This proposal addresses the need for additional confocal microscopy capacity and capability at the W.M. Keck Biological Imaging Facility located within the Whitehead Institute. This core facility provides confocal training and service to 20 Whitehead labs, 39 from MIT and 4 from other institutions totaling 3732 hours of use between 1998 and 2003. The existing ZeissLSM510-Meta is based on point-scanning technology and is thus fundamentally limited in image acquisition speed. After evaluating several competing technologies that would address the need for additional confocal capacity as well as the ability to image fast biological processes, we determined that the second generation spinning-disk system from Perkin Elmer, the UltraViewRS, was most appropriate. This system will allow users to collect time-resolved 2D or 3D confocal data at over 10x the rate of the existing LSM510. While the LSM510-Meta will remain a core technology within the Facility, especially suited to imaging thick fixed tissue samples, the complementary capability provided by the UltraViewRS will enable an additional and broad range of important biological investigations to be undertaken. In the project descriptions section, we have described studies already underway in 7 Whitehead and 3 MIT laboratories that require these enhanced confocal capabilities. These projects include investigation of cytoskeletal structure during cell motility, embryonic development, microbial pathogenesis, protein conformational diseases, senescence, membrane dynamics, and erythropoiesis. The proposed system will allow excitation of relevant live-cell probes including all major GFP variants as well as commonly-used chemical probes and fixed-cell fluorophores. Apart from the enhanced speed of data collection, the proposed system also benefits users via reduced sample photo-damage, which is especially useful for those investigators studying embryonic development. Two other key system components, an environmental chamber and a motorized XY-stage, will provide much needed live-cell capabilities to the Facility and allow efficient use of the instrument through automated multi-point data collection regimes. The Whitehead Institute has an excellent track record of maintaining and further investing in NIH-funded shared instrumentation as documented in the ever-increasing use of the resources within the W.M. Keck Biological Imaging Facility.
