This revised Shared Instrumentation Grant application requests funds to be used in the purchase of a Leica TCS SP2 AOBS Laser Scanning Confocal Microscope. This instrument will be integrated into the H. Lee Moffitt Cancer Center & Research Institute Analytic Microscopy Core Facility. The new confocal will replace an 8-year old aging confocal system that does not meet the needs of our PHS funded investigators. The existing confocal is an aging 2-PMT / 2-Laser system that does not contain the need excitation and detection capabilities to required for multicolor quantitative work required by our investigators. The proposed Leica confocal system is modern 4-PMT system coupled with 5 laser lines sources (Near UV 405, Blue, Green, Orange and Red) to give our 7 PHS funded investigators the needed resources to advance their research efforts. Specific, factors in selecting the Leica confocal were the AOBS and the SP spectral detector. The Leica AOBS system utilizes a filterless design to produce brighter images when compared to other confocal systems we evaluated for this proposal. Unlike other spectral confocal systems that use a grating based detector, the AOBS SP spectral detector uses a prism for greater collection efficiency allowing detection ranges to be freely defined by the user. Another """"""""value added"""""""" feature that system will bring to the shared resourced facility are the spectral unmixing capabilities that can separate fluorochromes with very close or even overlapping emission ranges. Similarly the software driving this microscope was deemed as providing the most user friendly interface of the three confocal systems evaluated. The revised proposal is enhanced by a strengthened institutional supporting this instrument. The cost of this instrument will be matched by Moffitt Cancer Center with the purchase of a yearly $25,000 service contract, a dedicated space to house the instrument and salary support for Analytic Microscopy Core Facility. In addition, the Moffitt Cancer is committing to purchasing ancillary items such as live cell imaging equipment, collection of Leica add on software modules and the Imaris Bitplane Suite of software for 3D colocalization and volume rendering analysis. ? ? ?
