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Method Article

Mechanical Mapping of Spheroids Using Brillouin Spectroscopy

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DOI:

10.3791/67538

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December 12th, 2025

 ,  ,  ,  ,  ,  ,  ,  , 
1Centre for the Cellular Microenvironment, Advanced Research Centre, 11 Chapel Lane, University of Glasgow, 2LightMachinery Inc.

* These authors contributed equally

In This Article

Summary

This article describes a protocol for mechanical characterisation of living spheroids within a 3D matrix using Brillouin micro-spectroscopy. This all-optical method enables spheroid visualisation with microscale resolution and quantitative mechanical properties. This approach has implications for mechanical phenotyping; for instance, determining pathological states of tumor spheroids within a 3D microenvironment.

Abstract

Brillouin spectroscopy, an emerging technique gaining significant interest in biomedical science, allows researchers to gather information related to mechanics and structure by interrogating the viscoelastic and architectural properties of specimens in a non-destructive, contact-free manner. This approach evaluates the mechanical properties of 3D samples by measuring the interaction of visible light with thermally induced acoustic waves/phonons. The information that Brillouin spectroscopy provides has potential for in vivo assessment of biophysics and potential diagnosis of disease pathologies. A significant advantage of Brillouin spectroscopy is the capability to assess microscale mechanics inside a biological sample; other conventional techniques that can achieve this resolution, such as atomic force microscopy, can only probe samples in 2D since they require direct contact. This work describes the application of Brillouin micro-spectroscopy to investigate the biomechanics of living spheroids embedded within a 3D hydrogel matrix. Encapsulation of cellular spheroids within a 3D microenvironment establishes a spheroid system that closely recapitulates the interface between cells and the extracellular matrix in vivo. In our protocol, we describe spheroid sample preparation and measurements of Brillouin spectra with sequential fluorescence imaging. Additionally, we discuss procedures for spectral data analysis and technical details about the optical system.

Introduction

Culturing cells in 3D models is advantageous compared to the typical 2D cell culture methods, as it better recapitulates tissue structures and cell-cell interactions found in vivo. Spheroids are 3D cell aggregates that facilitate complex cell-cell interactions; these interactions are compromised in cells grown as a monolayer on tissue culture plastic. Spheroids can be grown within a scaffold that mimics the microenvironment where the cell originated from. For example, hydrogels are a popular biomaterial used to culture cells/spheroids in as they allow precise control of parameters such as stiffness, degradability, pore size, and the incorporation of growth factors1 (Figure 1A). Mechanical cues often precede biochemical changes in disease pathologies such as cancer and fibrosis2,3, therefore, acquiring the biomechanical signature in clinically relevant 3D models of disease is instrumental for developing diagnostic interventions to inform therapeutic practice and understanding the underlying pathophysiology4.

Brillouin Microscopy (BM) is an imaging technique that allows the mechanical properties of biological materials to be measured in 2D planes5,6,7. BM has some key advantages over other more commonly used methods, such as atomic force microscopy (AFM) and elastography. It is well-documented that AFM can achieve high spatiotemporal mechanical resolution and that elastography can probe mechanics inside samples8,9 . BM does not require direct contact with the material under investigation, making it an ideal technique over AFM for samples/cells that are difficult to access, such as spheroids within hydrogels. Another key advantage of BM is that it does not require labelling of samples and can provide microscale resolution inside biological samples, which commonly used elastography methods cannot achieve7; more advanced optical elastography techniques can operate at microscale resolution10. It should be noted that the precise effective resolution in Brillouin imaging of heterogeneous biological samples is not trivial to define. Firstly, for a given illumination wavelength, it depends on the optical properties describing the focal spot size (numerical aperture, NA, of the objective lens and sample opacity). Additionally, the accuracy with which the mechanical properties of the sample can be probed, as well as the shape of the Brillouin spectrum, is influenced by the acoustic characteristics of the material (phonon wavelength and propagation length)11,12. The underlying phenomenon behind Brillouin spectroscopy is Brillouin scattering, where the incident monochromatic laser light interacts with spontaneous, thermally induced acoustic phonons within the biological medium. This results in inelastic scattering of light and (Brillouin frequency shift, BFS) in Figure 1B and is typically in the order of several GHz and depending on whether the photon loses, or gains energy, Brillouin Stokes and anti-Stokes peaks are detected in the spectrum of the scattered light, respectively. Through its dependence on the local sound velocity in the medium, the BFS is related to the elastic longitudinal modulus, which is in turn empirically correlated to the Young's modulus7,13; however, care must be taken when making comparisons between these moduli as (i) they are not directly related to each other, (ii) Young's modulus is usually in the Pa-kPa range for biological samples and soft matter whereas the longitudinal modulus obtained from Brillouin is in GPa range, and (iii) parameters such as refractive index changes and material density must be taken into account7. The Brillouin linewidth (in Figure 1B) is another measure obtained from BM. It is related to the longitudinal loss modulus, giving insights into the viscosity of the sample; these properties can be correlated with viscoelasticity14. Thus, BM allows for the assessment of the viscoelastic properties, where a larger BFS implies a stiffer material, and a larger linewidth implies a more viscous material (Figure 1B). It should be noted that elastic (Rayleigh) scattering, where the photons do not undergo a frequency shift, has a significantly larger likelihood of occurring compared to Brillouin scattering. Therefore, Brillouin spectrometers must be equipped to suppress the Rayleigh signal.

