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

利用活细胞成像，我们已经确定了许多与先天免疫相关的基本原理。例如，我们首次可视化了肺泡巨噬细胞，并表明它们可以通过Kohn孔从肺泡移动到肺泡，以持续维持体内平衡，并迅速清除任何吸入的细菌，否则这些细菌会不必要地招募中性粒细胞诱导炎症（Cell 2020）。我们现在想可视化是否固定这些巨噬细胞可能导致炎症，缺氧，改变代谢和纤维化。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微米，因此可以成像），并迅速帮助组织重新血管化，以实现适当的愈合。通过测量这个小区域的氧水平和体内平衡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组织检查等），将大大提高我们对中性粒细胞和巨噬细胞在这一过程中的重要性的理解。心脏中的类似模型会导致相反的结果，即损伤不断增加，最终形成疤痕。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，导致缺氧和纤维化。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和使用Imaris分析）表明，不同形式的胶原沉积形成不同的纤维化病变。我们建议将荧光寿命成像显微镜（FLIM）添加到我们现有的徕卡双光子深潜SP8显微镜上，这样我们就可以使用现有设备无法实现的非荧光方法来测量新陈代谢和纤维化。FLIM使研究人员能够研究生物体中细胞的完整性，这是基于荧光的系统无法做到的。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，并在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。通过我们组织的年度加拿大光学显微镜课程，由专家举办的培训课程将教育学员这些新方法。我们也向其他团体开放所有新平台。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，可能会允许对家养动物和人类进行非侵入性成像，以提高诊断水平，并可能让人们更好地了解许多生物过程。

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.