Laser Scanning Confocal Microscope/Multicolor Spectra

University Of Colorado Denver, Aurora CO

Investigators

Abstract

DESCRIPTION (provided by applicant): We request funds to purchase a Zeiss LSM 510 Meta confocal laser scanning microscope. Seven Major Users and five Minor Users, well funded by NIH (e.g., Principal Investigators on 18 R01 s, 1 PPG, 2 Center Grants, 3 R32 Training grants), describe 18 research projects that span a broad range of biological questions and will collectively utilize all of the capabilities of the instrument. The applicants represent seven different departments in the School of Medicine. The Dean has made a commitment of $250,000 for support of the instrument, which will be housed in and managed by a core Light Microscopy Facility. Most Users have had extensive experience with various imaging techniques, and have acquired sound preliminary data that illustrate both the need for this equipment and the feasibility of the proposed experiments. A comprehensive inventory of existing equipment and careful scrutiny of proposals make clear that none of the projects can be accomplished with current equipment at the Health Sciences Center or any other campus in reasonable proximity. A stable and time-tested management plan that has emerged over the past 10 years of continuous administration of shared optical facilities by the same personnel will insure that the instrument will be well maintained, that it will become self-supporting, and that it will be used intensively by well-trained scientists drawn both for the User Group and, over time, from the larger population of scientists in the region. The specific proposals all relate to existing funded R01 research grants, but were not originally included as part of those applications because of the lack of equipment to perform them. Almost all projects involve studies of trafficking of molecules and organelles in living cells, using Green Fluorescent Protein and its analogs as markers. The most important attribute of the requested instrument is the ability to produce emission spectra through 'lambda stack' acquisition and 'linear unmixing' of multiple fluorophores, characteristics that were documented first hand by Users in pilot studies. Other projects involve tracking movements of molecules by Fluorescence Recovery After Photobleaching (FRAP), and protein-protein interactions with Fluorescence Resonance Energy Transfer (FRET). Extensive User experience with these techniques insures that the hypotheses put forward are testable, and that results will be reliable.