BM has been used to characterize the mechanical properties of various tissues, biomaterials, and 3D cell culture models. For example, BM was utilized to differentiate between melanoma and the surrounding healthy tissue, showing that the non-cancerous tissue was significantly softer than the melanoma15. Rad et al. showed that increasing the polymer concentration of hydrogels resulted in increased BFS and linewidth. At the lowest polymer concentrations, hydrogels with cells showed larger decreases in Brillouin frequency shifts over seven days compared to empty hydrogels, which was proposed to be due to cellular remodeling of the hydrogel. This was not observed at 10% polymer concentration, which underscored a possible lack of cell viability found in these hydrogels16.

Of most relevance to the present study, BM has been used to investigate the mechanical properties of spheroids17,18,19,20. In particular, the effect of the local microenvironment on spheroid mechanics can be detected using BM20. Compared to tumor spheroids cultured in compliant hydrogels, spheroids grown in stiff hydrogels exhibited a higher BFS, which is indicative of an increased elastic modulus20. Spatial variation of mechanical properties within a spheroid can also be characterized with BM. For example, the BFS of tumor spheroids has been found to increase towards the spheroid core20,21. Furthermore, a decrease in BFS correlates with increased invasiveness of tumor spheroids20. BM has been used to investigate the mechanical changes of tumor spheroids in response to osmotic shock22. In the study, spheroid growth was monitored over 5 days, finding that the initial mechanical differences between cancer and healthy spheroids observed with BM were lost with time. Another study found an increase in BFS of the spheroid after 7 days which was attributed to increasing spheroid size and number of cells16.

Furthermore, multimodal investigations, employing Brillouin microscopy alongside AFM and rheology, have been carried out to probe the mechanical properties of cells and hydrogels16,23,24. Rad et al. used rheology to contextualize the trends measured with BM, establishing a strong positive correlation between the longitudinal modulus and storage shear modulus with respect to the polymer concentration of hydrogels16. Brillouin microscopy and optical tweezer micro-rheology were used to measure the mechanics of cancer cells cultured in 3D hydrogels23. In this study, decreased mechanical heterogeneity was found among cells in spheroids compared to single cells grown suspended in hydrogels.

In this work, a protocol to investigate the mechanical properties of live spheroids embedded in hydrogels with a commercial Brillouin microscope is detailed (Figure 1). Berghaus et al. described a protocol detailing the steps to build a high-resolution Brillouin spectrometer and a method to calibrate it with reference materials25. Further, a protocol by Shi et al. describes the use of Brillouin microscopy to monitor the changes in the mechanical properties of embryonic tissue26. We use mesenchymal stem cell (MSCs) spheroids in poly(ethylene) glycol (PEG)-maleimide hydrogels to mimic the bone marrow niche and investigate the mechanical properties of this environment. This 3D model has the potential to uncover the mechanical signature of bone marrow diseases, including leukemia, multiple myeloma, and secondary bone cancer. The protocol is therefore directly transferable to 3D models of other diseases where disrupted mechanobiology may have an important role in the pathology. The spheroid and hydrogel preparation, fluorescence imaging, and Brillouin microscopy techniques are detailed. The recommended procedures, possible drawbacks, and potential improvements of the technique are discussed. The detailed experimental setup description is presented in Figure S1 and within the Supplementary Materials and methods section related to the experimental setup.

