Spinning disk confocal / FRAP microscope for quantitative live cell imaging

描述（由申请人提供）：我们正在申请资金购买一个国家的最先进的转盘共聚焦显微镜系统，专门设计用于活细胞成像的哺乳动物细胞和组织。该仪器将由七名NIH资助的研究人员及其实验室组成的核心小组使用，以促进14个NIH资助的项目。该显微镜系统将用于实验的主要类型包括活细胞和组织中荧光标记蛋白质的多维成像，通过FRET分析蛋白质活性，通过FRAP或光活化分析蛋白质和细胞器动力学，以及活细胞或组织中笼状化合物的光消融或释放。我们所要求的系统包括一个Nikon Ti-E倒置显微镜支架上的Yokogawa CSU-X1旋转磁盘扫描仪，该扫描仪具有内置的焦点反馈系统，可以在高分辨率下成像数小时，而没有明显的焦点漂移。该显微镜还将配备一个环境室，一个机动阶段，和CCD相机的高分辨率和高灵敏度成像。要求油浸和水浸物镜以最高可实现的光学分辨率从2D或3D环境中的活细胞或组织中最佳收集数据。固态激光线要求在440，488，514和561 nm，以允许所有常用的荧光蛋白变体的成像。此外，该仪器将配备一个脉冲染料激光为基础的光漂白/光烧蚀模块扩展其实用性超出简单的观察活标本。目前，没有旋转盘显微镜系统存在于UCSF具有FRAP或光活化能力，这严重限制了可以进行的实验类型。预计主要用户将使用该仪器约80%的时间。然而，由于该仪器将被安置在UCSF Parnassus生物成像开发中心并通过该中心进行管理，因此UCSF社区内的其他NIH资助的研究人员将获得培训和使用。

DESCRIPTION (provided by applicant): We are requesting funds to purchase a state-of-the-art Spinning Disk Confocal Microscope System specifically designed for live cell imaging of mammalian cells and tissues. The instrument will be used by a core group of seven NIH-funded investigators and their laboratories to facilitate fourteen NIH- funded projects. The principle types of experiments for which this microscope system will be used include multidimensional imaging of fluorescently tagged proteins in living cells and tissues, analysis of protein activity by FRET, analysis of protein and organelle dynamics by FRAP or photoactivation, and photoablation or release of caged compounds in living cells or tissues. The system we are requesting includes a Yokogawa CSU-X1 spinning disk scanner on a Nikon Ti-E inverted microscope stand, which has a built-in focus feedback system allowing imaging for many hours at high resolution with no discernable focus drift. The microscope will also be equipped with an environmental chamber, a motorized stage, and CCD cameras for high resolution and high sensitivity imaging. Oil- and water- immersion objectives are requested for the optimal collection of data from live cells or tissues in a 2D or 3D environment at the highest achievable optical resolution. Solid-state laser lines are requested at 440, 488, 514, and 561 nm to permit imaging of all commonly used fluorescent protein variants. In addition, this instrument will be equipped with a pulsed dye laser-based photobleaching / photoablation module extending its usefulness beyond simple observation of live specimens. Currently, no spinning disk microscope system exists at UCSF that has FRAP or photoactivation capabilities, which severely limits the types of experiments that can be performed. It is expected that the major users will utilize the instrument for ~80% time. However, because the instrument will be housed in and managed through the UCSF Parnassus Biological Imaging Development Center, training and use will be available to additional NIH-funded investigators within the UCSF community.