Upgrading an existing NIKON A1-confocal with LUNV Laser Launch and DU-G detector

University Of Tx Md Anderson Can Ctr, Houston TX

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Abstract

PROJECT SUMMARY/ABSTRACT This request is for upgrading an existing A1 NIKON confocal with a LUNV Laser Launch and DU-G detector unit. The A1 confocal is currently located in the BSRB Microscopy Facility, in the Genetics Department at UT-MD Anderson Cancer Center, under the leadership of Dr. Lozano. The BSRB Microscopy Facility was founded in 1998 and expanded its capabilities to cope with high demand to its current configuration. Our current A1 confocal was acquired in 2011 and it has been upgraded over the years and currently is integrated into a NIKON Ti microscope body equipped with motorized stage and CO2 stage-top incubator. Our system is composed of 7 laser lines: 405nm, 561nm and 647 nm are solid state lasers, and 453nm, 477nm, 488nm and 514nm are delivered by an Argon Ion Gas laser. Although the system is under service contract, the multi-line argon laser is no longer being supported by NIKON. A replacement option is available for the 488nm laser only with a solid state single-line laser, but since the existing laser launch only supports four lasers, we will not be able to replace the 453nm and 514nm lines, which we currently use. Thus, in order to keep our current functionality, we need to replace our existing laser launch with a new one capable of running at least 6 single- line solid state lasers. This new laser launch would also provide bright illumination with improved stability and reduced maintenance. Although our investigators are experts in good sample preparation, many of the projects involve samples that are limited in brightness for various reasons. Examples include: Dr. Behringer's group (major user) works with a variety of endogenous expressed fluorescent proteins (requiring multiple laser lines including 453nm and 514nm), that are sometimes weakly expressed, to study defects in reporter mice reproductive tract development; Dr. McCrea's group works with 3D image rendering of dendrites (about 2 mm thick) and performs co-localization analysis of two novel protein complexes PdLim5:delta catenin and Mag- 1:delta catenin within dendritic processes. He also plans to do image-based Fuorescence Resonance Energy Transfer (FRET) studies to look at the activity and spatial distribution of RhoA, Cdc42 and Rac1 (by utilizing established GTPase activity FRET biosensors) at the dendrites as part of his recent NIH R01 submission. Dr. Andrew Gladden (junior faculty) currently looks at FRET signals of established vinculin tension sensor (VinTS) at the focal adhesions, which are very small in size and restricted to the base of the cell. Dr. Galdden also does multi-color live cell imaging of three-dimensional lumen and organotypic cultures. The majority of the projects cited in this application require confocal sectioning microscopy with optimal spatial resolution, multiple excitation lines covering the 405nm-640nm spectral range (including 453nm and 514nm lasers), high depth penetration as provided by laser excitation, high detection efficiency, and improved sensitivity. Therefore the acquisition of solid-state lasers and hybrid GaAsP detectors are necessary to provide adequate excitation illumination and to improve quantum efficiency and the quality of our data.