Development of a Combined Fluorescence, Optical Diffraction Tomography and Brillouin (FOB) Microscope for the Quantitative Investigation of Phase Transitions in Cells

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Advances in our understanding of physiological and pathological processes in biology and biomedicine have often been driven by the availability of novel technological capabilities. The need for novel technologies is particularly urgent in the field of biological phase separation and transition. Phase separation is emerging as an entirely new way to organize the cytoplasm of cells, but it has also been associated with devastating neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Prion-like RNA-binding proteins such as Fused in Sarcoma (FUS) are now considered key players of phase transitions in cells. In vitro reconstitution experiments have shown that these proteins initially phase separate to form physiological condensates with liquid-like properties, but these mature into more solid-like structures that cause disease.While these recent discoveries have been breath-taking, the methods currently available to study phase transitions in cells, such as fluorescence recovery after photobleaching (FRAP) or the observation of droplet fusion (or lack thereof) using optical tweezers, are mostly qualitative, indirect and time-consuming, thus constituting a serious impediment for future progress. It is the central objective of this project to develop a new combined fluorescence, optical diffraction tomography and Brillouin (FOB) microscope to address this need, and to use FOB microscopy to study physiological and pathological phase transitions in vitro and in vivo. FOB microscopy will permit the quantitative imaging of 3D distributions of mass density, longitudinal modulus and viscosity inside living cells and with optical resolution. Here, we will first build the FOB microscope and identify the physical signatures associated with phase transitions of synthetic systems (Aim I), then verify these signatures in prion-like protein droplets in vitro (Aim II), and finally use FOB microscopy to study the connection between phase transitions of these proteins and functional changes in cultured cells and in motor neurons (Aim III). The quantitative characterization of condensates formed by wild-type and mutated proteins will reveal the connection between physical signatures measured by FOB microscopy, the molecular mechanisms underlying their conversion from a physiological to an aberrant disease-causing state, and the ultimate functional changes leading to disease pathology. In future, FOB microscopy can then also be used to screen for ways to alleviate the disease consequences.Once established, FOB microscopy will be made available to all groups within the SPP2191 and the wider community to enable novel insight into the physical mechanisms at work in living cells. Thus, FOB microscopy will address the urgent need to extract quantitative physical information at the mesoscale from cells, and will lay the groundwork for analysing physiological and aberrant phase transitions in various other in vitro systems cell types, and disease models.