cell adhesion protein
   1. Sterilize the PDMS ring by exposing it to UV light for 15 min (Figure 4A). Perform all subsequent steps of the protocol under the biohood to ensure sterility.
   2. Rinse the plate under the hood with sterile PBS.
   3. Incubate a cell-adhesion protein^11,14,15 by depositing a 100-200 µL drop of the solution in the center of the ring (Figure 4A).
      NOTE: Commonly, a solution of 10 µg/mL of fibronectin is appropriate¹¹. Other adhesion proteins of interest can be used to coat the PDMS (e.g., laminin, keratin¹⁵; specific treatments/protocols might be required). Incubation for fibronectin coating is recommended overnight at 4 °C; the alternative method is 1 h at 37 °C.
   4. Draw the perimeter of the drop with a permanent marker from the bottom side of the PDMS membrane. This helps locate the sample in the following steps.
2. Cell seeding on the PDMS ring (Figure 4A).
   1. If needed, transfect the cells of interest with a fluorescent protein previous to the stretching experiment (for transient transfections, 1-3 days before the experiment, depending on the transfection method and characteristics) or use specific protocols to visualize the fluorescent markers of interest.
      NOTE: For initial experiments, it is convenient to use a plasma membrane marker such as GFP-mem^11,12 to visualize cell membrane response and potential ruptures. The marker is easy to transfect, and it is advised to stretch the transfected cells at increasing strain levels to assess the strain threshold that will lead to plasma membrane rupture. See the plasmid map in Supplementary Folder 5. This depends on the cell type and will be lower in single-cell experiments compared with cell monolayers. This membrane marker is compatible with cell stretching, contrary to some membrane-inserting dyes that were observed to lead to plasma membrane rupture at low stretch levels.
   2. On the day of the experiment, warm cell media and prepare CO₂-independent media.
      NOTE: If the microscope has CO₂ control compatible with the stretch system, the experiments can be performed in regular cell culture media (this would be a necessary setup for delicate samples that cannot survive in CO₂-independent media). CO₂-independent supplemented with an anti-bleaching agent is recommended (e.g., for GFP proteins, rutin supplement²¹ at 10 mg/mL).
   3. Trypsinize the cells and count them.
   4. Seed an appropriate number of cells in a small volume (20-50 µL) with standard cell media to promote cell attachment and keep in a CO₂-humidity controlled incubator.
      NOTE: For flat and large fibroblasts and single-cell experiments, 5000 cells; numbers depend on the cell type and the experiment itself.
   5. After 20-30 min incubation (check initial cell attachment), gently add 500 µL media on top of the cells. Place back into the incubator to allow for further cell spreading.
      NOTE: The seeding time depends on the cell type and on the experimental requirements; the formation of a monolayer with overnight incubation is also possible. The volume of culturing media must be adjusted accordingly. Secondary cell staining: once attached, cells can be stained with a dye such as SpyDNA (see Table of Materials) to visualize the nuclei (Supplementary Figure 5) or another staining to visualize any structure of interest. Always beware that live-cell staining or protein over-expression by transfection can affect cell response to stretch, especially at the cell cortex, the plasma membrane, or mechanosensitive proteins/structures.
   6. Exchange to CO₂-independent media and immediately use in the stretching device.
      NOTE: Now, the sample is ready for a stretch experiment.
3. Cell stretching experiment: example of a single stretch-release cycle and imaging of plasma membrane (PM) to capture dynamical PM remodeling of fibroblasts.
   1. Mount the stretch ring on the microscope stage as in section 6, step 6.2.
   2. Find a cell of interest, record its position, and take an image of the pre-stretched cell (Figure 4B).
   3. Apply the desired vacuum that, according to the calibration curve, matches the desired substrate strain.
   4. To find the stretched cell, first, refocus in Z and then move in the X-Y direction to find and center the cell back to the field of view.
   5. Record images during the desired duration (Figure 4B).
      NOTE: Initial experiments are commonly needed to determine the cell response to tensile strain depending on its type (on average). Stretch the cells at different substrate strains to determine the maximum extensile load they can bear. Plasma membrane rupture will occur, as visualized through the plasma membrane fluorescent membrane marker, if the strain is excessive for the cell.
   6. For cell release, go back to the cell position at rest and turn the acquisition mode of the microscope ON to ensure that acquisition can start as soon as the sample has been refocused.
   7. Release the stretch and quickly refocus manually in Z.
   8. Record images of the released cell for the desired time (Figure 4B). Due to the flexible nature of the PDMS membrane, the sample easily defocuses with time. Therefore, manually adjust the focus throughout the image acquisition.
      NOTE: To successfully refocus during the imaging after release, images must be visualized often enough; a 3-5 min video imaging every 3 s is reasonable. The PDMS substrate is not compatible with the microscope's perfect focus system, so manual adjustment by the user for fast acquisition is usually required. Software focusing may be settled, but beware of possible errors and large stage movement that may damage the objective by hitting a stretch post part. The user can decide to perform multiple stretches of the sample depending on the experiment.
4. Post-processing: Align the images using Fiji
   1. Open the images at rest and strain.
   2. Use the Fiji plugin template matching and select a region of the cell to align (Figure 4C).
      NOTE: Relative displacements of the aligned strain image are rendered by Fiji, and images are cropped and aligned. Download the template_matching plugin from this link : https://sites.google.com/site/qingzongtseng/template-matching-ij-plugin