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Protocol

1. 2D cell culture

NOTE: Where possible, ensure that all cell culture work is performed in a sterile environment using a tissue culture standard laminar flow hood to avoid contamination. MSCs were used in the present protocol, but other cell types can be utilized to create spheroids, depending on the specific aims of the study.

  1. Culture MSCs in Dulbecco's Modified Eagle Medium (DMEM) cell culture medium, with sufficient volume to cover the cells, containing 4.5 g/L D-Glucose, + L-Glutamine supplemented with 10% fetal bovine serum (FBS), 100 µM sodium pyruvate, 1% non-essential amino acids (NEAA), 1% penicillin and streptomycin. Incubate cells at 37 °C and 5% CO2.
  2. Passage cells at 70-80% confluency, by incubating with 0.05% trypsin ethylenediaminetetraacetic acid (EDTA) (the volume of trypsin added should be 1/3 of the DMEM volume) for 3 min at 37 °C and 5% CO2 to detach cells from the plate. To stop the reaction, add culture media containing FBS (at least matching the volume of added trypsin). Count cells using a haemocytometer.

2. Spheroids preparation

NOTE: Where possible, ensure that all cell culture work is performed in a sterile environment using a tissue culture standard laminar flow hood to avoid contamination. In this study, microwell plates were used to create MSC spheroids. These plates are comprised of 24 wells, and each well contains 1200 microwells, 400 µm in diameter. This allows the preparation of a large number of spheroids (Figure 2).

  1. To prepare the microwell plate, pre-treat the wells by adding 500 µL of anti-adherence rinsing solution to each well. Centrifuge plate at 1,300 g for 5 min at room temperature. Under a microscope at 20x magnification, check whether there are any air bubbles present. If there are, repeat the centrifugation step. Remove the anti-adherence rinsing solution.
  2. To wash the plate, add 1 mL of phosphate-buffered saline (PBS) and aspirate. Do this twice to ensure the rinsing solution has been completely removed.
  3. Determine the number of cells per spheroid and the number of spheroids per gel. This will depend on individual research questions. For example, we used 100 cells per spheroid and 100 spheroids per gel. Therefore, for this protocol, only one well of the microwell plate was required, as this creates 1200 spheroids in total. To calculate the number of cells required for one well, we did the following calculation: 1200 x 100 = 1.2 x 105. Calculate the volume of cell suspension containing 1.2 x 105 cells. Centrifuge the cell suspension at 300 g for 5 min at room temperature and remove supernatant.
  4. Resuspend the cells in 1 mL of DMEM (+4.5 g/L D-Glucose, + L-Glutamine) supplemented with 100 µM sodium pyruvate, 1% NEAA, 1% penicillin and streptomycin. Add 1 mL of cell suspension to one well of the microwell plate and gently pipette up and down to disperse cells evenly throughout the well. Be careful not to introduce air bubbles. Centrifuge the plate at 100 g for 3 min at room temperature.
    NOTE: At this stage, we do not add FBS to the media. This is to prevent the cells from attaching to the tissue culture plastic.
  5. Observe the plate under the microscope to ensure the cells are evenly distributed and have sunk to the bottom of the microwells. Incubate cells at 37 °C and 5% CO2. Observe spheroid formation 24 h later.
    NOTE: Spheroids can be left in the plate for up to 48 h, depending on the cell type and desired compactness of the spheroid.
  6. To harvest the spheroids from the plate, first slowly and gently remove the culture media, aiming not to disturb the spheroids. To prevent spheroids from sticking to plasticware, coat pipette tips and tubes using PBS with 1% FBS. To avoid the spheroids getting stuck in the pipette tips, cut the tip to widen the opening. Maximum recovery tips can be used during these steps as an extra measure to prevent the loss of spheroids. Add 500 µL of PBS to each well of the microwell plate and pipette up and down to dislodge spheroids. Collect the PBS with suspended spheroids in a tube (Figure 2).
  7. View the plate under the microscope at 20x magnification and repeat this step until most of the spheroids have been successfully removed.
  8. To count the number of spheroids in the suspension, transfer 50 µL from the suspension into a well of a 96-well plate. Count the number of spheroids in the 50 µL. To aid counting, a cross can be drawn on the bottom of the well using a permanent marker. Calculate the number of spheroids using equation (1):
    Formula for calculating spheroid count in suspension, involving volume and count in 50 μL.

