Microscope for Multicolour Single Molecule Fluorescence Detection, Imaging, and (Cross-)Correlation Spectroscopy

单分子荧光成像和光谱系统将由商业组件构建，以提供重要的新功能，从而推进生物医学，生物技术，环境和材料科学主题的领先研究和培训计划。主要组件包括多激光引擎和光束组合器，高灵敏度EMCCD相机和三色图像分离器，雪崩光电二极管，光子计数电子器件，倒置显微镜框架和光学，过滤器，以及集成组件和组装整个系统所需的光学机械。所提出的系统将是独特的多色单分子测量的能力，在两个成像格式与固定化荧光团和非成像格式与自由扩散荧光团。所提出的设计提供了最先进的能力和灵活性，以支持多个用户和应用程序。具体而言，该系统将提供多达三种颜色的单分子荧光成像，多达四个平行颜色通道的荧光猝发符合测量，荧光相关光谱（FCS）和多达四色荧光互相关光谱（FCCS）。据信，拟议中的系统将是加拿大独一无二的。该系统将用于开发超灵敏的生物分析，包括极其罕见的基因突变分析和复杂细胞过程的成像探针;研究大气气溶胶颗粒中的扩散和化学反应;对蛋白质折叠动力学进行高分辨率结构分析;以及对生物分子结合过程和多蛋白质复合物的形成进行表征。该系统将成为UBC高级光谱和成像研究实验室（LASIR）的一部分。除了在多个领域开展前沿研究外，拟议的系统还将加强50多名现有研究生、本科生和博士后学员的研究和培训，并在未来六年内培训200多名学员（预计）。

A single molecule fluorescence imaging and spectroscopy system will be built from commercial components to provide important new capabilities that will advance leading research and training programs with biomedical, biotechnological, environmental, and materials science themes. The main components include a multi-laser engine and beam combiner, a high-sensitivity EMCCD camera and three-colour image splitter, avalanche photodiodes, photon counting electronics, and an inverted microscope frame and optics, filters, and optomechanics needed for integrating components and assembling the full system. The proposed system will be unique in its capacity for multicolour single molecule measurements in both an imaging format with immobilized fluorophores and a non-imaging format with freely diffusing fluorophores. The proposed design offers both state-of-the-art capability and flexibility to support multiple users and applications. Specifically, the system will offer single molecule fluorescence imaging with up to three colours in parallel, fluorescence burst coincidence measurements with up to four parallel colour channels, fluorescence correlation spectroscopy (FCS), and up to four-colour fluorescence cross-correlation spectroscopy (FCCS). It is believed that the proposed system would be one-of-a-kind in Canada. The system will be used to develop ultra-sensitive biological assays, including assays for extremely rare genetic mutations and imaging probes for complex cellular processes; to study diffusion and chemical reactions in atmospheric aerosol particles; for high-resolution structural analysis of protein folding dynamics; and for characterization of biomolecular binding processes and the formation of multi-protein complexes. The system will be part of the Laboratory for Advanced Spectroscopy and Imaging Research (LASIR) at UBC. In addition to enabling cutting-edge research in multiple areas, the proposed system will enhance the research and training of more than 50 current graduate student, undergraduate student, and postdoctoral trainees, and more than 200 total trainees (projected) over the next six years.