Olympus TIRF Microscope for Simultaneous Multichannel Live cell Imaging

描述(由申请人提供)：由6名NIH资助的研究人员和达特茅斯医学院病理、生化、医学、微生物学和免疫学系的一名初级研究员组成的多样化小组申请资金，从奥林巴斯美国公司购买最先进的多色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405/491/561，并能够FRAP/光激活；一个连接到DualView分束器的超高速/高灵敏度sCMOS相机(Andor Neo)；为奥林巴斯采集/分析软件优化的完全集成的变形器，优化用于1)快速/超灵敏4D四通道采集；2)FRAP和光激活；3)粒子跟踪；新的最先进的工作站将补充我们经常使用的蔡司LSM 510元和老化的单通道手动TIRFM系统，同时在多通道采集、速度和灵敏度方面进行重大升级。重要的是，多色TIRFM系统将为S经常使用的光学细胞成像设备提供新的/独特的全内部荧光能力，该设备由&gt；75个实验室和约300名用户组成。我们的研究人员都是由美国国立卫生研究院资助的，他们致力于一系列细胞生物学和生化问题的研究，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。要圆满完成NIH赞助的这些用户项目的既定目标，需要在具有高空间和时间分辨率并降低光毒性的活细胞中检查动态细胞过程。本提案中描述的全内反射显微镜是一种通过使用经过验证的技术来满足这些需求的最先进的仪器。在要求的配置中，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/高级活细胞显微镜能力，这些能力目前在达特茅斯是不足的/不存在的。它将是唯一一种服务于整个达特茅斯生命科学界的同类工具。新系统将在诺里斯·科顿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，以确保收回供应和维护资金。

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.