Microscopy Infrastructure for Controlling and Measuring Dynamic Cellular Processes

点击翻译按钮获取中文摘要

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. **