3. Preparation of PEG hydrogels

NOTE: Where possible, ensure that all cell culture work is performed in a sterile environment using a tissue culture standard laminar flow hood to avoid contamination. In this study, PEG-maleimide hydrogels were used, but other types can also be utilized. PEG-maleimide hydrogels were formed using the thiol-Michael addition reaction27. The crosslinkers used were a mixture of PEG-dithiol and protease-degradable peptide, flanked by two cysteine residues (VPM peptide, GCRDVPMSMRGGDRCG). The gels in the present study used 70% PEG-dithiol and 30% degradable VPM peptide.

  1. Make a 200 mg/mL stock solution of VPM peptide and add 2.4 µL of peptide per gel. Make a 200 mg/mL stock solution of PEG-dithiol and add 5.6 µL per gel.
  2. Resuspend the spheroids in PBS before mixing with the crosslinkers (Figure 2). Mix the spheroid suspension with the PEG-dithiol (5.6 µL per gel) and VPM (2.4 µL per gel). This solution is termed Solution B.
    NOTE: In this protocol, 100 spheroids were added per gel.
  3. Make a 250 mg/mL PEG-Maleimide stock solution. Add 50 µg of fibronectin per gel. Mix the PEG-Maleimide and fibronectin solution; this is termed Solution A. Pipette Solution A (40.3 µL for 100 µL gels) onto the bottom of a 12-well tissue culture plate. Add Solution B (59.7 µL for 100 µL gels) at a molar ratio 1:1 maleimide:thiol to ensure full crosslinking; ensure that gelation occurs instantaneously. The final volume of the hydrogels is 100 µL.
  4. Incubate gels for 30 min at 37 °C and 5% CO2 to allow gelation. Add complete culture media (DMEM supplemented with 10% FBS, 100 µM sodium pyruvate, 1% NEAA, and 1% penicillin and streptomycin) to the well, ensuring the gel is fully submerged (Figure 2). Incubate the gel at 37 °C and 5% CO2 until ready to take measurements under the Brillouin microscope.
    NOTE: The duration of spheroid culture within the hydrogels prior to Brillouin measurements will be dependent on the cell type and the research question. For example, in this study, the spheroids are typically cultured within hydrogels for up to 7 days, during which the spheroids are monitored by Brillouin microscopy. Viscoelastic properties of the hydrogels with/without spheroid encapsulation were characterized and show no significant impact of spheroids on gel mechanics (Figure S2).

4. Fluorescence staining and imaging of spheroids in hydrogels

NOTE: In this study, cell nuclei and actin filaments comprising the cytoskeleton were fluorescently stained and imaged prior to Brillouin measurements.

  1. To stain actin, add live actin stain to culture media at 1 in 500 dilution 3 h before imaging. Keep the sample out of light by wrapping in foil. Incubate the gel at 37 °C and 5% CO2.
  2. To stain the nuclei, add 2 drops/mL of live nuclear stain to the culture media surrounding the gel 40 min before imaging. Return the samples to the incubator.
  3. Acquire fluorescence images as soon as possible after the staining incubation time is complete. Verify that the appropriate optical path (exposing the microscope camera) and the correct filter wheel position are selected. Dim the lights in the room and capture the background. Switch on the light-emitting diode (LED) light source and choose the wavelength depending on the staining protocol (here, 385 nm was used for nuclei and 621 nm for actin imaging). Acquire fluorescent images of spheroids at 20x magnification with preferred software. Turn off the LED source when finished.

