A micromechanical cell stretching device compatible with super-resolution microscopy and single protein tracking

Cell mechano-sensing is based on biomolecule deformations and reorganizations, yet the molecular mechanisms are still unclear. Super-resolution microscopy (SRM) and single protein tracking (SPT) techniques reveal the dynamic organization of proteins at the nanoscale. In parallel, stretchable substrates are used to investigate cellular responses to mechanical forces. However, simultaneous combination of SRM/SPT and cell stretching has never been achieved. Here, we present a cell stretching device compatible with SRM and SPT, composed of an ultra-thin Polydimethylsiloxane (PDMS) layer. The PDMS sheet is gliding on a glycerol-lubricated glass cover-slip to ensure atness during uniaxial stretching, generated with a 3D-printed micromechanical device by a mobile arm connected to a piezoelectric translator. This method enables to obtain super-resolved images of protein reorganization after live stretching, and to monitor single protein deformation and recruitment inside mechanosensitive structures upon stretching. This protocol is related to the publication ‘Cell stretching is amplied by active actin remodeling to deform and recruit proteins in mechanosensitive structures’, in Nature Cell Biology.


Introduction
Growing evidence demonstrates that macromolecular assemblies driving critical cellular functions are regulated by mechanical forces. However, the exact molecular mechanisms of force-sensing within most macromolecular assemblies in cells are still unknown. Several innovative techniques allow to measure and generate forces on proteins in vitro or within cells [1][2][3][4] , but they cannot probe protein mechanical responses within crowded macromolecular structures inside the cell or con ned at the interface with the extracellular environment.
In parallel, stretchable substrates of Polydimethylsiloxane (PDMS) have been coupled with optical imaging to investigate cell responses to external forces 1,11,12 . However, the simultaneous combination of SRM or SPT with cell stretching is extremely challenging, since it requires to combine glass-like optical properties with mechanical stability of the imaged plane during substrate deformation.
SMLM/SPT require the optimal signal to noise ratio of single molecules emission to attain the best spatial resolution (typically 10-50 nm) [5][6][7] . SMLM/SPT techniques are thus ideally performed in the total internal re ection uorescence (TIRF) or oblique illumination modes using high numerical aperture (NA) short working distance oil immersion objectives matching the index of refraction of glass slides [5][6][7]13,14 .
In addition, SMLM/SPT techniques rely on object reconstruction or tracking from thousands of imaging planes, which implies perfect mechanical stability of the sample while imaging. This is incompatible with large deformations and displacements of the substrate in the axis (Z) and plane (XY) of observation during stretching.
Similarly, coordinate targeted STED/RESOLFT nanoscopy techniques perform better using high numerical aperture (NA) short working distance oil immersion or glycerol immersion objectives. Compared to SMLM/SPT techniques, STED like techniques will be less sensitive to drift, as they have low pixel dwell times 15,16 . However, mechanical drift stemming from multiple factors (e.g., motorized and piezoelectric stages) can greatly compromise the performance of the STED system, degrading signal-tonoise ratio and spatial resolution 15,16 . Thus, perfect mechanical stability of the sample has to be ensured throughout acquisitions. Once again, this could be incompatible with large XYZ deformations during stretching.
In most commercial con gurations, cell stretching is performed using macroscopic devices and images are acquired after xation, or thick elastomeric substrates are stretched in combination with low NA objectives and upright microscopes 17,18 . These low magni cation con gurations are quite permissive to slight defocusing 19 . Various custom-made devices could potentially enable simultaneous stretching and live cell imaging 1,11,[19][20][21][22] . However, they are either limited to low-magni cation imaging or are incompatible with continuous automatic focusing during stretching Here, we present a cell stretching device compatible with SRM and SPT which enables to study the nanoscale reorganizations and deformations of protein assembly or individual proteins inside mechanosensitive structures 23 (Fig. 1a). This device is composed of an ultra-thin Polydimethylsiloxane (PDMS) layer (10 µm) providing glass-like optical properties compatible with SRM and SPT (Fig. 1b,c). To simultaneously enable substrate stretching and ensure atness upon deformation, this ultra-thin PDMS layer is deposited on a glycerol-lubricated glass cover-slip (Fig. 1b,c). Glycerol allows the deformable substrate to oat freely while avoiding PDMS adhesion and refractive index mismatch. A drop of glycerol is sandwiched between the plasma cleaned PDMS sheet and glass coverslip forming a glycerol layer of ∼ 0.7 µm in thickness (Fig. 1c) To manipulate the thin PDMS substrate and avoid any distortions along the optical path, the size of the observation chamber is kept as small as possible (∼9 mm2 (Fig. 1b)), and its mechanical stability is reinforced by adding a thicker (40 µm) Gel-Pak frame on top of the thin PDMS sheet (Fig. 1c). To generate uniaxial stretches on the glass-PDMS assembly, we design a 3D-printed micro-device (Fig. 1a,b). The micromechanical device consists of a xed (holding) arm and a mobile (stretching) arm, connected to a piezo motor, positioned on opposite sides of the observation chamber on the PDMS frame (Fig. 1a,b). A clamp xes the glass-PDMS slide to the base of the device and to the holding arm (Fig. 1a). The observation chamber or the whole microchip can be lled with culture or observation medium.
Concerning the rst approach, we can perform SPT acquisitions in cells before and after large (10-50%) or small (2-5%) stretches to study the effect of external stress on protein dynamics and diffusive properties (Fig. 2a). In addition, we can also perform simultaneous live cell stretching (2-5%) and SPT to study acute mechanical response of individual proteins: 1) force-dependent protein unfolding or deformations (Fig. 2b); or 2) force-dependent protein recruitments and reorganizations. Finally, the device can also be used to acquire STED SRM images of live cells that experience stretching (Fig. 2c). Regarding the second approach, the device can be used to perform SRM (e.g. DNA-PAINT, STED, STORM) in xed cells after live stretching (2-50 %) followed by rapid xation and labelling for SRM (Fig. 2d, Assembling the micromechanical device Glass-PDMS assembly 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 xed (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 xation, the design of the holding arm is modi ed 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 les (.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 nal 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 nishing the printing, wash and cure the pieces according to the requirements of the resin. 3Dprinted 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) laments 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 preparation
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 asks. Mounting the stretching device for live stretching or large stretches followed by rapid xation The following steps are common for mounting and preparing the micromechanical device for stretching experiments in live cells or xed cells combined with super resolution microscopy and single protein tracking. 7. Before acquiring any cells, test whether the device is working properly and de ne parameters for speci c stretching percentages. For that, select a region with a good density of uorescent beads and acquire a snapshot. Measure the distance between two beads in the same horizontal axis (D before ). 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 (D after ). If D after is larger than D before , 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 uorescence 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.

