Mechanical Mapping of Spheroids Using Brillouin Spectroscopy

We are using Brillouin spectroscopy to non-invasively assess cell mechanics to understand the role of mechanobiology in disease progression. Non-invasive technologies, such as Brillouin spectroscopy, allow us to characterize mechanics in three dimensions, but without disturbing the sample. To begin, pretreat the wells of the microwell plate by adding 500 microliters of anti-adherence rinsing solution to each well.

Centrifuge the plate at 1, 300 g for five minutes at room temperature. After eliminating air bubbles, remove the anti-adherence rinsing solution from the wells. Next, add one milliliter of PBS to each well and aspirate it completely.

After repeating the PBS wash, determine the number of cells per spheroid and the number of spheroids per gel according to the experimental design. Pellet the cells and resuspend them in one milliliter of supplemented DMEM. Then add one milliliter of this cell suspension into one well of the microwell plate and mix well.

Centrifuge the plate at 100 g for three minutes at room temperature. Observe the microwell plate under the microscope to confirm even distribution of cells and that they have settled at the bottom of the wells. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for 24 hours to allow spheroid formation.

To harvest the spheroids, gently and slowly remove the culture media without disturbing the spheroids. Cut the PBS/FBS coated pipette tip to widen the opening and add 500 microliters of PBS to each well to dislodge the spheroids. Collect the PBS containing the spheroids into a tube.

Then transfer 50 microliters of the spheroid suspension into one well of a 96-well plate. To aid counting, draw a cross on the bottom of the well using a permanent marker and count the number of spheroids in this volume under the microscope. Use the equation to calculate the total number of spheroids.

Open the SpectraLok software and wait until all connected devices are detected. Then select the spectrometer camera. Install the acrylic cube on the microscope sample holder with the clear side facing the objective lens.

Lower the objective lens as much as possible and center the cube beneath it. Ensure that the optical path in the microscope is set so that the spectrometer camera is exposed by turning the rotary port selector to L.In the SpectraLok Camera window, set the sensor Exposure time to 500 milliseconds and the Software Gain to 100. Press Capture, and slowly move the objective lens upward.

Continue raising the lens until the Brillouin peaks become visible and their intensity stabilizes, indicating that the focal spot is positioned within the acrylic cube. Now, open the Pump Killer, or PK control window. Zoom in on one of the brightest Brillouin orders and adjust the pressure actuator position in increments of 100 micrometers by clicking move rel to minimize the Rayleigh peak intensity.

If the Rayleigh signal increases, click Reverse to change the adjustment direction. Once the Rayleigh peak intensity decreases, fine-tune the etalon pressure in smaller increments of 10 micrometers. In the Camera window, zoom in on one of the Brillouin orders.

Press S to visualize stripes across which the LUT is applied to the camera image. Open the Settings window, and press Quick Calibrate to automatically recalculate the optimal horizontal offset of the stripes. After verifying the stripe alignment, adjust the collimator lens axes to maximize the coupling efficiency of light from the Pump Killer to the spectrometer.

Then open the Spectrum window and click Unwrap to retrieve the spectrum. In a homogeneous acrylic cube, aim for complete suppression of the Rayleigh signal and adjust until the Brillouin signal is clearly visible. To save the optimized parameters, click Refresh All, and then Save All in the PK control window.

Finally, in the main window, click Save in the File I/O section to store the configuration. After opening the scanning software, install the glass-bottom Petri dish containing the hydrogel spheroid sample in place of the acrylic cube. Remove the Petri dish lid, add sufficient media to submerge the gel, and place a cover slip on top to prevent gel movement.

Locate the spheroid by first blocking the laser beam. Switch the microscope optical path to the eyepiece and turn on the white light illumination. Using the joystick, manually control the sample stage to locate and focus on the spheroid.

Acquire a brightfield image of the spheroid and adjust the illumination brightness by rotating the control wheel. Switch the optical path to R to expose the microscope camera and set the exposure time as required. Next, set the microscope port to L and capture the background with the laser still blocked.

Unblock the laser, and depending on the desired signal to noise ratio, select an exposure time between 200 and 500 milliseconds. From the unwrapped spectrum, select the Brillouin peak fitting range. Adjust the threshold value to approximately half the Brillouin peak intensity, and exclude any Brillouin peaks with amplitude below the threshold.

Apply wavelength correction to the laser according to all identified Brillouin peak pairs. Finally, choose the raster scan geometry by adjusting the scan width and step size, and start the scan to record the measurements. Representative images of a mesenchymal stem cell spheroid embedded in hydrogel showed distinct Brillouin shift and Brillouin linewidth maps along with corresponding brightfield, nuclei, and actin filament fluorescence images.

The low resolution Brillouin frequency shift map was segmented into gel and spheroid regions based on the contour manually traced from the brightfield image. Ambiguous pixels resulting from down-sampling were excluded from analysis while clear binary masks for the gel and spheroid regions were created to extract BFS values. Comparison of high and low resolution BFS maps demonstrated that core sampling provided mean BFS values comparable to high resolution scans, indicating consistent measurement of spheroid bulk mechanics.

Our protocol will provide a platform that will enable researchers to study many diseases linked to mechanobiology. It will be interesting to see if changes in Brillouin shift that correlate with cell mechanical properties are effective markers for the stages of disease progression. We will assess how drug treatments influence spheroid mechanics over timescales that mimic disease progression.