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.

Quantifying cellular forces and biomechanical properties by correlative micropillar traction force and Brillouin microscopy

“…The noncontact, label-free and high-resolution capabilities of Brillouin microscopy have stimulated a wide range of applications, including the assessment of the cellular mechanical properties and their response to external stimuli [38][39][40][41], the mapping of the spinal cord stiffness in zebrafish larvae [42] and the investigation of the Alzheimer's plaque viscoelasticity [43]. Moreover, Brillouin microscopy holds promise to become a potential diagnostic instrument for diseases such as atherosclerosis [44], keratoconus [45], cancer [46], meningitis [47] and amyotrophic lateral sclerosis [48,49]. To this aim, significant research efforts have also focused on the instrumental advancement to achieve a high spectral contrast [38,50,51] and extinction ratio [52,53], as well as to decrease the data acquisition time [54,55], which are key ingredients to enable in-vivo measurements of living biosystems.…”

On the actual spatial resolution of Brillouin Imaging

“…Using 2σR as lateral dimension of the scattering volume and considering as a first approximation that the contributions to the spatial resolution add in quadrature (exact for Gaussian distributions), the lateral acoustic contribution can be found as 2σ E = 2√(σ B ) 2 − (σ R ) 2 . While this value is always utterly negligible for the glass-water interface, for the PET-Glycerol one studied by 1.2 NA objective 2σ E results The present analysis is of significant relevance for a critical assessment of a number of recent works 15,21,[24][25][26][27] , where submicrometric features are discriminated on the basis of Brillouin signature. While the comparison with different optical techniques indicates that the observed elastic modulation is indeed related to structural changes, the measured values of the elastic constant should be considered as spatial averages, whose extent depends on both NA of the optics and on the propagation length of phonons, in turn depending on the investigated material.…”

Long-term Brillouin imaging of live cells with reduced absorption-mediated damage at 660nm wavelength

“…An additional advantage of the 660 nm illumination could materialize in experiments in which Brillouin maps are combined with fluorescence imaging [37,38]. As fluorescent labels are a source of absorption and can significantly enhance the laser light damage to the live samples, care should be taken to avoid fluorescently labelling live samples with fluorescent dyes that have excitation near the Brillouin illumination wavelength.…”