5. Brillouin microscopy of spheroids

  1. Switching on the devices and laser emission
    1. Switch on the devices: the computer, spectrometer, microscope cameras, the microscope, the laser cooling fan, and laser emission by turning on the key on the controller. Ensure that this is done 15 min prior to spectrometer optimization to ensure the laser has thermalized. Verify that the emitted laser power is as expected with a power meter (100 mW in our experiment).
  2. Turn on the temperature-control application (see Table of Materials) and verify that the etalon temperature is optimized by measuring the beam power after the etalon. If it is significantly lower than expected (after accounting for the optical density (OD) filter used), change the target temperature by 1 °C and observe if there is an increase in power with time. If there is a decrease, reverse the direction of change. Once the power is close to expected, tune by small increments (0.1 °C) until the maximum is reached.
  3. Setting the objective lens focal spot inside the acrylic cube
    NOTE: Suppression of elastic scattering and coupling from the "pump killer" to the spectrometer is optimized with a sample of stiff, homogenous reference material prior to each experiment. In this study, an acrylic cube is used.
  4. Open SpectraLok software and wait until all devices are detected. On the left side of the main window (Figure 3), select the spectrometer camera.
    1. 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 under it. Ensure that the optical path in the microscope is chosen so that the spectrometer camera is exposed (set the rotary port selector to L).
      NOTE: In our setup, the L port is connected to the confocal circulator, while R is connected to the microscope camera port.
    2. In the SpectraLok Camera window, set the sensor exposure time to 500 ms and the software gain to 100. The software gain setting only enhances the contrast for visualization purposes.
  5. Unblock the laser shutter.
    NOTE: As the laser emission is on, take care to never put head/eyes in the laser beam path.
    1. Press capture, and move the objective lens upwards. There will be a significant increase in signal intensity, and a saturated pattern (Figure 4A) will appear. This is caused by a strong reflection of laser light at the air-acrylic cube interface. Moving the lens further up, the signal intensity will decrease, and Brillouin peaks will become apparent. Continue repositioning the objective lens until the intensity of the Brillouin peaks is constant (Figure 4B). This corresponds to the focal spot located entirely within the acrylic cube.
  6. Optimizing the etalon pressure to minimize residual elastically scattered light
    NOTE: The purpose of the following steps is to suppress the elastically scattered Rayleigh signal so that only the frequency-shifted Brillouin light reaches the spectrometer camera.
  7. In SpectraLok software, open the Pump Killer (PK) control window (Figure 5).
  8. Zoom in on one of the brightest Brillouin orders and adjust the pressure actuator position in increments of 100 µm by clicking move rel to minimize the Rayleigh peak intensity. If the Rayleigh signal is increasing, reverse the direction of change by clicking reverse. Subsequently, fine-tune the etalon pressure in increments of 10 µm.
    NOTE: In the acrylic cube, the Rayleigh light can be completely suppressed; this may not be the case in a heterogeneous biological sample.
  9. Optimizing the stripe overlay to "unwrap" spectral data
    NOTE: The following procedure ensures that the (x, y) pixel coordinates of the camera sensor are appropriately mapped to wavelength via application of the look up table LUT.
  10. In the camera window, zoom in on one of the Brillouin orders.
  11. Press s to visualize stripes across which the LUT is applied on the camera image (Figure 6A). Open the Settings window and press quick calibrate to recalculate the optimal horizontal offset of the stripes. If the sensor is saturated, reduce the exposure time before this step.
    1. In the camera window, verify that the purple stripes are well overlaid with the central region of Brillouin peaks (Figure 6B).
