Olympus TIRF Microscope for Simultaneous Multichannel Live cell Imaging

描述（由申请人提供）：一个由6名NIH资助的研究人员和一名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初级研究人员组成的多元化小组要求提供资金，从Olympus America购买最先进的RTIRFM活细胞成像系统。该系统集成了奥林巴斯IX-81倒置显微镜，一个自动化的全内反射显微镜（TIRFM）照明（Olympus Celltirf），配备有三个激光器405/491/561，并且能够进行FRAP/光活化;超快/高灵敏度sCMOS相机（Andor Neo）耦合到DualView分束器;完全集成的MetaMorph for Olympus采集/分析软件，针对1）快速/超灵敏的4D四通道采集; 2）FRAP和光活化; 3）颗粒跟踪进行了优化;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，同时在多通道采集，速度和灵敏度方面进行重大升级。重要的是，该系统将为光学细胞成像设备提供新的/独特的总内部荧光能力，该设备经常被多个学校和部门使用，包括超过75个实验室和约300个用户。我们的研究人员，所有通过NIH资助，致力于一系列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。这些用户的NIH赞助的项目的既定目标的圆满完成需要在活细胞中检查动态细胞过程，具有高的空间和时间分辨率和降低的光毒性。本建议书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。在所要求的配置中，RTTIRFM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，这些功能目前在达特茅斯是缺乏的。它将是服务于整个达特茅斯生命科学界的唯一同类仪器。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现有的完善的再充电系统，以确保回收用于供应和维护的资金。

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.