Adding FLIM to Image and Analyze Metabolism Hypoxia, Fibrosis and beyond during an Immune Response

将 FLIM 添加到图像中并分析免疫反应期间的代谢缺氧、纤维化等

• 批准号: RTI-2021-00098
• 负责人: Kubes, Paul
• 金额: $ 10.51万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=718026
• 关键词: Adding; FLIM; Image; Analyze; Metabolism

Using live cell imaging we have identified numerous fundamental principles related to innate immunity. For example, we for the first time visualized alveolar macrophages and showed they can move from alveolus to alveolus via the Pores of Kohn to constantly maintain homeostasis and to rapidly scavenge any inhaled bacteria that would otherwise unnecessarily recruit neutrophils inducing inflammation (Cell 2020). We would now like to visualize whether immobilizing these macrophage may lead to inflammation poor oxygenation altered metabolism and fibrosis. In another study we identified that neutrophils (Science 2017) and peritoneal macrophages (Cell 2016 and now funded by NSERC) come into a site of focal liver injury (500 um in size so it can be imaged) and quickly help to re-vascularize the tissue for proper healing. Understanding when the area is healed by measuring both oxygen levels and homeostatic metabolism in this small local area (need imaging- as too small to try to take biopsies etc) would greatly improve our understanding of the importance of the neutrophil and macrophage in this process. Similar models in the heart lead to opposite effects of an increasing injury that eventually scars. In an infectious wound we see overgrowth of blood vessels when the immune system is perturbed and no healing. A readout of the metabolic status of parenchymal cells will help determine when interventions disrupt homeostasis of the tissues leading to poor oxygenation and fibrosis. Presently, we are unable to measure metabolism or collagen subtype deposition, but preliminary data (testing FLIM and analyzing with Imaris) suggest different forms of collagen deposition forming different fibrotic lesions. We propose to add Fluorescence Lifetime Imaging Microscopy (FLIM) onto our existing Leica Two Photon Deep Dive SP8 microscope so that we can measure metabolism, and fibrosis using non-fluorescence based approaches that are not possible with existing equipment. FLIM is allowing investigators to study the integrity of cells in living organisms that fluorescence based systems fail to do. While our fluorescence based systems allow us to track and delineate molecular mechanisms understanding immunity, its impact on tissues especially the local environment are desperately needed. FLIM will provide us with this key information and in the future may allow imaging of immune cell subsets independent of fluorescence. We also require appropriate analysis software to analyze the data we acquire. Training sessions run by experts through our annual Canadian Light Microscopy Course that we organize will educate the trainees in these new approaches. We make all new platforms available to other groups as well. Ultimately, this non-flourescent approach may open up a whole new field of imaging in Canada, may allow for non-invasive imaging in domesticated animals and humans to improve diagnostics and may allow better understanding of many biological processes.

使用活细胞成像，我们已经确定了许多与先天免疫相关的基本原则。 例如，我们第一次可视化肺泡巨噬细胞，并显示它们可以通过Kohn孔从肺泡移动到肺泡，以不断保持体内平衡，并快速清除任何吸入的细菌，否则这些细菌将不必要地招募中性粒细胞诱导炎症（Cell 2020）。 我们现在想看看固定这些巨噬细胞是否会导致炎症、缺氧、代谢改变和纤维化。 在另一项研究中，我们发现中性粒细胞（Science 2017）和腹膜巨噬细胞（Cell 2016，现在由NSERC资助）进入局灶性肝损伤部位（大小为500 um，因此可以成像），并迅速帮助组织血管化以进行适当愈合。 通过测量这个小的局部区域的氧气水平和稳态代谢来了解该区域何时愈合（需要成像-因为太小而无法进行活检等）将大大提高我们对中性粒细胞和巨噬细胞在此过程中的重要性的理解。 心脏中的类似模型导致相反的效果，即不断增加的损伤最终会留下疤痕。 在感染性伤口中，当免疫系统受到干扰而无法愈合时，我们会看到血管过度生长。实质细胞代谢状态的读数将有助于确定干预何时破坏组织的稳态，导致氧合不良和纤维化。 目前，我们无法测量代谢或胶原亚型沉积，但初步数据（测试FLIM并使用Imaris分析）表明不同形式的胶原沉积形成不同的纤维化病变。我们建议在现有的Leica Two Photon Deep Dive SP 8显微镜上增加荧光寿命成像显微镜（FLIM），以便我们可以使用现有设备无法实现的非荧光方法测量代谢和纤维化。 FLIM允许研究人员研究活生物体中细胞的完整性，而基于荧光的系统无法做到这一点。 虽然我们基于荧光的系统使我们能够跟踪和描绘理解免疫的分子机制，但它对组织特别是局部环境的影响是迫切需要的。 FLIM将为我们提供这一关键信息，未来可能允许独立于荧光的免疫细胞亚群成像。我们还需要适当的分析软件来分析我们获得的数据。 由专家通过我们每年举办的加拿大光学显微镜课程举办的培训课程将教育学员这些新方法。我们也向其他团体提供所有新平台。 最终，这种非荧光方法可能会在加拿大开辟一个全新的成像领域，可能会允许在驯养动物和人类中进行非侵入性成像，以改善诊断，并可能更好地了解许多生物过程。