  12. Optimizing the PK-spectrometer coupling
  13. Adjustment of the collimator lens axes allows maximizing the efficiency of the coupling of light from the PK to the spectrometer.
  14. Open the spectrum window and click unwrap to retrieve the spectrum (Figure 7).
  15. Open the PK control window. With increments of 0.0005°, iteratively adjust the collimator axes 1 and 2 to maximize the Brillouin signal. If needed, the etalon pressure control can be further optimized. In a homogenous acrylic cube, the Rayleigh signal can be completely suppressed (Figure 7A). While accurate Brillouin measurements with some residual Rayleigh signal are feasible (Figure 7B), in cases of poor suppression (Figure 7C), elastically scattered light obscures the Brillouin signal.
  16. Verify that the achieved Brillouin signal is comparable to reference amplitude. For example, in our system, at exposure of 500 ms and with OD 0.3 the Brillouin peak intensity amplitude is approximately 3000 counts. To save changes of the PK parameters to the configuration file, in the PK window, click refresh all and save all. Furthermore, in the main window, press save in the File I/O section.
  17. Switching to scanning software and verifying that the parameters are still optimized
  18. Close SpectraLok and open the scanning software. In the present protocol, LabVIEW-based software was used and is described in more detail in Figure S3. To set the scan geometry, this proprietary user interface (UI) is used to control the Prior XY-Z translation stage; however, other methods can be employed.
    1. Install the glass-bottom petri dish with the hydrogel-spheroid sample instead of the acrylic cube (Figure 8). To prevent dehydration of the gel, the incubator is set at 37 °C and 95% relative humidity. Remove the petri dish lid and add media to submerge the gel. Add a coverslip to the top of the gel to prevent movement of the gel.
    2. Locate the spheroid. With the laser beam blocked, switch the microscope optical path to the eyepiece. Turn on the white light illumination. Using the joystick, manually control the sample stage, locate and focus on the spheroid.
    3. Acquire a brightfield image of the spheroid. Adjust the illumination brightness by rotating the control wheel. Set the optical path to R, thus exposing the microscope camera. Exposure time can be adjusted as needed to obtain a high contrast image (i.e., 50 ms).
    4. Verify that the spectrometer and unwrapping parameters are still optimized. Set the microscope port to L and capture the background with the laser blocked. Unblock the laser by turning the wheel to firing. Based on the desired signal-to-noise ratio, select the exposure time. Exposure time typical to these measurements is 200-500 ms.
    5. If some time has passed (i.e., several hours) since initial calibration or there has been a mechanical disturbance of the system, the etalon pressure setting or the PK-spectrometer coupling may no longer be optimal. Repeat steps 5.3-5.5 to re-optimize the aforementioned, if necessary.
    6. From the unwrapped spectrum, select the Brillouin peak fitting range. Adjust the threshold value to about half the intensity of the Brillouin peaks. Disregard the Brillouin peaks with amplitude below this threshold. Correct the laser wavelength according to all of the identified Brillouin peak pairs.
  19. Setting the multi-dimensional scan geometry
  20. Choose the raster scan geometry by adjusting the width of the scan region and the step size.
    NOTE: For spheroids of size approximately 70 µm, for a low-resolution scan, we typically use a scan area of 150 µm × 150 µm and a step size of 10 µm. This yields a low-resolution 2D map in approximately 3.4 min.
    1. Start the scan.
  21. Switching off the Brillouin system
    1. Close the software and turn off the computer. Turn off the laser emission using the key on the controller. Leave the shutter wheel at the blocked position. Switch off the laser fan cooling. Turn off the microscope and spectrometer cameras. Finally, turn off the microscope.

