Fabricating a 10 µm PDMS elastic substrate
1. Using the Graphtec Cutting Plotter, pre-cut an elastomer Gel-Pak frame (40 µm) to the size of the glass coverslip with a squared (3x3 mm) observation chamber.
a. Note: Layout for cutting can be drawn on the software provided by the company or using a plug-in for Adobe Illustrator® and CorelDraw®.
2. Mix PDMS and curing agent in a 10:1 ratio.
3. Centrifuge for 5 min at 2500 rpm to remove air bubbles
4. Spin the PDMS on a silicon wafer to a final thickness of 10 µm.
5. Pre-cure the PDMS for 25 min at 70°C
6. Place the pre-cut Gel-Pak frame in contact with the 10 µm PDMS
7. Curate the whole assembly overnight at 70°C
8. Cut around the Gel-Pak frame with a blunt pair of and gently detach the PDMS substrate from the wafer.
Assembling the micromechanical device
1. Plasma clean 24x40 mm glass coverslips.
2. Spin coat each slide with glycerol to form a uniform glycerol layer of ∼0.7 µm thickness.
3. Plasma clean the PDMS substrate for 1 minute, on the side that will glide on the glycerol layer.
4. Immediately after, lay the PDMS substrate onto the glycerol-coated coverslip.
Printing the 3D micro-device
1. Design the fixed (holding) arm, the mobile (stretching) arm and the clamp composing the device on a CAD software.
a. Note: For experiments that require sustained stretching after fixation, the design of the holding arm is modified to include a threaded hole, while the stretching arm is enlarged to include two grooves. Two screws allow to clamp the stretching arm onto the holding arm and sustain the stretching.
2. Export the files (.obj or any 3D printing compatible format) and upload them onto PreForm software. Layer thickness should be intermediate or small to avoid imperfections in the final structure. As for the resin, we recommend using Grey Pro resin because it offers high precision, moderate elongation, and low creep (https://formlabs.com/eu/materials/engineering/#grey-pro-resinGrey Pro Resin). This material is ideal for concept modeling and functional prototyping, especially for parts that will be handled repeatedly.
a. Note: Ensure that the support material is correctly placed to avoid collapsing of the piece during the printing process.
3. After finishing the printing, wash and cure the pieces according to the requirements of the resin. 3D-printed micro devices can be re-used several times, especially if they are composed of a resin conceived for engineering.
a. Note: Although we use SLA 3D printing for our micro-devices, we have also tested 3D micro-devices printed with fused deposition modelling (FDM). It requires polylactic acid (PLA) filaments and it can be easily implemented in a lab, besides having shorter printing times. However, we found that the devices are less resistant and less durable than the ones printed with SLA.
Attaching glass-PDMS assembly to 3D-printed micro-device
1. Cut glass coverslips in small parts with high precision knife.
2. Using superglue or Dow Corning™ High-Vacuum Grease, attach small glass parts to holding and stretching arm.
3. Stick double sided tape to the glass parts on the holding and stretching arm.
4. Place the glass-PDMS assembly inside the clamp and attach them to the holding arm
5. Attach the stretching arm to the glass-PDMS assembly using double-sided tape.
Cell electroporation (24 to 48h before imaging)
Actively dividing immortalized MEFs are cultured in DMEM supplemented medium (see solutions for cell culture) in 75 cm2 flasks.
1. Detach cells with trypsin/EDTA solution (1.5 mL). Inactivate trypsin immediately after detachment by adding serum-containing DMEM (5 ml). Count cells and adjust cell suspension volume to keep 1–2 million cells per tube per experimental condition.
2. Centrifuge cells at 300 × g for 5 min
3. Resuspend the cell pellet in transfection reagent and mix with the DNA plasmids.
a. Note: For sptPALM experiments, cells are usually co-transfected with DNAs encoding for the protein of interest, (3–5 μg per condition, e.g., Talin-C-tdEos), and for a GFP-coupled reporter of the structure of interest (1–2 μg per condition, e.g., GFP-paxillin for adhesive structures).
b. Note: For STED experiments, if necessary, cells are transfected with a GFP-coupled reporter of the structure of interest (1–2 μg per condition, e.g., tubulin-GFP for microtubules). Presence of a GFP-coupled reporter is only required for low-resolution images of the cells before and after stretching, especially when performing large stretches (e.g. , 30%)
c. Note: For DNA-PAINT microscopy, vimentin-Halo can be visualized with Cy3B-labelled DNA imager strands, added to the stretching chamber at variable concentrations (2-5 nM), as previously described 24.
4. For all cases, electroporate the cells with the Nucleofector™ 2b Device using the MEF T-020 program (Lonza Nucleofactor protocol)
5. After electroporation, replate the cells in a 6-well plate (about 0.3 million cells/well) in preheated culture medium and place them in a 37 °C incubator with humidified air containing 5% CO2
Coating the micromechanical stretching device (same day of the experiment)
1. Cover the micromechanical device with 10 μg/ml fibronectin solution (500-750 µl per device) and incubate at 37 °C for 1 to 1.5 hours.
