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

点击翻译按钮获取中文摘要

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.