8. Cell seeding on a prestretched substrate

1. Mount the stretched ring coated with the adhesion protein in the stretching device.
2. Stretch the PDMS substrate to the desired strain level.
3. Seed the cells as in section 7.
   NOTE: In this experiment, the seeding is not performed in sterile conditions, so the protocol may not be appropriate for all cell types.
4. After cells have adhered, exchange the media to a CO₂-independent media.
   NOTE: Choose fast adhering cells to minimize the adhering time.
5. After a desired incubation time, release the stretch and image a cell of interest.
   NOTE: In this step up, finding a same cell after stretch release is difficult, as the cell position at rest is unknown, and the PDMS substrate movement is not easily predictable.

9. Fixing stretched or relaxed cells

1. Fix cells in stretched states (work under chemical hood)
   NOTE: Paraformaldehyde (PFA) is a standard fixation agent; use it warm at 37 °C to ensure fast fixation. Alternatively, glyoxal solution²² permits a quick sample fixation with good sample preservation.
   1. Prepare fixation agent.
   2. Seed cells on the rings following step 7.2.
   3. Mount the stretching post and controller inside of a chemical hood and connect to the vacuum supply.
   4. Place the ring with seeded cells in the stretching post.
   5. To fix the stretched state, stretch to the desired strain. Remove cell culture media and fix by adding the 4% PFA solution to the cells for 10 min at 37 °C. Then wash with PBS, always holding the stretch. Slowly release the vacuum and wash with more PBS.
      NOTE: Do not forget to prepare a non-stretch control state (for this, a small PDMS piece can be cut and dried with a small amount of 96% ethanol solution underneath, on top of a glass coverslip). To image the fixed sample, it is recommended to stretch the sample back to the same magnitude. As PFA is toxic, adequate precautions must be taken in this step. It is convenient to use a small oven that can be kept under the hood, see Table of Materials).
   6. Image the sample, preferably applying the vacuum again, to the same level used when fixing, which ensures sample "reflattening" (Figure 4D).
2. Fix cell after stretch-release (work under chemical hood).
   1. Stretch the cells to the desired strain and time. Just before stretch release, remove most of the media without completely drying the sample and release the strain. Alternatively, apply a cyclic stretch of a specific duration.
   2. Quickly remove media and immediately fix with the fixative solution for 10 min. Wash with PBS and image.
      NOTE: The sample can also be seeded on a pre-stretched substrate and, after a user-defined seeding time, fixed post-stretch release as in step 9.2.2.
3. Immunofluorescence of fixed sample: follow standard protocols for immunofluorescence on either stretched or released; blocking with Fish Gelatin is preferred to BSA¹⁷.
4. Image the sample (Figure 4E).
5. Sample preservation
   1. Prepare mounting media for sample preservation (Mowiol or Prolong).
   2. Remove the PBS and add a drop (about 75 µL) of mounting media.
   3. Grab a coverslip with tweezers and delicately place it on top (avoid bubbles, which would hamper visualization).
   4. Dry for at least 48 h at room temperature (RT).
   5. Cut around the PDMS fixed sample and mount on another glass coverslip for later imaging.
      NOTE: This step is not compatible with post-imaging of the sample on the stretched membrane as mounting the PDMS membrane on a coverslip with the preservation media involves preservation of the membrane in its relaxed state only.

10. Fixation with the grid for imaging the same sample area in different modes - SEM imaging as an example

NOTE: This protocol can be used to image the same single cell in two different microscopes, fluorescence microscopy and scanning electron microscopy, as presented here. Correlative imaging requires advanced image processing that is not in the scope of this protocol. Other useful applications can be, for instance, visualization of the sample in live mode, further fixation under the hood, and visualization of the same cell after immunostaining (the protocol used for Supplementary Figure 5).

1. Prepare a stretch ring using the patterned PMMA plates from section 4.
2. Seed the cells as in step 7.2.
   NOTE: Beware that the coating solution must be carefully applied to ensure complete coverage of the PDMS, even in the small grid holes.
3. Fix as described in section 8 but using appropriate fixative and protocol compatible with the sample and electron microscopy imaging.
   NOTE: If using a mix of PFA + glutaraldehyde (e.g., 2%-2.5%), beware of autofluorescence coming from the presence of glutaraldehyde reagent and eventually perform a quenching step²³.
4. Image the sample in fluorescence mode and record the cell position (print the grid and note the position with a cross, for instance).
5. Once all images are taken, prepare the sample for EM by first cutting the PDMS around the sample (previously removing the lubricant).
6. Process the PDMS membrane + sample for scanning electron microscopy (which involves standard protocol of sample dehydration and metal sputtering)¹².
7. When imaging in the SEM microscope, find the grid positions and look for the cells previously imaged in fluorescent mode (Figure 4F,G).

