Separating water content from network dynamics in cell nuclei with Brillouin microscopy

TL;DR

This study uses Brillouin microscopy to separate water content from solid-network dynamics in living cell nuclei under osmotic compression. By combining Brillouin shift $\nu_B$ and linewidth $\Gamma_B$ with FXm volume measurements, the authors show that $\nu_B$ is mainly governed by water content while $\Gamma_B$ reveals network-driven dissipation, requiring a poroelastic (Biot-like) description with a quasi-constant friction $f$ (≈$8.5\times10^{12}$ N s m$^{-4}$). The nucleus behaves as a two-phase system (water + solid network) where an isostress mixing law describes $\nu_B$ at low $\phi$ and a poroelastic framework accounts for $\Gamma_B$ at higher $\phi$, providing a unified interpretation of hypersonic Brillouin data in cells. This work offers a framework to decouple water content from polymer-network mechanics in nuclei, with implications for studying molecular crowding and water transport in living cells.

Abstract

Probing forces, deformations and generally speaking the mechanical properties of cells is the hallmark of mechanobiology. In the last two decades many techniques have been developed to this end that are largely based on deforming the cells and measuring the reaction force. In cells, an alternative approach has been implemented mid 2010's, based on Brillouin Light Scattering (BLS) that produces a spectrum that can be interpreted as the response of the sample to an infinitesimal uniaxial compression at picosecond timescales. In all of these measurements, the response of the cell is quantified with a colloquial "stiffness" that encompasses both the contribution of load-bearing structures and volume changes, much to confusion. To clarify the interpretation of the hypersonic data obtained from BLS spectra, we vary the relative volume fraction of intracellular water and solid network by applying osmotic compressions to single cells. In the nucleus, we observe a non-linear increase in the sound velocity and attenuation with increasing osmotic pressure that we fit to a poroelastic model, providing an estimate of the friction coefficient between the water phase and the network. By comparing BLS data to volume measurements, our approach demonstrates clearly that BLS shift alone is mostly sensitive to water content while the additional analysis of the linewidth allows identifying the contribution of the biopolymer-based network dynamics in living cells.

Figures (3)

• Figure 1: a. Sketch of the mixing law (left) and poroelastic (right) models for a mixture of a solid network (orange) and a liquid phase (blue). In the mixing law model, the two components can be represented by springs in serie and the attenuation can be modeled by a Poiseuille flow. In the poroelastic model, the solid fraction forms a network permeated by the liquid phase. Friction, $f$, between the moving fluid and the elastic network couples their dynamics. b. Normalized cell volume measured with FXm (blue markers) and intranuclear solid volume fraction (orange markers) vs sucrose concentration (mM) and the corresponding fits (lines). c. FXm images of three cells at the isotonic condition and after a shock at $100$ mM. Scale bar: $20\;\mu$m. d. Bright-field image (top) and Brillouin frequency shift images at the isotonic condition (middle) and after a shock at $100$ mM (bottom). Scale bar: $10\;\mu$m.
• Figure 2: a. BLS set-up. The laser light is focus inside the cell through a glass cover-slip. b. Typical BLS spectra in a cell (dots) and the corresponding fits to a Lorentzian function (lines), before (blue) and after (green) a shock at $500$mM. c. Brillouin frequency shift (top) and linewidth (bottom) images in the sagittal plane of a typical cell. Scale bar: $10\mu$m. d. Bright-field image of a cell with DNA labelled with Hoechst stain (left), and corresponding BLS images of the shift (center) and the linewidth (right). Scale bar: $10\mu$m. e.Left panel: profiles across the nucleus of the BLS shift before (blue) and after (green) a shock at $500$mM. Orange dotted lines correspond to the cell and medium values. Grey line indicates the $\nu_B$-value where the cell thickness is measured. Centre panel: Cell thickness measured with FXm (black) and that measured from BLS depth-profiles (blue) vs intranuclear solid volume fraction. Right panel: Cell thickness measured with FXm vs. that obtained from BLS depth profiles (black dots). We plot the fit to a line with a slope of $0.98$ (orange dotted line).
• Figure 3: a BLS shift vs intranuclear volume fraction and fit to the isostress (green line) and poroelastic (red dotted line) models. b Linewidth vs intranuclear volume fraction, with fit second-order correction (green) of the viscosity and to the poroelastic model (red dotted line) calculated from Eq. \ref{['eq-gamma-poro']} with $f=8.5\times10^{12}$ N.s.m$^4$ (see red dotted line in Fig. \ref{['fig:4']}c). c. Friction $f$ obtained from the measured linewidth plotted in Fig. \ref{['fig:4']}b and Eq. \ref{['eq-gamma-poro']}. The mean $f=8.5\times10^{12}$ N.s.m$^4$, used for the fit to poroelastic model in Fig. \ref{['fig:4']}b, is indicated by a red dotted line. We probed between $20$ and $40$ cells for each volume fraction.