This proposal requests support for the acquisition of a confocal microscope and a remote access workstation to be added to an existing shared resource in the Dept. of Genetics at Case Western Reserve University. The system consists of a Leica TCS SP2 AOBS filter-free confocal mounted on a fully-motorized Leica DM IRE2 inverted microscope, environmental chambers, a vibration-free workstation, computers and software. This system will serve two functions: 1) it will replace an outdated and inadequate BioRad MRC600 confocal that has an unreliable stepper motor, limited capabilities in terms of image acquisition, and no 3-D reconstruction ? & remodeling software, and 2) it will update our facilities so that live-cell imaging can be performed. ? Since none of the microscopes in the current facility have environmental controls or motorized parts we are limited in our ability to perform live cell studies and complex research tasks that rely on high measuring accuracy and processing of large amounts of data. The Leica AOBS was chosen over other confocals since it: a) has 30-50% higher sensitivity for fluorescence emission than any other system; b) needs far less damaging illumination light power which is an advantage for live cell applications; c) allows deeper imaging in highly scattered specimens; d) can use any combination of 9 laser lines; e) allows simultaneous imaging of 4 fluorophores; and f) has software that eliminates crosstalk of completely overlapping emission spectra. The workstation will be housed in the current shared facility for examination of chromosomes, living cells, embryos, and organisms labeled with multiple fluorescent probes. It will complement two wide-field microscope workstations already utilized at capacity. The imaging director and advisory committee will oversee management of the system. The University will provide support for space allocation, renovation, installation, maintenance, and salary for the imaging director. The projects outlined in this proposal depend upon co- Iocalizations where it is essential to maximize resolution and color separation of overlapping emission spectra, and require the ability to assay the subcellular distribution of molecules and perform 3-D reconstruction. These requirements necessitate a confocal microscope with the highest resolution, sensitivity, and flexibility. Based on technical considerations and comparisons between confocal systems, we have determined that the Leica AOBS confocal is the best choice to meet the imaging needs of the faculty at CWRU. ? ?
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