Advanced Multi-color Confocal and FRAP-SAC Microscope

描述（由申请人提供）：我们正在申请资金购买一台马里亚纳实时共聚焦SDC显微镜，该显微镜配备1)一台横河CSUX Nipkow扫描仪；2)电动球差校正装置；3)五条激光线，可以使用广泛的荧光团，包括用于FRET的荧光团；4)用于荧光靶漂白（或光活化）研究的高速激光照明；5)采用高速Photometrics Evolve EM-CCD相机的双摄像头/图像分配器系统；6)封闭式co2调节培养箱。这种共聚焦系统对于满足主要用户和其他教师的直接研究需求是必要的，因为它提供了上述新功能，特别是SAC， FRET， FRAP，以及在受控环境中同时进行两色成像。此外，马里亚纳实时共聚焦SDC显微镜将增加可用仪器的容量，这对于越来越多的活细胞成像研究至关重要。这些研究通常需要大量的时间，而这些时间在越来越多的情况下是不可用的，特别是在我们老旧的Fluoview系统退役之后。新仪器将被纳入集成光显微镜核心（ILMC）设施，该设施目前服务于芝加哥大学的165多个实验室，以及来自邻近校园的大量多个协作用户。几乎所有的设施使用者都是美国国立卫生研究院资助的研究人员，其中大部分是芝加哥大学癌症中心项目资助的成员。使用新平台进行的大部分研究将直接支持与癌症、消化系统疾病、神经发育和残疾相关的研究。除了增加最先进的高速双色成像功能外，该平台还将作为一个额外的多色共聚焦平台，用于需要大量设备访问时间的活体样品。在高速共聚焦成像期间，同时共同观察两个或多个荧光探针的能力是一项关键需求，由于硬件的限制，现有设备无法满足这一需求。通过包含培养系统和xy扫描阶段，该成像系统针对活细胞观察进行了优化，这将允许用户在延时研究期间自动对准备过程中的多个位置进行成像，通过减少重复长时间实验以增加观察细胞数量的需要，大大提高了数据吞吐量。重要的是，该软件是直观的，并提供了用于存储采集协议和宏的实用程序。该软件与ILMC中其他系统的相似性将允许现有用户轻松过渡到新平台。核心工作人员将为用户提供支持和培训，以便迅速接受拟议系统所带来的技术进步。

DESCRIPTION (provided by applicant): We are requesting funds to purchase a Marianas Real Time Confocal SDC microscope equipped with 1) a Yokogawa CSUX Nipkow scanner; 2) a motorized spherical aberration correction (SAC) device; 3) five laser lines to enable use of a broad range of fluorophores, including those used for FRET; 4) high-speed laser illumination for bleaching (or photoactivation) studies of fluorescent targets; 5) a dual camera/ image splitter system with high speed Photometrics Evolve EM-CCD cameras; and 6) an enclosed CO2-regulated incubator. This confocal system is necessary to meet the immediate research needs of the major users and other faculty, as it provides the new features described above, particularly SAC, FRET, FRAP, and simultaneous two color imaging within a controlled environment. In addition, the Marianas Real Time Confocal SDC microscope will increase the capacity of available instruments, which is critical given the increasing numbers of live cell imaging studies. These studies often require large blocks of time that have been unavailable in an increasing number of cases, particularly after the decommissioning of our aged Fluoview system. The new instrument will be incorporated into the Integrated Light Microscopy Core (ILMC) Facility, which presently serves over 165 laboratories at the University of Chicago, as well as a large number of multiple collaborative users from neighboring campuses. Nearly all facility users are NIH-funded investigators with the majority members of the University of Chicago Cancer Center Program Project Grant. The bulk of research conducted using the new platform will directly support investigations related to cancer, digestive disease, and neurological development and disabilities. Apart from adding state-of-the-art, high-speed, two-color imaging capabilities, the platform will perform as an additional multi-color confocal platform for live samples, which require large blocks of equipment access time. The ability to simultaneously co-observe two or more fluorescent probes during high speed confocal imaging is a critical need that is not met by existing equipment due to limitations of hardware. The imaging system is optimized for live cell observations by inclusion of an incubator system and xy scanning stage, which will allow users to automatically image multiple locations in preparations during time-lapse studies, greatly increasing the data throughput by decreasing the need for repeating long experiments just to increase the number of cells observed. Importantly, the software is intuitive and provides utilities for storage of acquisition protocols and macros. The similarity of the software to other systems in the ILMC will permit easy transition of existing users to the new platform. Core staff will provide support and training of users to quickly embrace the technological advances enabled by the proposed system.