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Results

Files obtained after a scan include spectra, scan fitting parameters (.csv), scan parameters and analysis, BFS and linewidth maps, brightfield image with the scan region of interest identified, and a screenshot of the UI window. For each point of the multi-dimensional Brillouin scan a BFS value is acquired. In Figure 9, an example high-resolution scan is displayed alongside the brightfield image, and fluorescence images showing actin filaments and nuclei. Note that in our system, the fluores...

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Discussion

We have established a protocol to use Brillouin microscopy for biomechanically imaging spheroids embedded in hydrogels with microscale resolution. The hydrogel can be distinguished from the live spheroid by differences in the BFS values obtained. In the representative results, we used MSC spheroids and PEG-maleimide hydrogels; however, this protocol is broadly applicable to other cell types (e.g., cancer cell lines). The cells in this study were imaged live and within a relevant 3D microenvironment, which demonstrates th...

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Disclosures

The authors note that T. Allen and G. Wardle are employees of LightMachinery, which manufactures and sells the Brillouin spectrometer used in this study. The company may benefit from the publication of this work. All other authors declare no competing interests.

Acknowledgements

The authors acknowledge the funding obtained from MechanoMeds Phase 2 grant, UKRI/EPSRC, grant number EP/X033554/1 to support the development of this method.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
4-arm PEG-Maleimide (20 kDa)Creative PEGWorksPSB-455https://creativepegworks.com/product/4-arm-peg-mal-mw-20k
AggreWell 400 platesSTEMCELL Technologies34411http://stemcell.com/products/aggrewell400.html
Air-spaced etalonLightMachineryHF-16074-660https://lightmachinery.com/spectrometers/brillouin-spectroscopy-excitation-laser-optimization/
Anti-adherence rinsing solutionSTEMCELL Technologies7010https://www.stemcell.com/products/aggrewell-rinsing-solution.html
Bold Line Top Stage IncubatorOkolabH301-NIKON-NZ100/200/500-Nhttps://www.oko-lab.com/live-cell-imaging/stage-top-digital-gas/chamber/nikon/h301-nikon-nz100-200-500-n
Cobolt Flamenco-100 660 nm laserHuebner0660-05-01-0100-700https://hubner-photonics.com/products/lasers/single-frequency-lasers/05-01-series/
Confocal circulatorLightMachineryHF-13311N/A
D-LEDi Fluorescence illumination system NikonMBF83000https://www.microscope.healthcare.nikon.com/en_EU/products/light-sources/d-ledi
EPI-FL moduleNikonTi2-LA-FL-3https://www.microscope.healthcare.nikon.com/en_EU/products/accessories/intermediate-modules
Fibronectin (human)YoProteins663https://www.yoproteins.com/fibronectin-human-5-mg-663.html
Gibco Dulbecco’s Modified Eagle Medium (DMEM)ThermoFisher11965092https://www.thermofisher.com/order/catalog/product/11965092
HyperFine Brillouin spectrometerLightMachineryHF-8999-PK-660https://lightmachinery.com/spectrometers/brillouin-hyperfine-spectrometer/
LED-DA/FI/TR/Cy5-B Quadruple band filter cubeNikonMXR00753https://www.microscope.healthcare.nikon.com/en_EU/products/accessories/fluorescent-filter-cubes
Mesenchymal stem cells (MSCs)PromoCellC-12974https://promocell.com/us_en/human-mesenchymal-stem-cells-hmsc.html
Motorized translation stage Prior ScientificH117E2NNhttps://www.prior.com/imaging-components/motorized-stages?filter=MicroscopeBrand-33
NucBlue™ Live ReadyProbes™ reagent (Hoechst 33342)ThermoFisherR37605https://www.thermofisher.com/order/catalog/product/R37605
Objective, S Plan Fluor 20X NA 0.45, airNikonMRH08230https://www.microscope.healthcare.nikon.com/products/optics/selector/comparison/-1708
ORCA-Fusion CMOS cameraHamamatsuC14440-20UPhttps://www.hamamatsu.com/eu/en/product/cameras/cmos-cameras/C14440-20UP.html
PEG-dithiol, 2 kDaCreative PEGWorksPLS-613https://creativepegworks.com/product/hs-peg-sh-mw-2k-10g
sCMOS camera Teledyne Vision Solutions01-IRIS-15-USB-M-16-Chttps://www.teledynevisionsolutions.com/en-gb/products/iris/?model=01-IRIS-15-USB-M-16-C&vertical=tvs-photometrics&segment=tvs 
SPY555-actin SpirochromeCY-SC202https://spirochrome.com/product/spy555-actin/
TemperatureAppTemperature control software
VPM peptide, GCRDVPMSMRGGDRCGGenScriptN/AN/A
Eclipse Ti-2A microscopeNikonEclipse Ti-2Ahttps://www.microscope.healthcare.nikon.com/en_EU/products/inverted-microscopes/eclipse-ti2-series 

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Spheroid Mechanics3D Hydrogel MatrixCell MechanicsNon-Invasive BiomechanicsFluorescence ImagingBrillouin ShiftSpheroid PreparationSpectral Data Analysis
Mechanical Mapping of Spheroids Using Brillouin Spectroscopy
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