Development of Brillouin Spectroscopy for Mechanotransduction Research

机械生物学是一个快速发展的科学领域，有可能显著提高我们对细胞如何在组织/器官水平上发挥作用的认识。更好地了解细胞如何对其局部机械环境（细胞外基质，流体流动等）作出反应，也将导致先进的生物医学应用，如改进的生物材料和细胞治疗。对于这种新的治疗策略，有必要为细胞提供一个有限的环境（生态位），以增强和调节细胞的增殖和分化。在这方面，生物材料技术目前在试图通过人工重建原生三维环境来满足这些要求方面处于领先地位。然而，了解这些细胞如何、为什么以及在什么环境下以特定谱系的方式分化，对于理解再生生物学和开发更好的组织工程和干细胞治疗方法至关重要。我们认为，由于缺乏适当的工具，这种潜在的生物学（即细胞对局部机械刺激的反应）尚未得到适当的研究，这可能会破坏当前的尝试：1)理解机械生物学；ii)使用有目的的生物材料重建干细胞生态位。因此，我们计划通过开发一种复杂的显微镜来了解底物硬度在维持/指导细胞表型方面所起的作用，这种显微镜可以量化细胞及其直接环境的力学特性，同时在基因和蛋白质水平上测量细胞对这种环境的反应。我们的新方法只使用光，因此不需要与样品直接物理接触，可以深入到组织结构中。最重要的是，它可以在活细胞的生理条件下进行，便于在动态条件下进行实时测量。这样的实验以前是不可能的。然而，基于我们最近成功地建立了一个共聚焦成像系统来测量三维组织刚度，我们认为这现在是可能的。利用布里渊光谱学原理，该系统以共聚焦光学排列方式检测样品中声子的非弹性散射光，以促进非接触、直接读出组织的机械性能。我们现在希望探索是否可以同时使用相同的共聚焦系统来激发探针组织内的荧光分子。因此，我们的目标是修改我们现有的机器，使荧光标记的蛋白质（来自细胞，组织或组织工程构建物）的共聚焦图像可以被解析和覆盖，同时测量所述细胞/组织的机械特性。最终，这将首次以前所未有的细节，直接、实时地描述细胞分子对不同局部机械环境的反应。

Mechanobiology is a rapidly developing area of science with the potential to improve significantly our knowledge of how cells function at a tissue/organ level. A better understanding of how cells react to their local mechanical environment (extracellular matrix, fluid flow, etc.) will also lead to advanced biomedical applications such as improved biomaterials and cell therapy. For such new therapeutic strategies, it is necessary to provide cells with a confined environment (niche) that enhances and regulates their proliferation and differentiation. In this regard, biomaterial technology currently leads the way in trying to fulfil these requirements by artificially recreating native-like three-dimensional environments. However, understanding how, why, and in what environment these cells differentiate in a lineage-specific manner is essential for understanding regenerative biology and developing better tissue engineering and stem cell therapy approaches. We believe that this underlying biology (i.e., the cell's responses to local mechanical stimuli) has not yet been properly investigated, due to a lack of appropriate tools, and that this is likely to undermine current attempts to i) understand mechanobiology; and ii) recreate the stem cell niche using purposeful biomaterials. Therefore, we plan to understand the role substrate stiffness plays in maintaining/directing cell phenotype at an unprecedented level by developing a sophisticated microscope that can quantify mechanical properties of the cells and their immediate environment whilst simultaneously measuring the cells' response to this environment at a gene and protein level. Our novel approach uses only light, and thus requires no direct physical contact with the sample and can probe deep into tissue structures. Most importantly, it can be performed under physiological conditions on live cells, facilitating real-time measurements under dynamic conditions. Such experiments have not previously been possible.However, based on our recent success in building a confocal imaging system to measure tissue stiffness in 3D, we think this is now possible. Using the principle of Brillouin spectroscopy, the system detects light that has been scattered inelastically from acoustic phonons in the sample in a confocal optical arrangement to facilitate a non-contact, direct readout of the mechanical properties of tissues. We now wish to explore if this same confocal system can be used simultaneously to excite fluorescent molecules within the probed tissues. As such, our aims are to modify our existing machine so that confocal images of fluorescently-labelled proteins (from cells, tissues or tissue-engineered constructs) can be resolved and overlaid with simultaneous measurements of the mechanical properties of said cells/tissues. Ultimately, this will allow, for the first time and with unprecedented detail, the direct, real-time account of a cell's molecular response to differing local mechanical environments.