This award will support the establishment of a multi-user two-photon excitation microscope facility in the Department of Cellular and Structural Biology at the University of Texas Health Science Center in San Antonio. The award will support the purchase of a Zeiss LSM 410 laser-scanning confocal microscope modified for non-descanned detection, and a Coherent Verdi 5 watt diode pumped solid state laser coupled to a Mira-900-F Femtosecond Modelocked Ti:Sapphire laser as the excitation source. The Ti:Sapphire laser will contain Coherent's new X-wave optic set, allowing the tuning of excitation wavelengths from 680-1000 nm without the need to change laser optics. This provides two-photon excitation from 340-500 nm, allowing most UV-and visible-wavelength absorbing dyes to be excited. Applications for this instrument include 1) Monitoring mitochondrial Ca2+ and pH during apoptosis without exciting nearby slices (such as in the overlying or underlying mitochondria). 2) Monitoring temporal and spatial interactions between apopototic regulatory proteins in single intact living cells using two-fusion FRET. 3) Identifying the critical residues of calreticulin which mediate inhibition of pulsatile Ca2+ release from the ER. 4) Examining the molecular control of Ca2+ signaling by Ca2+ binding proteins, Ca2+-ATPases, and mitochondria. 5) Evaluating genotypic abnormalities of precursors and stem cells in archived breast cancer samples at the level of RNA and protein. 6) Dissection of protein-protein interactions that underlie the endocytic part of the neurotransmission process. 7) Photoreceptor cell patterning. And 8) Simultaneous localization of different cells and cytokines/addressins/chemokines in thick sections of the CNS.
