Brillouin confocal microscopy for biomechanical studies of metastatic cascade in 3D microenvironments

 描述（由申请人提供）：细胞与周围微环境之间的机械相互作用在很大程度上被认为是在肿瘤进展、恶性转化和转移中关键性地调节细胞功能，但由于缺乏合适的测量工具，对其仍然知之甚少。虽然在单细胞测量力和变形方面取得了很大进展，但测量细胞内和细胞外模量仍然具有挑战性，特别是在3D微环境中。然而，已知细胞/ECM机械性质是重要的，因为它们将环境机械刺激的呈现与机械相关信号传导途径的激活联系起来。在过去的几年里，我们一直在开发一种全光学方法来应对这一挑战，称为布里渊显微镜。布里渊细胞显微镜有望在3D微环境中以高分辨率、非微扰、无接触的方式绘制细胞内和细胞外弹性模量。正如强有力的初步数据所示，我们已经实现了几个关键的里程碑，证明了我们在细胞和亚细胞水平上测量相关机械性能的能力。在这项研究中，我们将开发和验证我们的癌症相关研究的显微镜平台。目标1将专注于仪器的先进开发，以达到与接触式机械测试相当的灵敏度的快速机械成像。目标2将专注于直接验证我们的技术对黄金标准技术使用已知的属性和设置是相关的转移进展。最后，目标3将在肿瘤细胞外渗的背景下测试我们的技术，这是一个具有挑战性的实验环境，没有其他技术可以进行机械表征。实现外渗的足够速度和灵敏度将保证我们的技术在肿瘤机械生物学方面的广泛适用性，并生动地展示我们的技术可以提供的新信息类型。重要的是，布里渊技术将被集成到共焦显微镜中，为广泛使用的仪器添加机械模式。严格的技术开发、验证、基准测试和现场测试过程将产生一个具有前所未有的研究细胞基质生物力学能力的仪器平台，并准备被癌症生物学研究界广泛采用。

 DESCRIPTION (provided by applicant): The mechanical interplay between cell and the surrounding microenvironment is largely recognized to critically regulate cell function in tumor progression, malignancy transformation and metastasis but it remains poorly understood due to the lack of suitable measurement tools. While much progress has been achieved in the context of single-cell measurements of forces and deformation, measuring intracellular and extracellular moduli remains challenging, especially in 3D microenvironments. Yet, cell/ECM mechanical properties are known to be important because they link the presentation of an environmental mechanical stimulus to the activation of a mechano-related signaling pathway. In the past few years, we have been developing an all-optical approach to this challenge, named Brillouin microscopy. Brillouin cellular microscopy promises to map the intracellular and extracellular elastic modulus at high resolution, non-perturbatively, without contact in 3D microenvironments. As shown in strong preliminary data, we have achieved several key milestones that demonstrate our ability to measure relevant mechanical properties at a cellular and sub-cellular level. In this research we will develop and validate our microscopy platform for cancer-related studies. Aim 1 will focus on the advanced development of the instrument to reach rapid mechanical imaging at sensitivities comparable to contact-based mechanical tests. Aim 2 will focus on a direct validation of our technology against gold-standard techniques using properties and settings known to be relevant for metastatic progression. Finally, Aim 3 will test our technology within the context of tumor cell extravasation, a challenging experimental setting where no other technology can perform mechanical characterizations. Achieving sufficient speed and sensitivity for extravasation will guarantee the broad applicability of our technology for tumor mechanobiology and vividly demonstrate the type of novel information that our technology can provide. Importantly, Brillouin technology will be integrated into confocal microscopes to add a mechanical modality to widely-used instruments. The rigorous process of technology development, validation, benchmarking and field-testing will yield an instrumental platform with unprecedented capabilities to study cell-matrix biomechanics and ready to be widely adopted by the cancer biology research community.