Development of Brillouin Spectroscopy for Mechanotransduction Research

用于力传导研究的布里渊光谱学的发展

• 批准号: BB/N021576/1
• 负责人: Che Connon
• 金额: $ 19.21万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FN021576%2F1
• 关键词: Development; Brillouin; Spectroscopy; Mechanotransduction; Research

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.

力学生物学是一个快速发展的科学领域，有可能显着提高我们对细胞如何在组织/器官水平发挥作用的了解。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）做出反应也将带来先进的生物医学应用，例如改进的生物材料和细胞疗法。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），以增强和调节它们的增殖和分化。在这方面，生物材料技术目前在通过人工重建类似原生的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、为何以及在什么环境下以谱系特异性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，并且这可能会破坏当前的尝试：i）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基质刚度在以前所未有的水平维持/指导细胞表型中所起的作用，该显微镜可以量化细胞及其直接环境的机械特性，同时测量细胞对这种环境的响应基因和蛋白质水平。我们的新颖方法仅使用光，因此不需要与样品直接物理接触，并且可以深入探测组织结构。最重要的是，它可以在生理条件下对活细胞进行，促进动态条件下的实时测量。此类实验以前是不可能的。但是，基于我们最近成功构建了共焦成像系统来测量 3D 组织硬度，我们认为现在这是可能的。该系统利用布里渊光谱原理，在共焦光学装置中检测样品中声子非弹性散射的光，以促进非接触式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焦系统来激发被探测组织内的荧光分子。因此，我们的目标是修改我们现有的机器，以便可以解析荧光标记蛋白质（来自细胞、组织或组织工程构建体）的共焦图像，并与所述细胞/组织的机械特性的同步测量重叠。最终，这将首次以前所未有的细节直接、实时地描述细胞对不同局部机械环境的分子反应。