2. Discard the fibronectin solution and wash 4-5 times with PBS.
1. Wash cells twice with PBS after removing the culture medium
2. Incubate with trypsin–EDTA solution (0.3 ml per well) for 1–3 min at 37 °C for detaching cells.
3. Inactivate trypsin with trypsin inactivation medium (1 ml per well) and count the cells (use any conventional cell counting method).
4. Centrifuge at 300 ×g for 5 min and resuspend cells in warm Ringer medium (1–2 ml). Allow cells to recover for 20-30 min inside the incubator at 37 °C with 5% CO2
5. Spread the cells on the device under a density of 70000-80000 cells per device.
Mounting the stretching device for live stretching or large stretches followed by rapid fixation
The following steps are common for mounting and preparing the micromechanical device for stretching experiments in live cells or fixed cells combined with super resolution microscopy and single protein tracking.
1. Prepare a 1:500 solution of TetraSpeck™ 0.1 µm fluorescent beads in warm Ringer solution and add 200-300 µL to each device to adsorb fluorescent beads on the substrate.
2. Mount the piezoelectric motor on a custom holder adapted to the microscope stage. Then, mount the holder on the microscope.
a. Note: Custom-made holders should be designed according to specific stage dimensions and can be either 3D-printed or 3D-milled. In this case, we have 3D-milled a PMMA holder for mounting our device on the motorized stage, since 3D-milled parts are often more resistant.
3. Connect the piezoelectric motor controller and launch the PI Mikro Motor software.
4. Mount the micromechanical device on the stage-adapted holder.
5. Attach the stretching arm to the linear stage with a screw
6. Adjust focus to the 3x3 mm observation 10 µm PDMS chamber.
7. Before acquiring any cells, test whether the device is working properly and define parameters for specific stretching percentages. For that, select a region with a good density of fluorescent beads and acquire a snapshot. Measure the distance between two beads in the same horizontal axis (Dbefore). Apply a test stretching by displacing the linear stage of the piezoelectric motor while keeping the same region in focus, either manually or with a custom-written Metamorph routine. Take another snapshot after the stretching and measure again the distance between the same two beads (Dafter). If Dafter is larger than Dbefore, then the device is working properly. After that, cell stretching can be performed.
a. Note: Stretching percentage is calculated by
, a formula which can be applied for all stretching experiments in order to determine stretching percentage.
b. Note: By knowing the displacement and the percentage for the test stretching, it is possible to determine with reasonable precision the necessary displacement to obtain a desired stretching percentage when performing actual cell stretching.
c. Note: Step size and speed of motor displacement should be kept consistent throughout all acquisitions.
Live cell stretching combined with super-resolution microscopy and single protein tracking
Stretching and live sptPALM
Cells are imaged at 37°C in the micromechanical device. Here, an inverted motorized microscope (Nikon Ti) was used, equipped with a CFI Apo TIRF 100x oil, NA 1.49 objective and a perfect focus system PFS-2), allowing long acquisition in TIRF illumination mode. For photoactivation localization microscopy, cells expressing mEos2/tdEos tagged constructs were photoactivated using a 405 nm laser (Omicron) and the resulting photoconverted single molecule fluorescence was excited with a 561 nm laser (Cobolt Jive). Both lasers illuminated the sample simultaneously. Their respective power was adjusted to keep the number of the stochastically activated molecules constant and well separated during the acquisition. Fluorescence was collected by the combination of a dichroic and emission filters (dichroic: Di01-R561, emission: FF01-617/73, Semrock) and a sensitive EMCCD (electron-multiplying charge-coupled device, Evolve, Photometric). The acquisition was steered by Metamorph software (Molecular Devices) in streaming mode at 50 Hz. GFP-paxillin was imaged using a conventional GFP filter cube (excitation: FF01-472/30, dichroic: FF-495Di02, emission: FF02-520/28, Semrock).
1. For simultaneous stretching and sptPALM with trapeze like patterns: select a cell and launch a PALM acquisition at high frequency (50 Hz) for the entire field of observation. The duration of the acquisition should comprise the entire trapeze pattern (at least 2500 frames).
2. Several hundred frames after, stretch by displacing the linear stage of the piezoelectric motor. Maintain the cell in the field of observation by compensating for XY displacements using manual repositioning (Nikon stage steered by a joystick) or automated stage repositioning (custom plugin developed in Metamorph). After the stretching has stopped, allow the plateau phase to last for 8-12 seconds before relaxation.
a. Note: When looking at focal adhesions (FAs), select cells with most of their FAs aligned almost parallel to the stretching axis in the field of observation.
b. Note: Imaging cells closer to the holding arm requires smaller XY repositioning while allowing to reach 6 % stretching.
