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

开发用于定量研究细胞相变的荧光、光学衍射断层扫描和布里渊 (FOB) 组合显微镜

• 批准号: 419138906
• 负责人: Professor Dr. Simon Alberti
• 金额: --
• 依托单位: Max-Planck-Institut für die Physik des Lichts
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/419138906?language=en
• 关键词: Development; Combined; Fluorescence; Optical; Diffraction

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.

我们对生物学和生物医学中的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推动的。在生物相分离和转变领域，对新技术的需求尤为迫切。相分离是一种组织细胞质的全新方式，但它也与肌萎缩侧索硬化症(ALS)等毁灭性的神经退行性疾病有关。Pron样RNA结合蛋白，如FUSE in Saroma(FUS)，现在被认为是细胞相变的关键角色。体外重建实验表明，这些蛋白质最初是相分离的，形成具有液体性质的生理性凝聚体，但这些凝聚体成熟后会形成更像固体的结构，从而导致疾病。尽管这些最近的发现是令人惊叹的，但目前可用于研究细胞相变的方法，如光漂白后荧光恢复(FRAP)或使用光钳观察液滴融合(或缺乏)，大多是定性的、间接的和耗时的，因此对未来的研究构成了严重的障碍。本项目的中心目标是开发一种新型的荧光、光学衍射层析成像和布里渊(FOB)联合显微镜来满足这一需求，并使用FOB显微镜来研究体外和体内的生理和病理相变。FOB显微镜可以定量成像活细胞内质量密度、纵向模数和粘度的三维分布，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特征(AIM I)，然后在体外PrP样蛋白液滴中验证这些特征(AIM 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功能变化之间的联系(AIM III)。对野生型和突变蛋白形成的凝聚体的定量表征将揭示FOB显微镜测量的物理特征、它们从生理状态转换到异常致病状态的分子机制与导致疾病病理的最终功能变化之间的联系。将来，FOB显微镜还可以用于筛选减轻疾病后果的方法。一旦建立，FOB显微镜将适用于SPP2191中的所有组和更广泛的社区，以使人们能够以新的方式深入了解活细胞中发挥作用的物理机制。因此，FOB显微镜将满足从细胞中提取中尺度定量物理信息的迫切需要，并将为分析其他各种体外系统、细胞类型和疾病模型中的生理和异常相变奠定基础。