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

我们对生物学和生物医学中生理和病理过程的理解的进步往往是由新技术能力的可用性所驱动的。在生物相分离和相转变领域，对新技术的需求尤其迫切。相分离是一种全新的组织细胞质的方法，但它也与破坏性的神经退行性疾病如肌萎缩侧索硬化症（ALS）有关。朊病毒样RNA结合蛋白，如融合肉瘤（FUS），现在被认为是细胞相变的关键参与者。体外重组实验表明，这些蛋白质最初发生相分离，形成具有液体性质的生理凝聚物，但这些蛋白质成熟后会形成更像固体的结构，从而导致疾病。虽然这些最新发现令人惊叹，但目前可用于研究细胞相变的方法，例如光漂白后的荧光恢复（FRAP）或液滴融合的观察使用光镊进行（或缺乏）测量大多是定性的、间接的和耗时的，因此对未来的进展构成严重障碍。本项目的中心目标是开发一种新的组合荧光，光学衍射断层扫描和布里渊（FOB）显微镜，以满足这一需求，并使用FOB显微镜在体外和体内研究生理和病理相变。FOB显微镜将允许活细胞内的质量密度、纵向模量和粘度的3D分布的定量成像，并具有光学分辨率。在这里，我们将首先建立FOB显微镜，并确定与合成系统的相变相关的物理签名（目的I），然后在体外朊病毒样蛋白液滴中验证这些签名（目的II），最后使用FOB显微镜研究这些蛋白质的相变与培养细胞和运动神经元中的功能变化之间的联系（目的III）。由野生型和突变蛋白形成的缩合物的定量表征将揭示通过FOB显微镜测量的物理特征之间的联系，其从生理状态转换为异常致病状态的分子机制，以及导致疾病病理的最终功能变化。在未来，FOB显微镜也可以用来筛选减轻疾病后果的方法。一旦建立，FOB显微镜将提供给SPP 2191和更广泛的社区内的所有团体，使新的洞察力在活细胞中工作的物理机制。因此，FOB显微镜将解决从细胞中提取中尺度定量物理信息的迫切需要，并将为分析各种其他体外系统细胞类型和疾病模型中的生理和异常相变奠定基础。

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.