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最先进的旋转盘共聚焦显微镜系统，专门设计用于哺乳动物细胞和组织的活细胞成像。该仪器将由7名美国国立卫生研究院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个美国国立卫生研究院资助的项目。该显微镜系统将用于的主要实验类型包括活细胞和组织中荧光标记蛋白质的多维成像，用FRET分析蛋白质活性，用FRAP或光激活分析蛋白质和细胞器动力学，以及光消融或释放活细胞或组织中的笼化化合物。我们要求的系统包括尼康Ti-E倒置显微镜支架上的横河CSU-X1旋转磁盘扫描仪，该扫描仪具有内置的焦点反馈系统，可在高分辨率下成像数小时，无明显的焦点漂移。该显微镜还将配备一个环境室，一个电动舞台，以及用于高分辨率和高灵敏度成像的CCD相机。需要油浸和水浸物镜，以便在2D或3D环境中以最高的光学分辨率从活细胞或组织中获得最佳的数据收集。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成像。此外，该仪器将配备基于脉冲染料激光的光漂白/光消融模块，将其用途扩展到简单观察活体标本之外。目前，在UCSF没有旋转盘显微镜系统具有FRAP或光激活能力，这严重限制了可以进行的实验类型。预计主要用户使用该仪器的时间约为80%。然而，由于该仪器将被安置在加州大学旧金山分校帕纳萨斯生物成像发展中心并由其管理，培训和使用将提供给加州大学旧金山分校社区内其他由nih资助的研究人员。

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.