1. For sequential large stretching and sptPALM (Before vs After): select a cell and, after acquiring an image of the GFP reporter, launch a PALM acquisition at high frequency (50 Hz) for the entire field of observation throughout 4000 frames.
2. Perform large stretching (10-50%) while following the cell displacement with the combination of a dichroic and emission filters (dichroic: Di01-R561, emission: FF01-617/73, Semrock). After stretching is finished, acquire an image of the GFP reporter (to have a perspective of the morphological changes) and launch another sptPALM acquisition.
Stretching and live STED
Cells are imaged at 37°C in the micromechanical device. Here, an inverted confocal microscope (Leica SP8 WLL2) was used, equipped with a HC PL APO CS2 motCORR 93X Glycerol, NA 1.3 objective. The confocal microscope was equipped of a white light laser 2 (WLL2) with freely tuneable excitation from 470 to 670 nm (1 nm steps). Scanning was done using a conventional scanner (10Hz to 1800 Hz). The confocal microscope was equipped with the STED module tunable to STED microscopy. A two-dimensional (2D) STED donut was generated using a vortex phase plate. This STED microscope was equipped with 3 depletion lasers: 592 nm, 660 nm and 775 nm For STED microscopy, cells were imaged with a combination of a WLL2 laser and a 775 nm depletion laser. Fluorescence was collected with an internal hybrid detector. The acquisition was steered by LASX Software (Leica).
1. Perform live labelling of the target protein on the micromechanical device. For actin or tubulin labelling, use SiR-Actin or SiR-Tubulin compounds, according to previous studies and manufacturer’s instructions 25.
2. After the labelling, wash the staining solution and incubate the device in warm Ringer solution until the experiment.
3. After bead incubation and mounting of the sample as previously described, select a cell and acquire a confocal image followed by a STED image on a sub region of the cell (pixel size has to be inferior to 20 µm).
4. Stretch the cell according to the desired percentage (e.g. 4 or 30%) and maintain the cell on the field of observation by observation by compensating for XY displacements using manual repositioning (Leica stage steered by a joystick)
5. After stretching, acquire a new confocal image followed by a STED image for the same sub region.
Super-resolution microscopy in fixed cells with large stretches
The following steps are required to perform large and sustained stretching followed by rapid cell fixation.
1. Warm 4% PFA with 0.25% Glutaraldehyde at 37°C.
2. After mounting the micromechanical device on the microscope, acquire several low resolution images of GFP markers for different cells.
3. Remove the entire module (device and motor in the stage-adapted holder) and stretch the cells outside the microscope. Immediately after stretching, remove the Ringer solution and fix the cells in warm 4% PFA with 0.25% Glutaraldehyde.
4. After fixation, rinse 3-4X with PBS.
5. Clamp the stretching arm to the holding arm using the thread and groove system and two M3 screws. Afterwards, remove the screw that connects the stretching arm to the motor. With this, stretching is sustained throughout all the subsequent labelling steps and super-resolution imaging.
6. Label target proteins according to the imaging technique.
7. After labelling, and before performing super-resolution imaging, acquire again several low resolution images of GFP markers for the same cells.
DNA- PAINT on fixed cells with large stretches
Cells are imaged at 25°C in Buffer C in the same microscope used for live sptPALM. Cy3B-labelled strands were visualized with a 561 nm laser (Cobolt Jive). Fluorescence was collected by the combination of a dichroic and emission filters (dichroic: Di01-R561, emission: FF01-617/73, Semrock) and a sensitive sCMOS (scientific CMOS, ORCA-Flash4.0, Hammatasu). The acquisition was steered by Metamorph software (Molecular Devices) in streaming mode at 6.7 Hz. Vimentin-GFP was imaged using a conventional GFP filter cube (excitation: FF01-472/30, dichroic: FF-495Di02, emission: FF02-520/28, Semrock). Super-resolution DNA-PAINT reconstruction and drift correction were carried out as described before, using the software package Picasso 24.
1. Dilute the desired imager strand in Buffer C. For instance, Vimentin-Halo was visualized with Cy3B-labelled DNA imager strands.
2. Incubate the micromechanical device with 200-300 µL of 90 nm gold nanoparticles, diluted in PBS with a 1:5 ratio, for 15 min at RT. Gold nanoparticles serve as fiducial markers
3. Wash 3X with PBS and 1X with Buffer C.
4. Add imager strands to the stretching chamber until reaching the ideal density of blinking events. Vimentin-Halo was visualized with Cy3B-labelled DNA imager strands, added to the stretching chamber at variable concentrations (2-5 nM), as previously described 24.
STED on fixed cells with large stretches
Cells are imaged at 25°C in PBS 1X in the same microscope used for live STED, with the same combination of a WLL2 laser and a 775 nm depletion laser.