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.
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|Edelblum, Karen L; Shen, Le; Weber, Christopher R et al. (2012) Dynamic migration of Ã½Ã½Ã½Ã½ intraepithelial lymphocytes requires occludin. Proc Natl Acad Sci U S A 109:7097-102|