Microscopy Infrastructure for Controlling and Measuring Dynamic Cellular Processes

要破译生物系统是如何组织和调控的，需要详细了解分子在何时何地发挥特定功能。许多因素具有多种功能，因此在高时空分辨率下检测蛋白质和细胞的活性和动力学至关重要。尖端的光操纵技术允许对特定细胞位置的分子进行有针对性的激活、去活、寡聚或消融，而其他地方的分子池则不受影响。因此，这些方法提供了对细胞和生物体内分子功能的无与伦比的分析。这项建议寻求建立新的光操纵模块和高分辨率的实时成像系统，这将是多伦多大学医学院核心所独有的。我们的跨学科小组寻求获得基础设施，以改进最近获得的共焦显微镜，以测量和控制各种活细胞和组织中的动态细胞过程。我们方法的关键是通过光来调节分子的活性、位置或动力学的能力。为了激活特定区域中的特定分子，所需的强烈光照必须限制在细胞内。为了最大限度地发挥多个蜂窝位置的光调制潜力，视场必须均匀照明，以允许不同区域之间的直接定量比较。我们的Andor旋转圆盘共聚焦显微镜配备了北极光照明器，这是第一个均匀照亮大视场的技术。此外，该系统还包括实时超分辨率技术SRRF Stream，它可以将分子的空间分辨率提高多达十倍。因此，Andor公司最新的数字微镜设备、马赛克和Micropoint模块的加入将为光激活和烧蚀实验提供最灵活的照明和精确的光传输。*在我们的节点中获得一个具有光操纵能力的显微镜对于我们团队由NSERC资助的研究是绝对必要的。加拿大新的环境和气候变化法规限制了在我们设施外对活的转基因生物和细胞进行实验，这使得我们设施内的基础设施的住房变得重要。我们的设施中不存在配备光操纵模块的系统。因此，如果没有这个系统，我们团队对细胞骨架进行DNA微消融、组织损伤和光遗传操作的能力将极其有限。这种新的最先进的设备将对细胞和生物体内的分子功能产生独特的见解，从而为我们的团队和UofT的其他研究人员提供特殊的技术优势，以保持他们的国际竞争力。此外，该设备将有助于培训众多高素质的人员，以解决细胞生物学的基本问题。**

Deciphering how biological systems are organized and regulated requires a detailed understanding of where and when a molecule exerts a specific function. Many factors have multiple functions, making it vital to examine the activity and dynamics of proteins and cells at high spatiotemporal resolution. Cutting-edge photomanipulation techniques allow the targeted activation, deactivation, oligomerization or ablation of molecules at specific cellular locations, whilst pools of the molecules elsewhere remain unaffected. Consequently, these approaches provide unparalleled analysis of molecular function within cells and organisms. This proposal seeks to establish novel photomanipulation modules coupled with a high-resolution live-imaging system, which would be unique to the University of Toronto Faculty of Medicine core.******Our interdisciplinary group seeks to obtain infrastructure to modify a recently acquired confocal microscope to measure and control dynamic cellular processes in various live cells and tissues. Key to our approach is the ability to modulate the activity, location, or dynamics of a molecule by light. To activate specific molecules in defined regions, the intense illumination required must be restricted within the cell. To maximize the potential of photo-modulation at multiple cellular locations, the field of view must be equally illuminated to allow the direct quantitative comparison between the different regions. Our Andor spinning disc confocal microscope is equipped with a Borealis illuminator, which is the first technology to equally illuminate large fields of view. Moreover, the system includes a real-time super-resolution technique, SRRF Stream, that enhances the spatial resolution of molecules by up to ten-fold. Thus, the addition of Andor's latest Digital Micro-mirror Device, Mosaic, and Micropoint modules will offer the most flexibility in illumination and precise light delivery for photoactivation and ablation experiments possible. ******The acquisition of a microscope in our node with photomanipulation capabilities is absolutely essential for our team's NSERC-funded research. New Environment and Climate Change Canada regulations limit experimentation on live transgenic organisms and cells outside our facility, making the housing of this infrastructure within our facility important. A system equipped with photomanipulation modules does not exist within our facility. Thus, without this system our team's ability to carry out DNA micro-ablation, tissue wounding, and optogenetic manipulation of the cytoskeleton will be extremely limited. This new state-of-the-art equipment will yield unique insights into molecular functions within cells and organisms, thereby providing exceptional technological advantages to our team and other UofT researchers to maintain their international competitiveness. Furthermore, the equipment will contribute to training of numerous highly qualified personnel to tackle fundamental problems in cell biology. **