Olympus TIRF Microscope for Simultaneous Multichannel Live cell Imaging

描述（由申请人提供）：达特茅斯医学院病理学、生物化学、医学、微生物学和免疫学系以及达特茅斯学院生物科学系的 6 名 NIH 资助研究人员和一名初级研究人员组成的多元化小组请求资金从奥林巴斯美国购买最先进的多色 TIRFM 活细胞成像系统。该系统集成了奥林巴斯IX-81倒置显微镜、自动多色全内反射显微镜（TIRFM）照明器（奥林巴斯Celltirf），配备三个激光器405/491/561，并能够进行FRAP/光激活；与 DualView 分束器耦合的超快/高灵敏度 sCMOS 相机 (Andor Neo)；完全集成的 MetaMorph for Olympus 采集/分析软件针对 1) 快速/超灵敏 4D 四通道采集进行了优化； 2) FRAP 和光活化以及 3) 粒子追踪；新的最先进的工作站将补充我们大量使用的 Zeiss LSM 510 META 和老化的单通道手动 TIRFM 系统，同时在多通道采集、速度和灵敏度方面实现重大升级。重要的是，多色 TIRFM 系统将为光学细胞成像设施提供新的/独特的总内部荧光功能，该设施经常被多个学校和部门（包括超过 75 个实验室和约 300 个用户）使用。我们的研究人员均获得 NIH 的充足资助，致力于研究一系列细胞生物学和生化问题，旨在了解基本细胞过程的分子机制，例如膜运输、内吞作用、病原体感染性和细胞骨架组织以及与质膜的相互作用。要圆满完成 NIH 资助的这些用户项目的既定目标，需要以高空间和时间分辨率以及降低的光毒性来检查活细胞中的动态细胞过程。本提案中描述的全内反射显微镜是一种最先进的仪器，它通过使用成熟的技术来满足这些需求。在所要求的配置中，多色 TIRFM 工作站将为我们不同的研究人员小组提供广泛的基本/高级活细胞显微镜功能，而这些功能目前在达特茅斯缺乏/不存在。它将成为服务整个达特茅斯生命科学界的同类仪器中的唯一工具。新系统将在 Norris Cotton 综合癌症中心光学成像核心设施 (OICF) 内方便地定位、管理和维护；它将被纳入OICF现有的、完善的充值系统中，以确保回收供应和维护资金。

DESCRIPTION (provided by applicant): A diverse group of 6 NIH funded investigators and a junior investigator within Departments of Pathology, Biochemistry, Medicine, Microbiology and Immunology at Dartmouth Medical School and Biological Sciences at Dartmouth College request funds to purchase a state of the art multicolor TIRFM live cell imaging system from Olympus America. The system integrates an Olympus IX-81 inverted microscope, an automated multicolor total internal reflection microscopy (TIRFM) illuminator (Olympus Celltirf) equipped with three lasers 405/491/561, and capable of FRAP/ photoactivation; an ultra-fast/highly sensitive sCMOS camera (Andor Neo) coupled to a DualView beam splitter; fully integrated MetaMorph for Olympus acquisition/analysis software optimized for 1) rapid/ultra-sensitive 4D four channel acquisition; 2) FRAP and photoactivation and 3) particle tracking; The new state-of-the-art workstation will complement our heavily utilized Zeiss LSM 510 META and aging single channel manual TIRFM system, while constituting a significant upgrade in multi channel acquisition, speed and sensitivity. Importantly, the multicolor TIRFM system will provide new/unique total internal fluorescence capabilities for the Optical Cellular Imaging Facility that s utilized regularly by multiple schools and departments comprising >75 laboratories and ~300 users. Our investigators, all well funded through the NIH, work on a host of cell biological and biochemical questions aimed at understanding molecular mechanisms of fundamental cellular processes such as membrane trafficking, endocytosis, pathogen infectivity and cytoskeletal organization and interaction with the plasma membrane. Satisfactory completion of the stated goals of the NIH-sponsored projects of these users requires examination of dynamic cellular processes in live cells with high spatial and temporal resolution and reduced phototoxicity. The total internal reflection microscope described in this proposal is a state of the art instrument tht satisfies these needs by using proven technology. In the requested configuration, the multicolor TIRFM workstation will afford our disparate group of investigators a broad range of basic/advanced live-cell microscopy capabilities that are presently deficient/absent at Dartmouth. It would represent the only instrument of its kind to serve the entire Dartmouth life science community. The new system will be conveniently located, administered, and maintained, in the Norris Cotton Comprehensive Cancer Center Optical Imaging Core Facility (OICF); and it will be incorporated into the OICF's existing, well established recharge system to ensure recovery of funds for supplies and maintenance.