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

用于研究细胞动力学的双摄像头采集旋转圆盘共焦显微镜系统

• 批准号: RTI-2023-00091
• 负责人: Botelho, Roberto
• 金额: $ 10.93万
• 依托单位: Ryerson University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748527
• 关键词: Dual; Camera; Acquisition; Spinning; Disc

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.

我们由 NSERC 资助的研究项目有一个共同的长期目标：剖析和更好地理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环境，以及它们如何相互沟通。在此框架内，更具体地说，我们研究细胞如何控制细胞器的基本问题，细胞器将细胞内的无数反应区分开来。安东内斯库, Botelho，Gupta，Mattiazzi Usaj），膜和分子货物如何在细胞器之间分类和运输（Botelho，Gupta，Antonescu），细胞如何响应生长因子，代谢因素和环境损伤（Antonescu，Mattiazzi Usaj，Olson，Sabatinos） ，细胞如何附着、迁移和处理物理力（Antonescu，Olson），细胞如何根据 DNA 损伤和免疫信号等环境因素保护、修复和重塑染色质和基因表达（Botelho、Mattiazzi Usaj、Sabatinos），以及尽管遗传同质性，所有这些事件如何导致表型异质性（Antonescu、Mattiazzi Usaj、Sabatinos）虽然我们显然有个人利益，但我们有共同的长期目标，从而导致多种合作。我们团队之间的共同监督、HQP 以及共享专业知识和研究工具是我们团队的一个核心和通用工具，即旋转圆盘共聚焦 (SDC) 荧光的使用。使用荧光探针和荧光蛋白质来可视化和量化亚细胞和细胞组织和动态，并揭示其行为和功能 SDC 显微镜是任何现代细胞生物学实验室的主力，它可以快速采集高分辨率和清晰的图像。重要的是，我们依靠活细胞 SDC 显微镜来获取动态信息，以响应药物治疗或遗传畸变等事件。然而，就其本质而言，活细胞 SDC 显微镜需要对一种情况进行成像。这对我们的研究和培训生产力造成了严重限制，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落射荧光显微镜升级为 SDC 显微镜系统。通过添加双摄像头采集 (DCA)，DCA-SDC 系统将在此次升级中添加新功能，使我们能够同时跟踪和量化两个荧光通道，非常适合解剖细胞器融合和裂变、内吞作用等快速过程。重要的是，第二个 SDC 系统将通过减轻现有 SDC 显微镜的巨大压力来提高我们的生产力，并通过为 HQP 提供更大的灵活性来执行其研究项目，从而使我们的研究项目更加公平。研究和培训。