The overall goal of this proposal is to include within the Microscopic Imaging Core of the UCLA Brain Research Institute a two-photon laser scanning microscope (TPLSM) equipped for investigation of physiological processes in living cells. The intention is to complement other imaging technologies, such as confocal microscopy and electron microscopy, which are already available within the Core. Many important new experimental avenues will be made possible by utilizing two-photon excitation in a cellular imaging system equipped for complex cellular manipulation using electrophysiology and microinjection. The TPLSM allows measurement of fluorescence within a diffraction limited volume, without the need for a confocal pinhole and with minimal photobleaching and photodamage outside the focal plane. This makes two- photon excitation ideal for measurements in living cells. In addition, the use of longer wavelengths for illumination allows imaging deep within tissue. The projects in this proposal come from a wide spectrum of neuroscience faculty with different research interests. Several of the major users propose to measure ion concentrations (Ca2+ or Zn2+) in cells within intact tissue. These include slices from various regions of the brain (Istvan Mody, Chris Colwell, Michael Levine), retina (Nicholas Brecha), neuroendocrine organs (Nancy Wayne, Jonathan Monck), and skeletal muscle fibers (Julio Vergara). Another user will study inspiratory-modulated synaptic inputs to various neuron types with fluorophores (Jack Feldman). Several users are interested in the organization of the nervous system during development and propose to use fluorescent labels (green fluorescent protein (GFP), carboxycyanines) to trace neural projections or synaptic connections (Anthony Campagnoni, Susana Cohen-Cory, Ellen Carpenter). Two users are tracing GFP targeted proteins expressed in specific neuronal populations in transgenic mice and C. elegans (Anthony Campagnoni, Alex van der Bliek). Another user proposes to use GFP to study receptor internalization in neurons within intact gastrointestinal tissue (Catia Sternini). The availability of the TPLSM will significantly enhance and expand the research directions of these investigators, most of whom have ongoing NIH-funded research programs.
