MULTIPHOTON CONFOCAL MICROSCOPE

描述（由申请人提供）：这是一个请求，以取代1999年购买的双光子共聚焦显微镜，采用新系统，结合多光子显微镜的几个进步。所要求的系统包括蔡司LSM 710 NLO共焦系统和安装在蔡司Axio Observer Z1倒置显微镜上的Coherent Chameleon Ultra II钛蓝宝石激光器。与我们目前的系统和其他多光子系统相比，所要求的系统提供了增量改进和基本技术进步。蔡司系统的显著优势包括：1）探测器具有更高的信噪比，2）更快的扫描速度，3）更通用的激光线选择，4）灵活的发射收集，以及5）减少与服务相关的停机时间的硬件和软件。它将允许我们的用户进行现有显微镜无法进行的双光子成像研究，包括1）从900-1080 nm激发（例如YFP，RFP，DiI），2）双激发，3）检测较弱的信号，以及4）成像较厚的样品（>100-200 5 m）。由于它使用的软件和配件与我们核心设施中的其他显微镜基本相同，因此它将为用户提供轻松的过渡，同时增加了几项重要的新功能。最后，该系统具有成本效益，因为我们可以利用现有的配件（例如镜头，DIC光学器件和用于活细胞成像的环境控制）。因此，所要求的仪器的费用大大低于同等新系统的费用。主要用户在光学成像方面具有广泛的专业知识，并有非常复杂的成像需求。例如，我们的主要用户将使用所需仪器在长时间间隔（1-2天）内对厚的活组织样本中的一个或多个荧光团进行4D成像（延时成像结合3D重建），这些荧光团具有可变的信号强度。这些研究对于在器官培养中的肾脏发育期间、在皮肤中的肿瘤形成期间以及在发育、成熟和/或老化的脑中可视化细胞分化、命运决定、迁移、分裂和变性是必不可少的。我们的主要用户是杰出的科学家，他们已经做出了基本的发现，包括运动分子动力蛋白，皮肤干细胞和细胞器运动的新机制。主要用户包括第一批研究体外肾脏发育的研究人员，从皮肤中分离干细胞，以及使用双光子显微镜观察神经元中的脊柱运动。毫不奇怪，所有主要用户的研究工作都得到了NIH赠款。目前，哥伦比亚大学没有显微镜具有所要求的仪器的能力。因此，这一设备将提高用户组完成正在进行的项目的能力，使新项目得以启动，并避免工作和费用的重复。该工具将被纳入自1997年以来一直由公共政策研究所指导的生产性核心基金，并得到重要的机构支持。作为我们的共聚焦和专业显微镜设施的组成部分，该仪器将提供给整个大学的主要用户和其他人，促进合作和思想交流。

DESCRIPTION (provided by applicant): This is a request to replace a 2-photon confocal microscope that was purchased in 1999 with a new system incorporating several advances in multiphoton microscopy. The requested system consists of the Zeiss LSM 710 NLO confocal system and Coherent Chameleon Ultra II titanium-sapphire laser mounted on the Zeiss Axio Observer Z1 inverted microscope. The requested system offers both incremental improvements and funda- mental technological advances compared to our current system and other multiphoton systems. Among the significant advantages of the Zeiss system are 1) detectors with improved signal:noise ratio, 2) faster scanning, 3) more versatile laser line choice, 4) flexible emission collection, and 5) hardware and software that reduce service-related downtime. It will allow our users to carry out 2-photon imaging studies that cannot be performed with the existing microscope including 1) excitation from 900-1080 nm (e.g. YFP, RFP, DiI), 2) multicolor exci- tation, 3) detection of weaker signals, and 4) imaging thicker samples (>100-200 5m). Since it uses largely the same software and accessories as other microscopes in our core facility, it will provide an easy transition for users, while adding several important new capabilities. Finally, the system is cost-effective, as we can take ad- vantage of existing accessories (e.g. lenses, DIC optics, and environmental control for live-cell imaging). As a result, the cost of the requested instrument is significantly less than that of an equivalent new system. The Major Users have extensive expertise in optical imaging, and very sophisticated imaging needs. For example, our Major Users will use the requested instrument to carry out 4D imaging (time-lapse imaging combined with 3D reconstruction) of one or more fluorophores, of variable signal strength, in thick, living tissue samples over long time intervals (1-2 days). These studies are essential for visualization of cell differentiation, fate determination, migration, division, and degeneration during kidney development in organ culture, during tumor formation in skin, and in developing, mature and/or aging brain. Our Major Users are preeminent scientists who have made fundamental discoveries including the motor molecule dynein, stem cells in skin, and novel mechanisms for organelle movement. The Major Users include the first researchers to study kidney development in vitro, to isolate stem cells from skin, and to visualize spine motility in neurons using 2-photon microscopy. Not surprisingly, the research efforts of all Major Users are supported by NIH grants. Currently, no microscope at Columbia University has the capabilities of the requested instrument. Therefore, this equipment will enhance the user group's ability to complete projects underway, allow new projects to be initiated, and avoid duplication of effort and expense. The instrument will be incorporated into a productive core facility that has been directed by the PI since 1997, and receives significant institutional support. As a compo- nent of our Confocal and Specialized Microscopy Facility, the instrument will be accessible to Major Users and others throughout the University, promoting collaboration and exchange of ideas.