Olympus TIRF Microscope for Simultaneous Multichannel Live cell Imaging

描述（由申请人提供）：一个由6名NIH资助的研究人员和达特茅斯医学院病理学、生物化学、医学、微生物学和免疫学以及达特茅斯学院生物科学系的一名初级研究人员组成的多元化小组向奥林巴斯美国公司申请资金，以购买最先进的多色TIRFM活细胞成像系统。该系统集成了一个奥林巴斯IX-81倒置显微镜，一个自动多色全内反射显微镜（TIRFM）照明器（奥林巴斯Celltirf），配备了三个激光器405/491/561，并能够FRAP/光激活；一个超快速/高灵敏度的sCMOS相机（Andor Neo）耦合到一个DualView分束器；完全集成的MetaMorph奥林巴斯采集/分析软件优化1)快速/超灵敏的4D四通道采集；2) FRAP和光活化；3)粒子跟踪；新的最先进的工作站将补充我们大量使用的蔡司LSM 510 META和老化的单通道手动TIRFM系统，同时构成多通道采集，速度和灵敏度的重大升级。重要的是，多色TIRFM系统将为光学细胞成像设备提供新的/独特的内部全荧光能力，该设备被多个学校和部门定期使用，包括750个实验室和300个用户。我们的研究人员都是由美国国立卫生研究院资助的，他们研究大量的细胞生物学和生物化学问题，旨在了解基本细胞过程的分子机制，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。圆满完成这些用户的nih资助项目的既定目标需要检查活细胞的动态细胞过程，具有高空间和时间分辨率，并降低光毒性。本提案中描述的全内反射显微镜是一种最先进的仪器，通过使用成熟的技术来满足这些需求。在要求的配置中，多色TIRFM工作站将为我们不同的研究小组提供广泛的基本/高级活细胞显微镜功能，这是达特茅斯目前缺乏的。它将是同类仪器中唯一一个服务于整个达特茅斯生命科学界的仪器。新系统将位于Norris Cotton综合癌症中心光学成像核心设施（OICF）中，便于管理和维护；它将被纳入OICF现有的完善的补给系统，以确保回收供应和维护的资金。

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.