We propose to acquire a Leica TCS SP2-MP Spectral Confocal Microscope to add to the facilities available in the Optical Biology Core Facility of the Chao Family Comprehensive Cancer Center at the University of California, Irvine. Confocal microscopy has been available as a shared facility at UCI since 1987, making it possible to study the tissue and subcellular localization of specific molecules in fixed tissues using fluorescent labels, often without the need for tissue sectioning. Recent advances in the design of genetically encoded fluorescent tags based on the jellyfish Green Fluorescent Protein (GFP) now make it possible to examine the localization of proteins in living, rather than fixed, tissue. An extension ofthis technology is to use pairs of mutant GFP tags that fluoresce at different wavelengths to detect fluorescence resonance energy transfer (FRET) between the two tags, revealing important informnation on molecular interactions between the tagged proteins in either fixed or live cells. Similar methods make it possible to study conformational changes in an individual protein within a cell by detecting changes in FRET between fluorescent labels at different locations in the molecule. Other applications of FRET microscopy allow monitoring of calcium levels, of protease activity, of membrane potential, and of neuronal activity patterns in live cells and tissues. These technologies promise to lead to entirely new research directions in developmental and cell biology and neurobiology. However, the approach will require the ability to discriminate excitation and emission wavelengths at a higher level of selectivity than is possible with available confocal microscopes. The proposed microscope solves this problem by using prisms rather than filters to select emission wavelengths. Preliminary data included in the application shows that two-photon excitation provides a dramatic improvement in separation of FRET signal from background, so this proposal includes a two-photon upgrade. The instrumnent will greatly facilitate not only FRET analysis but also applications requiring multiple labels, especially in living cells, and the two-photon excitation will provide a number of advantages including the ability to penetrate more deeply into tissue than is possible with conventional excitation. The instrument will be installed in the Optical Biology Core Facility and will be available to any qualified user on a recharge basis. Projects that are ready to use this instrument include studies of interactions between scaffolding proteins and signal transduction pathways controlling growth, interactions between signal transduction proteins and between transcription factors controlling patterning in embryos, monitoring of potassium and calcium channels, trafficking of the T cell receptor, association of glutamate receptor subunits, and many other protein interactions in development and disease.
