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 epifluorescence 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.
Highlights
The mechanical properties of tissues, single cells, and intracellular compartments are linked to their function, in particular during migration and differentiation, and as a response to external stress (Engler et al, 2006; Provenzano et al, 2006; Lo et al, 2000)
Quantitative analysis of (d) the refractive index and (e) the calculated longitudinal modulus taking into account the Brillouin shifts, refractive indices and absolute densities of 139 HeLa cells
Quantitative analysis of (d) the refractive index, (e) the Brillouin shift and (f) the calculated longitudinal modulus taking into account the Brillouin shifts, refractive indices and absolute densities of 22 polyQ granules
Summary
The mechanical properties of tissues, single cells, and intracellular compartments are linked to their function, in particular during migration and differentiation, and as a response to external stress (Engler et al, 2006; Provenzano et al, 2006; Lo et al, 2000). To measure the mechanical properties of biological samples, many techniques are available These include atomic force microscopy (Christ et al, 2010; Koser et al, 2015; Gautier et al, 2015; Franze et al, 2013), micropipette aspiration (Maître et al, 2012), and optical traps (Wu et al, 2018a; Litvinov et al, 2002; Bambardekar et al, 2015; Guck et al, 2001). These techniques can access the rheological properties of a sample and their changes under various pathophysiological conditions. Most of them require physical contact between probe and sample surface and none of them allows to obtain spatially resolved distributions of the mechanical properties inside the specimens
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