Fig. 1: Brillouin full widths at half maximum (FWHMs) obtained from different materials for viscosity calibration.

A left The Brillouin spectrum of a material is composed of a central narrow Rayleigh peak (due to photons scattered at the same frequency of the incident radiation) and two broader, identical Brillouin peaks (Stokes and Anti-Stokes), whose center is the Brillouin shift \({\nu }_{B}\) (red dotted lines) and width is \({\varGamma }_{B}\) (blue arrows). A right Brillouin triplet of panel A imaged with a VIPA spectrometer of free spectral range (FSR) = 15 GHz. The spectrometer repeats the Brillouin triplet every 15 GHz and introduces an instrumental broadening due to its finite point spread function (PSF): here, the Rayleigh width is \({\varGamma }_{{\rm{PSF}}}\) (purple arrows) and the Brillouin width is \({\varGamma }_{{\rm{experimental}}}\) > \({\varGamma }_{B}\). During data acquisition with a double-VIPA setup, only the Stokes (S) and Anti-Stokes (AS) Brillouin peaks of two adjacent diffraction orders (N and N + 1) are recorded, while their respective Rayleigh peaks (R_N and R_N+1) are suppressed (shaded areas). B top Brillouin spectra (only Anti-Stokes peak) obtained with SBM (dots) and the corresponding fits (continuous lines), from which we extrapolated \({\varGamma }_{B}\), i.e., the width unaffected by instrumental broadening. SBM laser source operated at 780 nm; to facilitate comparison with BM results, the spectra are here shown to an equivalent wavelength of 660 nm (described in the “Methods”). B bottom Brillouin spectra obtained with BM (dots) and the corresponding fits (solid lines), from which we extrapolated \({\varGamma }_{{\rm{experimental}}}\), i.e., the width affected by VIPAs broadening. The BM laser source was 660 nm: we used this wavelength as a reference for all the measurements. C Corresponding \({\varGamma }_{B}\) (red dots) and \({\varGamma }_{{\rm{experimental}}}\) (green dots) values for the different materials. Water, medium and LV medium all resulted in the same widths, and their values were redundant. Black dots are corrected \({\varGamma }_{{\rm{experimental}}}\) and obtained once properly deconvolved with a Gaussian \({\varGamma }_{{\rm{PSF}}}\). D left \({\varGamma }_{{\rm{experimental}}}\) vs \({\varGamma }_{B}\) for the four calibration materials, together with the different fits: linear (\(y=x+{\varGamma }_{{\rm{PSF}}}\): green line, yielding \({\varGamma }_{{\rm{PSF}}}\) = 0.291 GHz), root mean square (RMS: \(y=\sqrt{{x}^{2}+{\varGamma }_{{PSF}}^{2}}\): red line, \({\varGamma }_{{PSF}}\) = 0.532 GHz) and Voigt (Voigt1: \(y=\sqrt{\left({0.87* \left(\frac{x}{2}\right)}^{2}+{\varGamma }_{{PSF}}^{2}\right)}+1.0692* \left(\frac{x}{2}\right)\), in which PSF is a gaussian and Brillouin is a Lorentzian: cyan dotted line, \({\varGamma }_{{PSF}}\) = 0.425 GHz; Voigt2: \(y=\sqrt{\left({0.87* \left(\frac{{\varGamma }_{{PSF}}}{2}\right)}^{2}+{x}^{2}\right)}+1.0692* \left(\frac{{\varGamma }_{{PSF}}}{2}\right)\), in which the roles were reversed: blue dashed line, yielding \({\varGamma }_{{PSF}}\) = 0.440 GHz). The Gaussian fit resulted in a more significant description of the data. Black dashed line: y = x line, showing that \({\varGamma }_{{experimental}}\) > \({\varGamma }_{B}\) always. Data are shown as mean ± SD over n = 500 repeated measurements for BM and n = 100 for SBM. SBM data are shown in Supplementary Table 1. D right the Brillouin shifts of the calibration materials acquired with the SBM and BM matched, showing that the convolution operation does not affect Brillouin peak position but only its width. E Deconvolved widths of different liquids at room temperature (RT), heated at 37 °C or having different concentrations of Dextran, show that Brillouin linewidth is extremely sensitive to temperature (medium RT vs 37 °C: p = 0.003; LV medium RT vs 37 °C: p = 0.005; HV medium RT vs 37 °C: p = 0.001) and polymer concentration (water vs medium RT: p = 0.85; LV medium RT vs HV medium RT: p = 0.008). Medium: regular cell medium; LV medium: low viscosity medium, corresponding to culture cell medium with 6 kDa Dextran; HV medium: high viscosity medium, corresponding to cell medium with 500 kDa Dextran. F \({\eta }_{{bulk}}\) values, calculated from deconvolved widths of each material (y-axis), agree with theoretical values found in literature (x-axis). Dotted line: y = x line, showing a good correlation between experimental and theoretical data. Data are shown as mean ± SD, performed over n = 500 measurements. Statistical analysis has been performed using ordinary one-way ANOVA. **p < 0.01; ***p < 0.005; ns not significative.