Background-deflection Brillouin microscopy reveals altered biomechanics of intracellular stress granules by ALS protein FUS

Abstract

Altered cellular biomechanics have been implicated as key photogenic triggers in age-related diseases. An aberrant liquid-to-solid phase transition, observed in in vitro reconstituted droplets of FUS protein, has been recently proposed as a possible pathogenic mechanism for amyotrophic lateral sclerosis (ALS). Whether such transition occurs in cell environments is currently unknown as a consequence of the limited measuring capability of the existing techniques, which are invasive or lack of subcellular resolution. Here we developed a non-contact and label-free imaging method, named background-deflection Brillouin microscopy, to investigate the three-dimensional intracellular biomechanics at a sub-micron resolution. Our method exploits diffraction to achieve an unprecedented 10,000-fold enhancement in the spectral contrast of single-stage spectrometers, enabling, to the best of our knowledge, the first direct biomechanical analysis on intracellular stress granules containing ALS mutant FUS protein in fixed cells. Our findings provide fundamental insights on the critical aggregation step underlying the neurodegenerative ALS disease.

Fig. 1

Spectral contrast enhancement by background deflection. a Schematic of the spectrometer. The light to be analyzed is focused and coupled to the VIPA through an anti- reflection coated window. A mask with a rhomboidal aperture is placed before the Fourier lens to convolve the resulting diffraction pattern with the intensity transfer function of the VIPA etalon. As a result, the Stokes (SB) and Anti-Stokes (ASB) Brillouin spectral features gain high visibility despite the presence of strong elastic Rayleigh (R) peaks. b Interference patterns generated in response to a monochromatic light beam with (right) and without (left) the diffraction mask for different incident optical powers. In the standard configuration, a strong crosstalk line arises along the horizontal dispersion axis (dashed line). On the other hand, the elastic background is highly deflected by the mask, as further illustrated by the 3D plots (c). d Spectral intensity profiles along the dispersion axis (where the SB and ASB peaks are expected) for two consecutive interference orders. Whilst the standard single-stage VIPA spectrometer (black line) reaches a maximum contrast of ∼10³, our spectrometer (red line) gives ∼10⁷, which represents a 10,000-fold increase with respect to the standard case

Fig. 2

Validation of contrast enhancement. Representative 2D (a) and 1D (b) Brillouin spectrum of water. c Brillouin spectrum of an intralipid solution at concentration of 10%. In a standard VIPA spectrometer (black line), the Brillouin peaks are overwhelmed by the light scattered elastically. The spectrum, however, becomes visible (red line) when the background is deflected. d Background of the standard (black line) and deflected (red line) spectrometers averaged on a spectral range of 5–25 GHz as a function of the distance from a water-glass interface (dashed line)

Fig. 3

Biomechanical imaging of HeLa cells. The enhanced contrast enabled the acquisition of Brillouin images of single cells at different depths (∆z = 1 μm) in the case of uninduced (a) and doxycycline-induced (b) cells. Fluorescent images (c–d) showing the PABP (green) and DNA (blue; labeled by DAPI) merged staining. e–f Associated DIC images. Scale bar, 10 μm. g Box-and-whisker plot of the Brillouin frequency shift for different cellular compartments with (+) and without (−) FUSP525L expression. In both cases the expression of mutant FUS did not significantly altered the cytoplasm (Student’s t-test p = 0.22, N = 25 cells) and the nucleus (p = 0.37) properties (h) Box-and-whisker plot of the Brillouin frequency shift of living (−) and fixed (+) HeLa cells (N = 29, 3 experiments). Despite an overall increase in Brillouin frequency, different subcellular compartments are found to be similarly altered in living and fixed cells as indicated by the frequency ratio q

Fig. 4

Alteration of stress granule biomechanics. Representative Brillouin z-stack image of stressed HeLa cells without (a, Supplementary Video 1 for 3D reconstruction) and with (b, Supplementary Video 2) mutant FUS expression. Corresponding fluorescent (c–d; green: PABP; blue: DAPI) and DIC (e–f) images. Scale bar, 10 μm. While the nucleoli appear stiffer than the surrounding nucleoplasm in both cases, stress granules manifest a significantly higher (p = 2.5 × 10⁻⁸, N = 41 cells on three different experiments) Brillouin shift in response to mutant FUS expression, as illustrated in the box-and-whisker plot (g). h Representative Brillouin spectra of water (black) and stress granules with (red) and without (green) mutant FUS expression. Besides a higher frequency shift, the Brillouin peaks associated with mutant FUS expression manifests a larger linewidth (∆ν_B = 1.13 ± 0.02 GHz) than that without (∆ν_B = 0.86 ± 0.01 GHz), indicating a potential increase in the stress granule viscosity