Correlation of biomechanics and cancer cell phenotype by combined Brillouin and Raman spectroscopy of U87-MG glioblastoma cells

Abstract

The elucidation of biomechanics furthers our understanding of brain tumour biology. Brillouin spectroscopy is a new optical method that addresses viscoelastic properties down to subcellular resolution in a contact-free manner. Moreover, it can be combined with Raman spectroscopy to obtain co-localized biochemical information. Here, we applied co-registered Brillouin and Raman spectroscopy to U87-MG human glioblastoma cells in vitro. Using two-dimensional and three-dimensional cultures, we related biomechanical properties to local biochemical composition at the subcellular level, as well as the cell phenotype. Brillouin and Raman mapping of adherent cells showed that the nucleus and nucleoli are stiffer than the perinuclear region and the cytoplasm. The biomechanics of the cell cytoplasm is affected by culturing conditions, i.e. cells grown as spheroids are stiffer than adherent cells. Inside the spheroids, the presence of lipid droplets as assessed by Raman spectroscopy revealed higher Brillouin shifts that are not related to a local increase in stiffness, but are due to a higher refractive index combined with a lower mass density. This highlights the importance of locally defined biochemical reference data for a correct interpretation of the Brillouin shift of cells and tissues in future studies investigating the biomechanics of brain tumour models by Brillouin spectroscopy.

Keywords: Brillouin; Raman; glioblastoma; spheroids.

Figure 1.

Experimental set-up of the combined Brillouin and Raman system consisting of a 780.24 nm laser, a compact saturation spectroscopy module (CoSy), single/multi-mode fibres (SMF/MMF), Bragg gratings (BG), a polarizing beam splitter (PBS), a Fabry–Pérot interferometer (FPI), a photodiode (PD), neutral beam splitter (BS), cylindrical/spherical lenses (CL/SL) and virtually imaged phased arrays (VIPAs).

Figure 2.

(a) Bright field image of a living U87-MG cell; white box is 15 × 15 µm². (b) Raman cluster map of this cell consisting of four different clusters. (c) Mean Raman spectra of the three clusters associated with the cell: the nucleus (blue), the perinuclear region (red) and the cytoplasm (cyan). (d) Simultaneously acquired Brillouin shift and (g) Brillouin intensity map revealing the cellular structure. (e) Brillouin shift values for each pixel are assigned to the respective cell compartment obtained by cluster analysis of Raman spectra. (f) Examples of Brillouin single spectra of each cluster showing the reference methanol band at 3.81 GHz and the sample's band, which shifts to higher values and decreases when going from the grey to the blue cluster. (h) Brillouin linewidth map revealing the high viscosity of the nucleolus.

Figure 3.

(a) Example of a bright field image of a living U87-MG spheroid, where the black box indicates the measured area. Corresponding Brillouin maps of the same spheroid using (b) the Brillouin shift and (c) the Brillouin intensity as the contrast mechanism. Red arrows indicate the positions where the structural information between the maps differs.

Figure 4.

Cumulative frequency histograms (normalized) of the Brillouin shift values of all (a) living U87-MG cell (n = 11) and all (b) living U87-MG spheroid (n = 9) mappings, showing that there are different contributions fitted by Gaussian curves: the main contributions are at 5.3 GHz and 5.4 GHz, respectively. (c) Brillouin shift frequencies of the main contribution of the individual maps are significantly different (Mann–Whitney U-test; ***p < 0.001).

Figure 5.

Results of combined line scans on living U87-MG spheroids. Brillouin shift profiles (a,b) show regions with high frequencies which can be correlated with spectral patterns visible in the Raman heat maps (c,d), in which the area-normalized Raman intensity is colour-coded. Single Raman spectra at specific positions can be attributed to cytoplasm, lipids and protein-rich structures (e,f). Note that the Raman spectrum at 46 µm in line scan 2 actually shows a linear combination of lipid and protein bands indicating the presence of both within the measuring volume.

Figure 6.

(a) Example of a CARS image of a U87-MG spheroid cryosection revealing that there are several lipid droplets within the spheroids. (b) Quantification of the lipid droplet content for n = 7 CARS images revealed values between 0.2% and 1.3%, whereby the CARS image in (a) corresponds to image number 1.