Correlative all-optical quantification of mass density and mechanics of subcellular compartments with fluorescence specificity

Abstract

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples - so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample - a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

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

“…Indeed, the Brillouin shift of lipid droplets measured in our experiments (~5.5 GHz) is approximately 5% higher than that of the cytoplasm of adherent cells (5.22 GHz). This result agrees with previous studies on adipocytes, where lipid droplets display a Brillouin shift higher than cytoplasm (21,62), but a 10% lower stiffness by correcting the results for the index of refraction co-registered by optical diffraction tomography (21).…”

“…A region with highest values is located within the nucleus (Figure 2h). In agreement with (21), this was assigned to the high viscosity of the nucleolus.…”

“…In case of lipid droplets, the higher Brillouin shift does not indicate stiffness higher than the surrounding cytoplasm: the mass density of lipids is lower compared to cytoplasm ( ρ = 930 kg/m 3 (56) vs. ρ ≈ 1000 kg/m 3 (57)) while n is higher ( n = 1.41 (21) vs. n = 1.375 (58)), so that the two contributions do not compensate and the ratio is about 6% higher for lipids compared to the cytoplasm. Indeed, the Brillouin shift of lipid droplets measured in our experiments (∼5.5 GHz) is approximately 5% higher than that of the cytoplasm of adherent cells (5.22 GHz).…”

“…For cytoplasm, nucleus and nucleolus the changes of n and ρ are expected to compensate each other according to the two-substance mixture model (21,53–55). Therefore, the higher Brillouin shift measured in the cell nucleus and in the nucleolus of cells indicates a higher stiffness of these cellular compartments.…”

Multimodal microscale mechanical mapping of cancer cells in complex microenvironments

“…Therefore, Brillouin shift is used as a proxy measurement of the local longitudinal elastic modulus [44-46]. In most cells and tissues this assumption is valid, but there are exceptions like the lipid-rich samples where the mass density and index of refraction have dramatically different values [78, 79].…”