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

We study how cells respond and adapt to mechanical stimuli in physiological and pathological context. For this, we use the stretching device to apply external forces, a strain in this case, to cells.

We have found how cells and cell tissues react to specific mechanical stimuli and how they mechanically respond to other biological cues.

Our stretching device is very versatile compared to others as it offers the possibility to stretch cells cultured on polyacrylamide gels of different stiffnesses or to perform compression experiments.

[Narrator] To begin, place the PMMA rings on a sheet of white bench paper. Carefully go around the edge of the PDMS-coated plate using a blade to ensure a clean cut along the entire circumference. Use tweezers to lift the membrane at one edge, then gently peel it with the thumb to evenly detach half of the membrane from the plate. Use fingers to spread 96% ethanol evenly around the entire top surface of the PMMA ring. Carefully remove the PDMS membrane from the plate by lifting it from the previously lifted edge. Then, place it symmetrically onto the ethanol-covered PMMA ring, ensuring no wrinkles while avoiding stretching. Then, take another empty PMMA ring and place it on top of the mounting ring. Use metal clips to seal them together, sandwiching the PDMS membrane between the two rings. Position the bottom half of the stretch ring onto the mounting support. Place the PDMS ring sandwiched onto the stretch ring and push it down firmly until fully seated, ensuring the gasket is correctly positioned. Place the circular brass weights on top of the sample. Next, place the top half of the metal stretch ring on top, aligning the screw holes. Insert all screws and tighten them. Cut the PDMS around the ring gently using a blade. Cover the assembly with a Petri dish to protect the membrane from dust. Prepare one 250,000 dilution of 0.2 micrometer fluorescent beads in PBS using serial dilution. And place a 10 to 30 microliter drop of the bead solution at the center of the PDMS. Allow it to dry at a temperature no higher than 40 degrees Celsius to avoid post curing confounding issues. Now, mount the stretching device onto the microscope stage holder and secure the stretching post onto the microscope. Generously apply lubricant to the circular area of the post. Mount the prepared stretch ring onto the stretching post and screw the top cap to seal the assembly. Then, connect the rear of the vacuum control box to the vacuum source, followed by the front of the box to the stretch post. To perform strain calibration, choose a field of interest containing a high bead density and recognizable bead clumps for the user. Acquire an image at rest of the selected field of view and save the position. Apply stretch at a low vacuum level of negative 20 millibar, causing movement in all directions. Now, refocus in the Z plane and move the stage in XY directions to locate the same region of interest and acquire an image in the stretched state. Then, release the strain and repeat the steps with a higher vacuum level. Store acquired images in a folder organized by sample. Name, the reference bead image acquired without stretch as reference_str1 and the images stretched at increasing vacuum levels as stretched_str1, stretched_str2, stretched_str3, and so on. To quantify the strain, open MATLAB and load the gel strain code. After setting the parameters, define the parent, as well as the experiment folder paths and run the code. Then, open stretched_str1 as requested by the code, followed by the reference_str1. Now, calculate the median strain matrix for each vacuum level. After repeating this process for at least three samples, plot the median strain matrix versus vacuum level to obtain the calibration curve. To coat the PDMS ring, sterilize it under ultraviolet light for 15 minutes. Under a biosafety cabinet, rinse the plate under the hood with sterile PBS. After aspirating all the PBS used to rinse the plate. Deposit a 100 to 200 microliter drop of the solution in the center of the ring. Draw the perimeter of the drop with a permanent marker from the bottom side of the PDMS membrane and incubate it. On the day of the experiment, wash the adhesion protein with PBS. After trypsinizing the cells, proceed to cell counting. Seed an appropriate number of cells in a small volume of about 20 to 50 microliters with standard cell media to promote attachment and incubate it in a carbon dioxide incubator with humidity control. After 20 to 30 minutes, gently add 500 microliters of media on top of the cells and return the plate to the incubator to allow for further cell spreading. For a single stretch release cycle and imaging, mount the stretch ring onto the microscope stage as demonstrated earlier. After locating a cell of interest, record its position and capture an image of the pre-stretched cell. Apply the desired vacuum level corresponding to the required substrate strain based on the calibration curve to stretch the cell. To locate the stretch cell, first, refocus in the Z plane. Then, move in the XY directions to center the cell in the field of view. Record images during the desired duration. Navigate to the cell's original position and activate the microscope's acquisition mode to ensure that image capture begins immediately upon refocusing. Release the stretch and quickly refocus manually in the Z plane. Capture images of the released cell for the required time, manually adjusting the focus throughout the acquisition. For post-processing, open Fiji and load the images acquired at rest and during strain. Use the Fiji plugin template matching to select a region of the cell for alignment. Calibration ensured reproducibility and control over applied strain, confirming a nearly linear relationship between vacuum and strain up to 20%. Radial displacement analysis demonstrated isotropy and homogeneity of strain over the PDMS membrane. Fluorescence imaging of fibroblast cells under live cell stretching revealed membrane invaginations upon stretch release, which reabsorbed within three to five minutes. Restretching of fixed cells enabled imaging in a stretched state, preventing artificial folding artifacts. PDMS membrane with a patterned grid facilitated multimodal imaging, allowing localization of the same cells in fluorescence and scanning electron microscopy. Cells seated on soft PAA gels coated with fibronectin formed focal adhesions only when submitted to specific cyclic stretch cycles compared with non-stretched cells.