Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

This protocol dissects the individual mechanical properties of the cell nucleus that make it a mechanical gauge for cell polarization, differentiation, and migration. The main advantage of this technique is that it can measure mechanical properties and apply forces to subcellular compartments without external perturbation of the membrane or cortical cytoskeleton. The protocol can be easily adapted to manipulate beads inside other cells of other organism in order to study nuclear mechanics in different cell lines or animal models.

Alignment of the direct force sensor is critical to capture all the light creating and leaving the optical traps. This will ensure accurate light momentum measurements in viscoelastic medium of a cell. Begin the single-cell preparation by placing the sphere stage embryos four hours post-fertilization in a glass dish containing E3 media using a plastic Pasteur pipette.

Then select the embryos that are positive to the beads and express the fluorescent protein in case of mRNA injection. Use forceps to manually decurionate the embryos and transfer approximately 10 to 15 decurionated embryos to a 1.5 milliliter reaction container with the help of a glass Pasteur pipette. Remove the E3 media from the tube and add 500 microliters of pre-warmed carbon dioxide-independent tissue culture medium.

Gently shake the tube avoiding bubble formation and ensure that the contents of the tube become turbid as the cells dissociate with no big chunks visible by the eye. Then centrifuge the tube at 200 times G for three minutes. After centrifugation, remove the supernatant and collect the pellet to proceed with cell staining.

To label the nucleus, resuspend the cells by adding 500 microliters of the freshly prepared DNA Hoechst dye staining solution to the tube. Incubate the stained cells for seven minutes in the dark. Next, centrifuge the cells as described earlier and resuspend the pellet in either 50 microliters of DMEM for samples in suspension or 20 microliters of DMEM for cells in confinement.

For the preparation of the optical trappings or OT chamber for the experiments with the cells in suspension, cut a piece of double-sided scotch tape with an approximately 10 millimeter by 10 millimeter square hole in the center. Peel off one of the protective layers of the tape and place the uncovered side of the tape in the center of a number 1.5 H glass bottom dish. Press the tape gently to get all the surface of the tape adhere to the glass while avoiding air bubbles and peel off the remaining protective layer of the tape.

Incubate the surface with 100 microliters of Concanavalin A at 0.5 milligrams per milliliter for 30 minutes. Then remove the drop of Concanavalin A and rinse the surface with DMEM. Add 30 microliters of the solution containing the cells onto the cavity surface.

Then close the chamber very gently with a 22 by 22 millimeter cover glass. Use a scalpel or forceps if needed. Next, proceed for optical tweezers startup by turning on the laser at considerably high power for at least 30 minutes before the experiment to optimize the output power stability.

When done, turn on the electronics module of the optical micro manipulation and force measurement units. Before aligning the optical force sensor, put a droplet of water on the 60X 1.2 water immersion objective and place the chamber containing the cells on the stage. Focus onto the lower surface of the chamber where the cell samples will be placed.

After adding a droplet of immersion oil on top of the cover glass slide covering the sample, lower the collecting lens of the force sensor unit until it contacts the oil droplet. Following the standard procedure for the optical force sensor alignment, look at the sample plane image on the auxiliary camera to be used to position the OTs, then lower the optical force sensor until its field stop appears conjugated onto the sample plane. To check for air bubbles with the Bertrand lens, observe the imaging path through the auxiliary camera.

If any dirt or air bubbles are visible, clean the lens and chamber with dust-free lens tissue before repeating the procedure as described, adding more immersion oil. Using the lateral screws placed on the holder of the optical force sensor, center the field stop into the field of view. For accuracy, open the field stop almost to occupy the field of view visible on the auxiliary camera.

After placing the sample in the microscope and aligning the optical force sensor, use the rotating half-wave plate to set the initial trap power to 200 milliwatts if the stiffness of the nucleus or the intercellular structure investigated is not known. Once done, use the microscope stage software controller to look for a cell with one or two beads through transmitted Brightfield microscopy. In the numerical sheet, write the displacement and the time of each subsequent trajectory step.

Alternatively, load table S1 from the supplementary material. For a stress/relaxation experiment, program the trapezoidal loads to be applied. In the OT software, activate the optical traps.

For trapping a microsphere, set the image planes slightly above the bead with a microscope stage software controller. Click on the polystyrene microbead in the calibrated auxiliary camera imaging window. Successful confinement of the bead by the optical trap will strongly reduce the motion of the bead.

Click and drag the bead across the cytoplasm and place it at a distance of approximately two microns from the nuclear envelope. Ensure that the trajectory is set so that the bead indentation is perpendicular to the nuclear membrane. When the setup is ready, go to the imaging software and click on the acquisition button to start image acquisition.

Open the real-time force reading window, click on data, and save in the real-time force reading window to save. Start trap position and force measurement data. Initiate the previously loaded trajectory by right-clicking on the bead and selecting start trajectory.

Wait until the trajectory is finished and the system stabilizes before finishing the recording of the trap force measurement data. Stop image acquisition and plot the results in the post-processing software. In the representative analysis, an isolated zebrafish progenitor stem cell with a single microsphere close to the nucleus is displayed.

Distribution of polystyrene microsphere five hours post-injection inside an embryo was monitored. Brightfield and fluorescence image shows the beads are dispersed across the embryo tissue. Maximum projection of the confocal fluorescent Z-stack and a maximum projection of a sub-stack in the same region confirms that a large fraction of deep cells contained one to two beads.

At 24 hours post-fertilization, the beads were distributed across the entire body of the embryo. The Brightfield micrographs of the suspension cells on the confined cells with one or two injected beads were obtained. Furthermore, multiple fluorescent labels facilitated the investigation of different aspects of the cells, such as internuclear membrane, plasma membrane, DNA, and transgenic line.

Representative snapshots described Hoechst-labeled nuclei five seconds before/during and five seconds after indentation with an optically trapped microsphere. Intensity profiles along the indentation segment for three different frames and trajectories of the distal and proximal boundaries of the nucleus during an indentation experiment of suspended and confined cells were studied. Trapped trajectory and force experienced by the optically trapped microsphere during a repeated nuclear indentation experiment were measured.

Nucleus mechanics in the cells in suspension and under confinement confirmed that the confinement resulted in an expansion of the projected area and an insignificant change in nuclear stiffness. If beads are injected into the one cell stage embryo, they will most likely evenly distribute in the later stages. Remember that the bead must be stably trapped.

If the bead escapes the optical trap, redefine the trajectory or increase laser power. These experiments can easily be performed onto multiple microbeads inside one cell and extended to active microrheology measurements.