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 twodimensional (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 eld 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 xed cells with large stretches
The following steps are required to perform large and sustained stretching followed by rapid cell xation.
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 x the cells in warm 4% PFA with 0.25% Glutaraldehyde.
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 xed cells with large stretches
Cells are imaged at 25°C in Buffer C in the same microscope used for live sptPALM.

Cell preparation
If the device is well coated and cells are viable after electroporation, detachment and seeding, they should start to spread brie y after seeding. 1h later, spread cells should be visible in the observation chamber of 3x3 µm.
4. Preparing the stretching device for live stretching or large stretches followed by rapid xation Bead incubation can be quickly con rmed using GFP lter cube. Successful stretching and viability of the device can be con rmed by performing a test stretch and assessing that the same two beads are more distant between each other after stretching. If the device is assembled correctly, stretches closer to the holding arm will produce smaller displacements.
5. Live cell stretching combined with super-resolution microscopy and single protein tracking Successful combination of live sptPALM or STED with stretching is achieved if 1) focus is maintained throughout stretching, 2) Live XYZ repositioning is possible throughout stretching and 3) quality of either the single-molecule signal or the STED effect is assured throughout the entire acquisition.

Super-resolution microscopy in xed cells with large stretches
Sustaining the stretching after xation can be veri ed immediately after xation by assessing morphology of the 3x3 chamber on a bench top microscope. If the chamber remains deformed, stretching is maintained. This can also be con rmed when imaging the sample after labelling, by re-measuring the distance between the same two beads used to test the stretching before xation. For DNA-PAINT acquisitions to be successful, drift correction is a key aspect and has to be ensured.