Results

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...

Discussion

A large number of stretching devices have been used to apply mechanical stretch to biological samples, mimicking physiological conditions in the body^24,25. 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 microscopy^27,11.

The desi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetate mask	JD Photodata
Acrylamide	Biorad	161-0140	40% Acrylamide solution
APS	Sigma Aldrich	A3678	Ammonium persulfate for electrophoresis, ≥98%
Aptes	Sigma Aldrich	281778	(3-Aminopropyl)triethoxysilane
Autonomous microfluidic pump	Elveflow Cobalt	Cobalt Dual	Cobalt Dual
Bisacrylamide	Biorad	161-0142	2% Bis Solution
Blade	Fisher Scientific	11733919	Didactic Group Hoja didáctica X100 LAME BISTOURI STERILE N°23  LAMD23
Clips	Sprintis	AKXX000001	Bull Dog Clips, 32 mm, made of metal, capacity 10 mm, nickel-plated
Corona treater	Electrotechnicproducts	BD-20AC, SKU:12051A	BD-20AC LABORATORY CORONA TREATER
Cups	VWR	611-1368	VWR, Beaker Cups, Disposable, 250 mL
DAPI	Roche	10236276001	DAPI staining
Ethanol 96%	Panreac Química, SLU	131085-1212	Etanol 96% v/v para análisis, ACS- 2,5 L
Fibronectin	Sigma	F0895	Fibronectin, From Human plasma, liquid  0.1%
FluoSpheres Carboxylate-Modified Microspheres, 0.1 µm	Life Technology	F8801, F8803	Yellow green and Red fluorescent beads for resolution assessment
FluoSpheres Carboxylate-Modified Microspheres, 0.2 µm	Fisher Scientific	F8809, F8810	Yellow green and Red fluorescent beads for strain calibration
Glutaraldehyde	Sigma Aldrich	G6403	Glutaraldehyde solution 50% in H2O
Interferometer	Veeco	Wyko NT1100	Interferometer to measure PDMS membranes thicknesses
Isopropanol	Panreac Química, SLU	131090-1212	2-Propanol (Reag. Ph. Eur.) para análisis, ACS, ISO
Laiton weighs			Custom made
Lint free tissues	Berkshire	DURX670, DR670090920	Nonwoven-wipes
Lubricant (vaseline)	Sigma	16415	Vaseline
Mask Aligner	Süss MicroTec	MJB4	Mask aligner for photolithography on PMMA plates
Mowiol	Calbiochem - Merck	475904	MOWIOL 4-88 Reagent
NIKON microscope spinning disk confocal	Nikon	Eclipse Ti-E/B	equipped with Nikon Ti-S-ER  stage
NIKON Microscope Upright	Nikon	Eclipse Ni-E	equipped with Nikon NI-SH-E stage
Objective 60x	Nikon	CFI Apo NIR 60X W	NIR Apo 60X/1.0w DIC N2 WD 2.8
PBS solution	Gibco	14200067	DPBS, 10x
PDMS	Dow Corning	2401673921	Sylgard - 184 Silicone Elastomer KIT DN 109
Petri dish ring cover (big)	Proquinorte	93060	Plato de cultivo celular TPP SKU: 07177925 diametro 100
Petri dish ring cover (small)	Proquinorte	93060	Plato de cultivo celular TPP SKU: 07177924
Plasma cleaner	Harrick	PDC-002	Oxygen Plasma Generator
PMMA plates and rings			see excel specification table in supplementary documents
Prolong	Invitrogen	P36930 /P36934	ProLong Gold antifade reagent
Repel-Silane	GE Healthcare	17-1332-01	PlusOne Repel-Silane ES
Rings /rubber ring	Tecnifil		Custom made
Spin coater	Laurel	WS-650Sz	Spin coater
SPY650-DNA	Spirochrome	SC 501	SPY650-DNA
Steering spoon	VWR	231-0416	Eco short disposable spatula, double-ended spoon and scoop, PP and EcoPure additive
SU8 resin 2100	Microchem	Y111075	SU8-2100
SU8-Developer	Sigma-Aldrich	484431	Propylene Glycol Monomethyl Ether acetate
TEMED	Sigma-Aldrich	T9281-50ML	N,N,N',N'-Tetramethylethylenediamine, Bioreagent suitab. Electrophoresis reagent
Trichloro(1H,1H,2H,2H-perfluorooctyl)silane	Sigma Aldrich	448931	Trichloro(1H,1H,2H,2H-perfluorooctyl)silane
Tweezers	IESMAT	T5039	Dumont HP Tweezers 7 Stainless Steel. 0.17 x 0.10mm tip.(Agar Scientific Ltd)
Vacuum controller	GARCAP/Parker	990-005203-005	Controlador de vacio
Vacuum controller fluigent	Fluigent	LU-FEZ-N800	LineUp Flow EZ (n800mbar)
White bench paper	Raima	931.1001	Paper Raima 42 x 52