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，提供重要的新功能，将推进生物医学、生物技术、环境和材料科学主题的领先研究和培训计划。主要组件包括一个多激光引擎和光束组合器，一个高灵敏度的EMCCD相机和三色图像分配器，雪崩光电二极管，光子计数电子设备，以及一个倒置显微镜框架和光学，滤光片，以及集成组件和组装整个系统所需的光力学。所提出的系统将在其多色单分子测量的能力是独一无二的，在具有固定的荧光团的成像格式和具有自由扩散的荧光团的非成像格式。提议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。具体来说，该系统将提供多达三种颜色并行的单分子荧光成像，多达四种平行颜色通道的荧光爆发重合测量，荧光相关光谱（FCS）和多达四种颜色的荧光相互关联光谱（FCCS）。人们相信，拟议中的系统在加拿大将是独一无二的。该系统将用于开发超灵敏的生物分析，包括对极其罕见的基因突变的分析和复杂细胞过程的成像探针；研究大气气溶胶粒子的扩散和化学反应；用于蛋白质折叠动力学的高分辨率结构分析；并用于表征生物分子结合过程和多蛋白复合物的形成。该系统将成为UBC先进光谱和成像研究实验室（LASIR）的一部分。除了在多个领域进行尖端研究外，拟议的系统还将加强对50多名在职研究生、本科生和博士后学员的研究和培训，并在未来六年内增加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.