A Dual Camera Acquisition-Spinning Disc Confocal Microscope System to Study Cellular Dynamics

我们由NSERC资助的研究项目有一个共同的长期目标：剖析和更好地了解动态控制细胞功能的分子信号和过程。总的来说，我们的目标是了解细胞如何感知和适应他们的环境，他们如何相互交流，以及他们如何实现和适应他们的亚细胞组织以生长、生存和适应不同的情况。在这个框架内，更具体地说，我们研究了以下基本问题：细胞如何控制它们的细胞器，这些细胞器划分了细胞内的各种反应(Antonescu，Botelho，Gupta，Mattiazzi Usaj)，细胞膜和分子货物如何在细胞器之间分类和运输(Botelho，Gupta，Antonescu)，细胞如何对生长因子、代谢因素和环境侮辱做出反应(Antonescu，Mattiazzi Usaj，Olson，Sabatinos)，细胞如何附着、迁移和处理物理力量(Antonescu，Olson)，细胞如何保护、修复和重塑染色质和基因表达，环境因素如DNA损伤和免疫信号(Botelho，Mattiazzi Usaj，Sabatinos)，以及细胞如何保护、修复和重组染色质和基因表达以及所有这些事件如何导致表型异质性，尽管遗传同质性(Antonescu，Mattiazzi Usaj，Sabatinos)。虽然我们显然有各自的利益，但我们有共同的长期目标，导致我们的团队之间进行多次合作，共同监督HQP，并共享专业知识和研究工具。我们团队的一个核心和常见工具是使用旋转圆盘共聚焦(SDC)荧光显微镜，使用荧光探针和设计成荧光的蛋白质来可视化和量化亚细胞和细胞的组织和动态，并揭示它们的行为和功能。SDC显微镜是任何现代细胞生物学实验室的主力，它允许快速获取细胞的高分辨率和清晰图像。重要的是，我们依靠活细胞SDC显微镜来获取动态信息，以应对药物治疗或遗传异常等事件。然而，就其本质而言，活细胞SDC显微镜需要一次成像一种情况。这严重限制了我们的研究和培训生产率，因为我们的设施中只有一台SDC显微镜。我们需要帮助将现有的荧光显微镜升级为SDC显微镜系统。然而，我们建议通过添加双摄像头采集(DCA)来在此升级中构建新功能。DCA-SDC系统将允许我们同时跟踪和定量两个荧光通道，非常适合于以前所未有的速度和精度解剖细胞器融合和分裂、细胞吞噬和细胞骨架重塑等快速过程，同时获得高分辨率图像。重要的是，第二个SDC系统将通过缓解现有SDC显微镜的巨大压力来提高我们的生产率，并通过给予HQP更多的灵活性来执行他们的研究和培训，使我们的研究计划更加公平。

Our NSERC-funded research programs share a common long-term goal: to dissect and better understand the molecular signals and processes that dynamically control cellular function. Collectively, we aim to understand how cells sense and adapt to their environment, how they communicate with each other, and how they achieve and adapt their subcellular organization to grow, survive, and adapt to various situations. Within this framework, and to be more specific, we study fundamental questions of how cells control their organelles, which compartmentalize myriad reactions within cells (Antonescu, Botelho, Gupta, Mattiazzi Usaj), how membrane and molecular cargo is sorted and trafficked between organelles (Botelho, Gupta, Antonescu), how cells respond to growth factors, metabolic factors, and environmental insults (Antonescu, Mattiazzi Usaj, Olson, Sabatinos), how cells attach, migrate and handle physical forces (Antonescu, Olson), how cells protect, repair, and remodel chromatin and gene expression upon environmental factors like DNA damage and immune signals (Botelho, Mattizzi Usaj, Sabatinos), and how all these events can lead to phenotypic heterogeneity despite genetic homogeneity (Antonescu, Mattiazzi Usaj, Sabatinos). While we clearly have individual interests, we have mutual long-term goals, leading to multiple collaborations between our groups, co-supervision of HQP, and sharing of expertise and research tools. A central and common tool for our groups is the use of spinning disc confocal (SDC) fluorescence microscopy to visualize and quantify subcellular and cellular organization and dynamics using fluorescent probes and proteins engineered to fluorescence and reveal their behaviour and function. SDC microscopy is a workhorse for any modern cell biology lab, which allows for rapid acquisition, high-resolution and sharp images of cells. Importantly, we rely on live-cell SDC microscopy to acquire dynamic information in response to events like a drug treatment or genetic aberration. However, by its very nature, live-cell SDC microscopy requires imaging one condition at a time. This has imposed a severe limitation on our research and training productivity because we only have a single SDC microscope available in our facilities. We need aid to upgrade an existing epifluorescence microscope into a SDC microscope system. However, we propose to build new functionalities into this upgrade by adding dual-camera acquisition (DCA). The DCA-SDC system will allow us to track and quantify two fluorescent channels concurrently, ideally suited to dissect rapid processes like organelle fusion and fission, endocytosis, and cytoskeletal remodelling with unprecedented speed and precision, while obtaining high-resolution images. Importantly, a second SDC system will boost our productivity by relieving intense pressure on the existing SDC microscope and make our research programs more equitable by giving HQP more flexibility to perform their research and training.